Synthetic Polynucleotides

Synthetic Polynucleotides

A. M. MICHELSON, J. MASSOUL&, Institut de Biologie Physico-Chimique, Paris, France AND

W. GUSCHLBAUER~

Dkpartment de Biologie, Centre $Etudes Nuclkaires de Saclay, Gif-sur-Yvette, France

I. Introduction . . . . . . . . . . . . . . . . 11. Preparation of Polynucleotides . . . . . . . . . . . 111. Techniques for Investigating the Physical Chemistry of Polynucleotides . . . . . . . . . . . . . . . . A. MoleculariWeight Determination by Ultracentrifugation, Diffusion, and Viscosimetry . . . . . . , . . . . . B. Thermal Stability . . . , . . . . . . . . . C. Characterization of-Nucleotide Material by Ultraviolet Absorption Spectra . . . . . . . . . . . . . . . D. Optical Rotatory Dispersion and Circular Dichroism . . . E. Titrimetry. . . . . . . . . . . . . . . . F. Infrared Spectroscopy . . . . . . . . . . . . G. X-Ray Diffraction, Low-Angle X-Ray Scattering, and Light Scattering . . . . . , . . . . . . . . . . H. Nuclear Magnetic Resonance (NMR) and Electron Spin Resonance (ESR) . . . . . . . . . . . . . . . I. Electron Microscopy. . . . . . . . . . . . . J. Equilibrium Density Gradient Centrifugation . . , . . IV. Homopolynucleotides . . . . . . . . . . . . . A. Polyguanylic Acid , , . . . . . . . . . . . B. Polyinosinic Acid. . , . . . . . . . . . . . C. Polyuridylic Acid. . . . . . . . . . . . . . D. Polyadenylic Acid . . . . . . . . . . . . . E. Polycytidylic Acid . . . . . . . . . . . . . V. Polynucleotide Complexes . . . . . . . . . . . . A. Complexes between Poly G and Poly C , . . . . . . B. Complexes between Poly A and Poly U . . . . . . . C. Thermal Dissociation of Poly A . Poly U and Poly A * 2 Poly U D. Stability of Poly A . Poly U and Poly A . 2 Poly U at Alkaline E. F. G. H.

pH . . . . . . . . . . . . . . . . . . Stability of Poly A . Poly U and Poly A 2 Poly U at Acid pH Base Pairing in Poly A . Poly U and Poly A . 2 Poly U . . . Thermodynamics of the Interaction between Poly A and Poly U Alternating Copolymers. . . . . . . . . . . , 9

'Helen Hay Whitney Foundation Fellow. 83

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VI.

VII. VIII. IX. X. XI.

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I. Association of Poly A and Poly I . . . . . . . . . J. Associations Involving Hypoxanthine and Cytosine. . . . Role of Sugar Phosphate Backbone . . . . . . . . . A. The Phosphate Residues . . . . . . . . . . . B. TheSugsr. . . . . . . . . . . . . . . . C. Right- or Left-Handed Helices, Parallelism, andiAntiparalle1ism. . . . . . . . . . . . . . . . . . Reversibility . . . . . . . . . . . . . . . . Displacement Reactions. . . . . . . . . . . . . Polynucleotide Analogs . . . . . . . . . . . . . Theory and Practice of Ilelix-Coil Transitions . . . . . . Factors Governing Structure . . . . . . . . . . . References. . . . . . . . . . . . . . . . .

116 116 119 119 120 122 122 123 125 130 131 134

1. Introduction Synthetic polynucleotides are of considerable interest not only from the biological viewpoint of code cracking but also as grossly simplified models for the study of the numerous physical properties manifested by nucleic acids. This review is limited to physical studies of structure in synthetic polynucleotides, including homopolymers of naturally occurring nucleotides and of various analogs, but is by no means exhaustive. For earlier reviews see reference 1.

II. Preparation of Polynucleotides Perhaps the least understood of enzymes catalyzing the formation of high molecular weight polymers is polynucleotide phosphorylase ( 2 ) . Although the function and detailed mechanism of action of polynucleotide phosphorylases remain somewhat obscure, such enzymes, isolated from various bacterial sources, have proved to be of great utility for the preparation of polyribonucleotides from the appropriate ribonucleoside 5’-pyrophosphates. Large quantities of polynucleotides can be prepared relatively easily from commercially available diphosphates. I n general, primers are not mandatory, and the enzyme shows a very low substrate specificity with respect to the purine and pyrimidine base. A wide variety of polyribonucleotide analogs can thus be prepared by the action of polynucleotide phosphorylase on the analog ribonucleotide pyrophosphates. Examples include NMe-UDP, 5Br-UDP, 5F-UDP, N6-hydroxyethyl-ADP, and isoadenosine diphosphate (3,4 ) . Whereas homopolymers of A, C, I, and U have been available for some considerable time, the preparation of poly G remained extremely difficult until recently. Suitable modification of the incubation conditions has rendered the preparation in quantity of this polymer equally feasible (6). I n general, the products are somewhat polydisperse.

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Although preparation of an analog diphosphate followed by polymerization is useful in many cases, an alternate approach lies in the modification of a preformed “natural” polynucleotide. Of course, any such modifications must be achieved under experimental conditions that do not lead to extensive degradation of the polynucleotide chain. Bromination and methylation of various nucleic acids have been described ( 6 ) and from a preparative viewpoint the chemical methylation of homopolyribonucleotides has been extensively studied (7). Other treatments include the action of ultraviolet light (8),hydroxylamine (9),and nitrous acid ( l o ) , and the acetylation of cytosine amino groups with acetic anhydride (11). Apart from polynucleotide phosphorylase, other enzymes catalyzing polynucleotide formation have been used. DNA polymerases are of use not only for replication of given DNA’s but also for synthesis (under suitable conditions) of poly (dA-dT) (12) (alternating) and poly dG * poly dC (homopolymers) (15) in addition to a variety of deoxy analogs. PolydA has also been prepared using a primer ( 1 4 ) . Similarly, RNA polymerase can give rise to polyribonucleotides of known sequence by the use of small, chemically synthesized oligodeoxynucleotides ( 1 5 ) . As a result of “slippage” during transcription, high molecular weight polynucleotides are obtained. Physical studies of such products have not been reported in detail as yet.

111. Techniques for Investigating the Physical Chemistry of Polynucleotides This section very briefly reviews some technical aspects of the physical chemistry of polynucleotides. For more detailed descriptions of the techniques mentioned, reference is made a t appropriate points to pertinent books, reviews, and specialized articles.

A. Molecular Weight Determination by Ultracentrifugation, Diffusion, and Viscosimetry

Ultracentrifugation in combination with diffusion and viscosity measurements has been widely used in the field of nucleic acids and proteins to obtain information on the size and shape of such macromolecules. Several books (16-19) and specialized reviews (ZO-24) describe these techniques and their applications. A priori, any molecular weight determination on a polymer th a t is not physically and chemically uniform will yield an average molecular weight. I n the field of the nucleic acids, two kinds of molecular weight

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are frequently used : the number-average molecular weight, M,, and the weight-average molecular weight, M,. They are defined as follows :

and

where f ( M )dM represents the weight fraction of material with molecular d M , Ni the number of molecules of kind i weight between M and M (i-mers) present in the mixture, Mi the molecular weight of the i-mer, and g the number of grams of i-mer. The ratio Mw:M , is a measure of the homogeneity of a polymer sample (see Table 17-1 in ref. 19). I n the ideal case where all molecules have the same chain length and thus the same molecular weight, M , equals M,. For nucleic acids this can actually be the case and for tRNA this ratio is close to unity (25).I n the case of synthetic polynucleotides, this ratio will always be very much larger. The number-average molecular weight is generally determined by osmotic pressure (,%a). More frequently noted in the literature are molecular weights obtained by sedimentation velocity or sedimentation equilibrium measurements. While the latter measurements give the M,, the former give the actual molecular weight a t the sedimenting peak. Quite frequently only the sedimentation from velocity measurements is quoted. This has the following connection with the molecular weight (in a simplified form)

+

(%?): s20,w

=

M(l

- Pp) Nf

(3)

where szo,w is the sedimentation coefficient (at 2OoC and corrected for water), M the molecular weight, the partial specific volume of the solute, p the density of the solution, N = 6.02 X loz3 (Avogadro’s number), and f the frictional coefficient, which is a function of size, shape, and hydration of the sedimenting polymer. Because of the greatly different values f can take depending on these properties, the sedimentation

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coefficient will mean quite different things for structured and unstructured polymers. Certainly there exist correlations between szo and molecular weight for random-coiled and structured polynucleotides ; these calibration plots, however, are not necessarily applicable to any other polymer. Thus it is now well-established that, for instance, oligonucleotides of guanosine can easily associate and assume a considerably higher molecular weight than if each polymer chain were free; even GMP can form polymerlike gels. It has been noted that, in a sample of the copolymer of U and G, poly (U,G), with an szo,u, of 5.6, some 80% of the materials is dialyzable in 4 N urea. Thus the sedimentation constant can frequently be a source of error when highly polymerized polynucleotides are used.

B. Thermal Stability What is called, in nucleic acid chemistry, the “melting point” is an operational term for a temperature-dependent change in structure. Therefore the expression “transition temperature” is much more appropriate. It describes the temperature a t which a helical polynucleotide assembly rearranges itself either to a different helical arrangement or to the randomly coiled state. This thermal transition, long known in polymer and protein chemistry, was first shown to be a characteristic property of nucleic acids by Thomas (66).Since then, T, determinations have been used routinely to characterize nucleic acids and polynucleotides. This transition from one helical to a different helical form or to the coiled state is accompanied by changes in various physical properties of the polynucleotide, for example in ultraviolet light absorption or optical rotatory dispersion (ORD). Changes in infrared spectra, circular dichroism, and buoyant density also occur (see below). It has been established that the T, is dependent on ionic strength, divalent cations being more effective than monovalent salts (by a factor of about 104). Further, it is now quite clear that, below a limiting molecular weight, chain length becomes a determining factor. As already established for DNA, the content of G and C influences the thermal stability of polynucleotides. Theoretical considerations of helix stability in polynucleotides are discussed in Sections X and XI.

C. Characterization of Nucleotide Material by

Ultraviolet Absorption Spectra Nucleic acids generally possess an absorption band around 260 mp. This absorption band (which is determined by the purine and pyrimidine bases) is changed when the polynucleotides form secondary structures. The hypochromicity and hyperchromicity are functions of various en-

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ergy transitions arising from interactions among the bases. These interactions are weakened when the secondary structure is destroyed, and the increases (hyperchromicity) . Hyperchromicity can absorption near ,A be caused by increased temperatures, ionic changes, or radiation. Most frequently the thermal dissociation is studied. In the case of perfect alignment, as in DNA, an abrupt change in absorption occurs over a very narrow temperature range. This so-called “cooperative” effect shows that base pairs are not broken independently, but that an all-or-none process takes place; i.e., in a hydrogen-bonded helical complex containing homopolynucleotides, the entire chain is dissociated (by heat or changes in pH, for instance). The temperature a t which this fusion takes place is called the melting temperature or T,. It is generally defined as the temperature of the mid-point of the thermal transition. Felsenfeld and Sandeen (27) have introduced another definition

where ADi is the change in absorption in the temperature interval around Ti. This definition is particularly useful for cases with broad melting profiles. I n cases where the melting step occurs within a few degrees, T, and T%will be essentially equal. 1. DIFFERENCE SPECTRA It is now established that hyper- or hypochromicity a t a given wavelength is a function of the bases involved in the polynucleotide structure. It is for this reason that determinations of T,, till recently confined to measurements a t 260 mp, are now being extended over a wider range of wavelengths. To illustrate the possible differences as a function of composition, the difference spectra of the following reactions are shown in Figs. 1 and 2.

+ + + +

poly A * poly U + poly A p01y U poly A 2 poly U -+ poly A 2 poly U poly G . poly C + poly G poly C 2 p l y G poly C -P 2 p01y G p01y C

-

One can readily see that transitions involving complexes between A and U (128)are vastly different from those involving G and C (29), and further, that ,double- and triple-stranded transitions also present significant differences. Such differences have permitted the detailed study of thermal transitions of both complexes involving poly A and poly U. Difference spectra have also been used for the examination of secondary structure in nucleic acids (127,3e56). All these studies involve some method of fitting the difference spectra of A U and G C to a

+

+

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I

I

I

I

I

E

E

4

2 x

W

2

0

1

1

,

1

240

1

1

260

1

280

,

30

FIQ. 1. Molar difference spectra for poly A . polyU and poly A * 2polyU between 20 and 85°C [after Massouli6 et d.(98)l.

r

I

I

240

I

I

I

260

200

1

300

A (mp)

FIG.2. Difference spectra (not on a molar basis) for poly G . poly C and 2 poly G poly C [after Pochon and Michelson (39)1.

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difference spectrum of a given nucleic acid. These investigations have given good results for DNA, the structure of which is known to be completely helical, and have given rise to suggestive results for various RNA’s for which the secondary structures are as yet unclear. The seemingly trivial question of the “strandedness” of RNA’s, known in the case of a few RNA’s to be double-stranded, apparently cannot be solved by difference spectra (36,36). 2. MIXINGCURVES(METHODOF CONTINUOUS VARIATION) This method, originally described by Job (37) permits the determination of the stoichiometry of complexes formed between two components.

Fraction U

FIG.3. Typical mixing curve at 260 mp between poly A and polyU in 0.2 M Na+ [after Massouli6 (In)].

It consists in studying a characteristic property as a function of the concentration of the two components A and B in the mixture. I n the

case of polynucleotides the hyper- or hypochromism of the mixture is followed. I n practice, a series of mixtures is made in each of which the total concentration of both components is constant, and only the ratio [A]/[B] is changed. (As in most other instances, all concentrations refer to monomer units of the polymer.) A mixing curve is then obtained by plotting the change in absorption a t a given wavelength against the ratio of concentrations (Fig. 3). I n the case of no interaction between

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the two components the mixing curve becomes a straight line connecting the two points for the pure components. A sharp intersection between the two branches shows the stoichiometric mixture. Curved or flattened intersections indicate that the mixture is not thermodynamically equilibrated. Frequently such curved intersections sharpen considerably if the measurements are repeated some time later. Felsenfeld (38)investigated theoretical models for the interaction of linear polymers. His studies led to the conclusion that sharp intersections correspond to the geometrically (and thermodynamically) most favorable arrangement ; i.e., the maximum number of base pairs are formed. This implies automatically a “slipping” mechanism in which the chains already associated disintegrate and reform base pairs locally. This hypothesis has been confirmed by studies with oligonucleotides. “Mixing curves” were first used with polynucleotides to show the interaction of polyA and polyU to give a complex resembling DNA (39) and a second complex involving the binding of two polyU strands for each polyA strand ( 4 0 ) . Since then, the mixing curve has become a standard criterion for evaluating polynucleotide complexes. Recently the use of mixing curves has been refined by use not only of measurements near A,,, but also other, more characteristic wavelengths. It is thus possible to determine the formation of the two-stranded complex poly A poIy U a t 283.5 mp where the three-stranded complex shows neither hypo- nor hyperchromicity ( 4 l , 4 2 ) . a

3. CHARACTERIZATION OF NUCLEOTIDES AND POLYMERS BY ULTRAVIOLET ABSORPTIONSPECTRA

The fact that the various bases and their corresponding nucleosides and nucleotides possess different absorption spectra has long been used for the determination and characterization of nucleotide material. For summaries of spectra we refer to the work of Fox and Shugar (43, 44), of Voet et al. ( 4 5 ) ,and of Beaven, Holiday, and Johnson ( 4 6 ) .Recently, Vengstern and Bayev (47) have published a series of thirty-three spectra, including those of oligonucleotides a t various pH values. Kerr and Seraidarian (48) and Steiner (49) exploited the differences in absorbancy of nucleotides a t various pH values and used the absorbancy a t characteristic wavelengths to determine approximate base compositions in mixtures. Reid and Pratt (50) first used computers to resolve spectra of nucleic acid hydrolyeates into their components. A similar method, though less accurate, has been employed by Vasilenko et al. ( 6 1 ) . Recently, several attempts have been made to use ultraviolet absorption spectra for the deteflmination of base compositions of nucleic acids and

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even to determine certain nucleotide sequences. Lee et al. (5g) have used the least square method of Reid and Pratt (50) with a theoretical error estimation and claim to be able to resolve up to nine different components simultaneously. Guschlbauer et al. (53), using ultraviolet absorption spectra of polynucleotide and RNA hydrolyzates before and after UV irradiation (which changes the spectra of the pyrimidine nucleotides) , have been able to determine base compositions to an exactness of 1% or less. Pratt et al. (54) further developed the computational aspect by using linear programming to determine exactly the composition of oligonucleotides without having recourse to hydrolysis. D. Optical Rotatory Dispersion and. Circular Dichroism

Electromagnetic radiation, upon interaction with matter, can be absorbed, refracted, scattered, or diffracted. If polarized radiation, which is split into a left and a right circularly polarized component, interacts with a molecule no change will be observed if the component beams are transmitted with equal velocity. If the refractive indices of the medium for the left and the right circular polarized light of a given wavelength are different (only very small differences suffice), the two beams are transmitted with unequal velocities and are out of phase upon recombination after passage through the medium, and the plane of polarization is rotated. Thus optical rotation is observed if a medium transmits two circularly polarized component beams with unequal velocity. If the absorption of the left and right circular polarized light is also different, the recombined light beam, after passage through the medium, becomes elliptically polarized. This phenomenon is called “circular dichroism” and causes the so-called “Cotton effect” (Fig. 4). An extensive discussion of the interrelation of absorption, optical rotation, and circular dichroism will be found in the book of Kauzmann ( 5 5 ) . Biot (56) first observed (in 1817) that optical rotation depends on wavelength. The dispersion of the optical rotation was first stated mathematically by Drude in 1906 (67) in an empirical way:

where [a]is the specific rotation, K a constant, h the wavelength a t which rotation measurements were made, and hq the wavelength of the absorption maximum of the chromophore at which the dispersion curve shows zero rotation (Fig. 3). By using the [a]values outside the Cotton region one can determine A,, with reasonable accuracy. This is done by plotting

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l / [ a ] against X2. If this plot does not yield a straight line, higher-term equations of the form

Optical rotatory dispersion (ORD) has been widely used for structure and absolute configuration determinations in organic chemistry [for a summary, see the book of Djerassi (68)1. Neither Eq. ( 5 ) nor (6) will, however, satisfy optical rotation changes caused by structural assymetry, such as secondary or tertiary structure.

FIG.4. Optical rotatory dispersion of poly BrU in 0.1 M NaCI, 0.05 M Na cacodylate, pH 7, 0.01 M MgClz at 2 and 20°C [after Michelson ( 1 1 ) l .

Moffitt and co-workers (59-61) have derived several empirical equations; the most widely used one is the following:

Here [m’]is the mean residue rotation, n the refractive index, M , the mean residue weight, and and &, two constants, a,, consisting of two terms, one depending on the intrinsic residue rotation, the other, as well as a,, depending on the helical configuration. It still must be kept in mind that this equation is empirical and numerous theoretical studies

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(62-64)have been undertaken to interpret the Moffitt equation, which has proven extremely useful in protein and polypeptide chemistry. I n recent years ORD measurements have been extended to polynucleotides and nucleic acids (66). The availability of several new instruments that record ORD curves automatically has produced a numTinoco ber of basic publications from the laboratories of Yang (66-68), (69-70),and Zamecnik (71-73). As mentioned before, the differential absorption of left and right circular polarized light will give rise to circular dichroism. If this differential absorption can be determined, the rotational strength (RJ can then be calculated (74):

where EL and E R refer, respectively, to the molar extinction coefficients for left- and right-handed circularly polarized light a t frequency v ( h is Planck’s constant, c the velocity of light, and N Avogadro’s number). Circular dichroism was virtually unavailable until recently. Grosjean and Legrand (76) designed an electrooptical apparatus for the measurement of circular dichroism, and Holzwarth (76) has developed an adaptation for the Beckmann DK-2A spectrophotometer. The apparatus of Grosjean and Legrand (Roussel-Jouan dichrograph) as well as an adaptor for the Cary 14 spectrophotometer for circular dichroism measurements are now commercially available. Brahms (74)has described a series of pioneering investigations on the circular dichroism of polynucleotides and nucleic acids (77-84).

E. Titrimetry

I

The simplicity and reproducibility of spectrophotometric titration has resulted in a wide use of this technique for polynucleotides and their complexes, and for nucleic acids. The titration is performed in a spectrophotometer cuvette with a fitted glass electrode, a magnetic stirrer, and a thin tube connected to a milliliter syringe (86). Changes in absorbancy and in pH are recorded as a function of added titrant, thus providing a convenient method for the determination of apparent pK values. These are characteristic of the material and of the salt concentration.

F. Infrared Spectroscopy If the incident electromagnetic radiation has a wavelength in the micron range, its energy will be sufficient to cause vibrations in the molecular structure of a molecule. Infrared ( I R ) spectra measure the separation between vibrational energy levels. The theoretical background

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of IR spectra can be found in the books of Tanford (19), Kauzmann (55),and Bellamy (86). I n polynucleotide chemistry, the use of IR spectra is rather limited, although they are very widely and routinely used in organic structure research. Apart from studies by Angel1 (87), Tsuboi et al. (88-90),and Miles (91-100) on bases, nucleosides, and nucleotides, Miles et al. have used this technique to study polynucleotide structures. Detailed discussion of their results is found in Section V, dealing with polynucleotide interactions. Many less popular techniques have also been utilized for the study of polynucleotides. Their lack of popularity is often due either to the fact that the equipment involved is very expensive or that extensive experience is needed to obtain meaningful results. Frequently a purity of material is required that is not easily obtainable from biological sources. However, the application of these techniques is growing as more problems in biochemistry necessitate novel approaches.

G. X-Ray Diffraction, Low-Angle X-Ray Scattering,

and light Scattering These three techniques have many physical principles in common but the first two provide more precise information. Only a very short introduction to these techniques is given. A brilliantly written and frequently amusing review with examples from “everyday” Cambridge life for noninitiated members of the nucleic acid field has been written by Crick and Kendrew (102).Kendrew (105) has also presented the mathematical foundations of X-ray diffraction. Low-angle X-ray scattering has been reviewed in this series by Luzeati (104). Luzzati (106) and Kratky (106) have discussed the mathematical and technical details of this method. Further treatises on these subjects are those of Porod ( l o r ) , Witz (108), and Stokes (109). Light scattering has been extensively discussed by Tanford (19) and by Geiduschek and Holtzer (110). Any electromagnetic wave can be scattered, X-rays by the electron density of a crystal (or any other molecule for that matter), light by whole (and preferentially large) molecules. This can be deduced from s = 2 sin

e/x

(9)

Here s is the direction of the propagation of a scattered ray. For a sphere with radius r the intensity distribution of scattered radiation I ( s ) will be given by sin sr - sr cos sr

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It can be shown that, for all practical purposes, only measurements in the range where

rs = X

(11)

are useful. Since s is limited by definition [see Eq. (9)] to values equal to or smaller than 2/h, this means that the resolving power will be reached with r = X/2. This limits the applicability of X-ray scattering to the angstrom range, and that of light scattering to the range of thousands of angstroms. 1. X-RAYDIFFRACTION

If molecules are arranged in ordered patterns, as in a crystal, X-rays will be scattered by the electron density of the molecules and will be caught on the photographic plate in an ordered manner. This diffraction pattern is called the “reciprocal lattice.” Each spot will correspond to a wave in a wave analysis of an electron density (Fourier analysis). The position of every spot is indicative of the wavelength and the direction of the wave. Crystal patterns show ordered arrays of spots, which is not the case with powder or fiber diagrams. I n powder diffraction patterns, the totality of the small crystallites gives rise to a pattern of all crystallites together. They show sets of more-or-less sharp concentric rings, the radii of which correspond to the spacings of the principal lattice planes in the crystals. Fiber patterns are generally less perfect than crystal patterns. They can be thought of as a crystal that continuously revolves around one (the helix) axis. From fiber diagrams, one can generally obtain the crystallographic repeat unit along the fiber axis. The dimensions of the unit cell can only rarely be obtained. Frequently, this information, as well as other crystallographic parameters, can be obtained by model building, calculation of the X-ray diffraction pattern (Fourier synthesis) of the constructed model, and comparison with the pattern obtained. By this method, often all possibilities except one can be excluded. 2. LOW-ANGLE X-RAYSCATTERING

The distinction between X-ray diffraction and low-angle X-ray scattering is by no means arbitrary, but is based on the fact that pure liquids (and many crystals) show no X-ray pattern below an angle of about 10”. This is apparent from Eq. (9). For a value of B = 10” and X = 1.5 A, r will be about 4 A. This value of r will increase rapidly with decreasing 8 , thus leaving the region of atomic dimensions. On the other hand solutions of high molecular weight substances will show this low-

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angle X-ray pattern. This phenomenon, first discovered by Guinier (1111, has recently become very useful in the study of macromolecules. The low-angle X-ray pattern can be exploited to determine the radius of gyration of the macromolecule and, if measurements are made on an absolute basis, yields the mass per unit length, a parameter highly useful in determining the “strandedness” of a polymer helix (11.2). For the mathematical details of this difficult and demanding method we refer to the reviews by Luazati (105), Kratky (106), and Wita (108). 3. LIGHTSCATTERING

This technique, widely used for the determination of M , in protein chemistry, has shown relatively little value for polynucleotides. Extensive reviews are found in the paper by Geiduschek and Holtzer (110), and in Chapter 17 in the book of Tanford (19). Peterlin (113) has compared light scattering with low-angle X-ray scattering.

H. Nuclear Magnetic Resonance (NMR) and Electron Spin Resonance (ESR)

Both these techniques are of recent date and their application to polynucleotide studies is still very limited, partly because of the costly equipment involved. Jardetaky and Jardetzky (114) first used NMR to study basic resonance assignments of nucleosides, while Schweitzer et al. (116) studied various substituted purines. Jardetzky (116) and McDonald et al. (117) also have applied NMR to the study of the structure of poly A, poly U, poly C, poly I, poly A * poly U, and DNA. I n a recent study, McDonald et al. (118) used NMR for the investigation of the secondary structure of tRNA. They were able to distinguish between the rotational liberation of the bases and that of the ribose units during denaturation. ESR has been used mainly by Blumenfeld and by Rahn et al. (119, 119a) for the study of basic assignments of purines, pyrimidines, and nucleosides.

1. Electron Microscopy Technical improvements in electron microscopy have greatly increased the interest for investigations of macromolecular structure. Hall (120) has pioneered this technique in the nucleic acid field. Recently, Beer and his collaborators (121-124) investigated the possibility of selective labeling of individual bases in the polynucleotide chain with a view to sequence determination.

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J. Equilibrium Density Gradient Centrifugation Centrifugation in a density gradient of cesium chloride was introduced by Meselson, Stahl, and Vinograd (125) for the determination of the buoyant density of deoxynucleic acids. Doty, et d.have established (126-129) a linear relationship between G plus C content and buoyant density. The fact that polyribonucleotides band a t considerably higher buoyant densities has inhibited the use of this technique for the study of polyribonucleotides and their complexes. However there have been several studies on hybrids between deoxyribo- and ribopolynucleotides (130, 131).

IV. Homopolynucleotides The various random coil and structured forms of polynucleotides containing a single naturally occurring purine or pyrimidine base are discussed in this section. Under suitable conditions of pH or salt concentration, all such polymers form hydrogen-bonded, multistranded, secondary structures.

A. Polyguanylic Acid The highly marked tendency of guanosine residues to aggregate and form gels was long evident to those who worked chemically with guanosine and guanosine phosphates. However, the first reliable investigation producing clear-cut results was that of Gelled ‘et al. (132,133) who reinvestigated an observation made by Bang in 1910 on gel formation with guanylic acid (134),Formation of organized structures, with a T, z 12OC in concentrated solutions of G5’P and G3’P (but not of G2’P), a t pH 5 was followed by changes in UV absorption and optical rotation. X-ray diffraction studies of fibers drawn from the respective gels indicated a linear aggregate of stacked tetramers with rotation about the helix axis in the case of the 5’-phosphate. ORD studies of 5’-GMP gels have also been reported (135). I n view of the above remarks, i t is not surprising that oligoguanylic acids aggregate readily. Such effects have been described for chemically synthesized oligoriboguanylates and oligodeoxyguanylates, one of the most striking results being a very marked increase in pK value (136). More detailed studies of oligoriboguanylates showed that, whereas the aggregation is rather slow, the thermal stability (T, = 23OC for GpG in 1 M NaC1) is marked (1S7). Various alleged preparations of polyG have been described but i t appears that the only reliable preparation involves E . coli polynucleotide

SYNTHETIC POLYNUCLEOTIDES

99

phosphorylase in the presence ( 5 ) of Mn++ (instead of Mg++)a t 60°C. This polymer possesses a secondary structure with an extremely high stability, indicated both by the thermal resistance to transition to a random coil, and by the large shift in pK value (to 11.3) of the bases (29, 138). Whereas alkaline dissociation is abrupt and cooperative (characteristics of the transition, structure + nonstructure) , acidic titration is noncooperative and shows no indication of loss of secondary structure (29). Indeed, the optical rotatory dispersion curve remains virtually unchanged a t pH 2 (no precipitation occurs) (139) and even with a proton on each base the T, is > 100°C in 0.15 M NaCl a t pH 2.5 (I@). Unlike oligo G’s (or small poly G’s obtained by a brief alkaline treatment but liberating no oligonucleotides), which form very viscous solutions a t 7 mg/ml (possibly as a result of multistranded aggregation) , poly G does not show either extreme viscosity or a tendency to aggregate and precipitate at neutral pH, as has been reported for an alleged poly G (141). There is a t present no evidence indicating the number of strands involved in the secondary structure. It is probable that poly dG also has a very strong secondary structure, which inhibits action as a template for RNA or DNA polymerase. Whereas poly d C is active with both these enzymes, poly dG is not (14.2,

-

143)

B. Polyinosinic Acid P o l y I forms a secondary structure that probably contains three strands with an interbase distance of 3.4 A (14.4). As with other helical structures containing uncharged bases, the T, increases as a linear function of the logarithm of the salt concentration. Poly dI is slightly more stable than poly I (T, = 50°C compared with 42°C in M NaCI) (146). The increase in thermal stability for a tenfold increase in salt concentration is 31.5”C1 compared with a typical double-stranded complex such as poly dI * poly dC, which shows only a 17°C increase (146). This suggests that poly dI, like poly I, is a triple-stranded complex, the effect of salt being presumably more marked with the higher concentration of negative charges present in a triple compared with a double helix (Fig. 5). The ORD of polyI is exceptional in that the general appearance is reversed: two troughs and one peak are present between 240 and 300 mp instead of two peaks and one trough shown by all other polynucleotides (and nucleic acids) examined (141). Formation of a complex with poly C or poly A inverts the ORD profile. Since an inverse ORD profile does not necessarily indicate a change in the “handedness” of the helices, this may be attributed to the difference in base interactions (141).

100

A. M. MICHELSON, J . M A S S O U L I ~AND

01

I

-3

GUSCHLBAUER

1

I

I

-2

w.

-I

0

tl

log [Nd]

FIG.5. Variation of T, with ionic strength of various two-stranded complexes. Symbol 0-0

(>-Q

Q-Q

+-+ x-x

A-A

v-v

0-0

0-0 A

rG . dC

D

rG rC

v

Complex

References

dG . dC dI .dBrC dI . dC dI * dI * dI rI rI rI rI . dC rI rC dI . rC rI rMeC

199, 869 199,807 199,206 146,806 606,868 606 806 606

-

888

+ EDTA

867

SYNTHETIC POLYNUCLEOTIDES

101

C. Polyuridylic Acid Secondary structure in poly U is apparent a t low temperatures (147, 148, 150). The formation of this structure can be followed not only by changes in ultraviolet absorption, but also by the variation of the Cotton effects (149) with temperature, and by UV circular dichroism ( 7 8 ) . Nevertheless, nothing is known about the number of strands in the structure or whether (if a double-stranded structure is formed) the strands are parallel or antiparallel. It is considered that polyU is completely devoid of any kind of organized structure a t temperatures above 15°C (148). However, the same authors found no anomalous titration behavior. This is not the case; polyU shows a very marked variation of pK as a function of ionic strength as well as an anomalous ORD. Recent work indicates the presence of considerable base stacking in singlestranded poly U (150).

D. Polyadenylic Acid Titration of polyA shows a very marked shift in pK value of the bases to a higher value. In acid solution (pH 5) , poly A possesses hydrodynamic properties that are characteristic of a rigid molecule (151), and ultraviolet absorption-temperature profiles indicate a cooperative loss of secondary structure over a narrow temperature range (152-155). The stability of the structured form is greater the lower the pH or concentration of salt. Decrease in salt concentration increases the apparent pK value and hence the pH difference between the pK and the ambient pH (Fig. 6). The T, is a direct function of this difference. Crystallographic studies indicate a double-stranded structure with parallel chains and an interplanar distance of 3.8 A. Hydrogen bonds occur between the 6-amino group and N7 of the bases; the proton a t N1 is not involved but probably stabilizes the double helix by electrostatic interaction with phosphate groups (156, 157). Formation of the “acid” form of poly A has also been followed by small-angle X-ray scattering techniques. The rodlike molecules have a mass per unit length similar to that in fibers of DNA (158).As might be expected, significant differences in optical rotatory dispersion and viscosity, as well as ultraviolet absorption, can be seen for the transition between the molccule a t neutrality and the double-helical “acid” form of polyA (151). I n neutral solution, poly A shows hydrodynamic properties characteristic of B random coil (42, 69, 74, 80, 159, 158). Whereas in doublestranded secondary structures involving the amino group, the bases are protected from attack by formaldehyde, the neutral form of poly A read-

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A . M . MICHELSON, J .

MASSOULIB

AND

w. GUSCHLBAUER

PY

FIG.6. Variation of T, with pH of homopolymer complexes at various ionic strengths. Symbol

+-+ x-x

v

A-A

A-A

v-v .-a

0-0 0-0

Complex

“a+]

References

dC * dC dC . dC dBrC . dBrC rC . rC rC ’ rC rMeC . rMeC rA . rA rA ’ rA rA . rA

0.1 M

146 268

~

0.4M 0.4M 0.15 M 0.1 M 0.15 M 0.03 M 0.15 M 0.5 M

268 268 167 260

166 166 166

SYNTHETIC POLYNUCLEOTIDES

103

ily reacts with this reagent (159).Nevertheless, a variety of optical properties such as the variation of ultraviolet absorption with temperature (159),the optical rotatory dispersion (69,160, 16l>,and circular dichroism (7‘8, 83) indicate, as was previously demonstrated with oligonucleotides, that there is a marked interaction (169-164) among successive bases in the chain leading to a single-stranded, stacked, helical structure. The same conclusion has also been drawn from NMR studies of poly A (117).Small-angle X-ray scattering techniques also indicate, over short regions, a rodlike structure with a mass per unit length corresponding to one nucleotide per 3.5 A (158).Nevertheless, the structure, as shown by the hydrodynamic properties, is nonrigid (152).The disorganization of structure with increase in temperature is noncooperative (49,69,159,159) and is probably independent of hydrogen-bond formation between adenine bases (hydrogen-bonded water molecules are, of course, present). Further, the transition appears to be independent of the salt concentration (159).The structure is random with respect to total conformation, but ordered in terms of short-range interactions (158). The thermodynamic analysis of such a conformation has received attention lately (49,169, 163,164),since an evaluation of the “stacking” energy can be thus obtained. The standard free-energy change obtained for single-stranded oligo- and polyadenylic acid a t neutral p H and 0°C is about 1 kcal/mole in favor of base stacking, about one in eight of the bases being unstacked (83) a t this temperature, thus giving considerable flexibility to the molecule (159,162). At 20°C, about two thirds of the bases are in the stacked conformation a t any given instant. [See also the paper by Ts’O, Helmkamp, and Sander (165)who arrive at a similar proportion of structural interaction by less refined techniques.]

E. Polycytidylic Acid As with poly A, an “acid” double-helical form of poly C can be obtained (165,166). However, the structure is somewhat different. Studies of thermal stability as a function of p H and ionic strength of the medium suggested that the system of hydrogen bonds involves a shared proton between each pair of cytosine bases (167).X-ray diffraction studies of fibers of polyC drawn from acidic solution led to the same conclusion (168).The chains are parallel with an interplanar distance along the helix axis of 3.11 A. The thermal transition can be followed by changes in the ultraviolet absorption and in optical rotation (169).The corresponding structure is also formed by polydC and, a s in the case of the polyribonucleotidc, the thermal stability of the secondary structure is greater the lower the pH and the ionic strength of the medium (145).

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Again there is an uptake of one proton for two cytosine residues on passing from the neutral form to the ordered helical structure. It may be noted that the pH of formation in a given solvent is higher than that for polyrC and, in conformity with this, the thermal transition occurs a t higher temperatures than with polyrC (respective pK’s in 0.05 M Na+ are 5.8 and 7.4) (Fig. 6). The structure of polyC a t neutral pH has also been examined. As with poly A, heating an aqueous solution a t pH 7 produces a noncooperative continuous increase in ultraviolet absorption (166) in the region of A,,,. Circular dichroism studies suggested that a t neutral pH poly C possesses a helical conformation ( 7 4 ) . Optical rotatory dispersion studies were also claimed t o prove the presence of a highly ordered secondary structure a t pH 7 (169). Since similar results were obtained with formaldehyde-treated poly C, hydrogen bonds were considered to be eliminated. I n fact, treatment with formaldehyde does not necessarily destroy hydrogen-bond-forming capacity. As suggested in earlier publications, it was considered that base stacking could contribute to hypochromism. The statement that p l y C a t p H 7 exists as a highly ordered asymmetric structure is erroneous if taken in a hydrodynamic sense and the conclusion that so-called hydrophobic forces are the predominant stabilizing factor is, rather strangely, immediately contradicted in the sentence following this claim when it is stated that hydrophobic forces are less effective a t lower temperatures. If this were so, then clearly the postulated helical structure should be more stable a t high temperatures than a t low, contrary to experience. It was also inferred that the helical structure is not necessarily completely rigid, but an interrupted helix made up of smaller helical regions. This static concept is untenable and, as in the case of poly A, one must consider poly C to be a random coil with continuously fluctuating regions of helicity arising from base-base interactions in constant change.

V. Polynucleotide Complexes A. Complexes between Poly G and Poly C Although the interaction of polyA and polyU has been studied in detail for some considerable time, studies on the formation of a complex between poly G and poly C have only recently become possible. Much of the earlier work relates to complex formation between oligo G’s (or short polydisperse poly G) (170) and poly C. Lipsett has examined the interaction of GG and GGG with poly C (171, 172). At neutral pH, a complex with the stoichiometry 1G:lC is rapidly formed, followed by slower formation of 2G: 1C. However a t slightly acidic pH ( A pH 6 ) a

105

SYNTHETIC POLYNUCLEOTIDES

third complex, probably containing a t least some protonated C residues, is formed with a ratio 1G:2C. The interaction of poly G (as opposed to oligo G ) and poly C gives somewhat different results (29). Mixing curves under various conditions and a t various wavelengths indicated forination of a 1:l coinplex only, with a stability greater than that of poly G, as shown by increased thermal stability and also by alkaline dissociation, which occurs cooperatively a t pH 12.27 for poly G * poly C in 0.15 M NaCl (compared with 11.43 for poly G). I n order to achieve separation of the strands a t neutral pH, 80% methanol was used, giving a T, of 89°C. Under these conditions, the dissociation is irreversible, until the methanol is removed. (The complex with poly BrC is even more stable.) The triple complex, 2G:lC, could not be obtained. However, when the polyG was briefly treated with alkali to reduce the molecular weight, the complex 2G:lC could be readily obtained. Similarly mixing curves of poly G with short poly C (average chain length z 15) indicate formation of a 2 G : l C complex. A possible explanation is simply that, with two extremely long polymers, rigidity of a double-helical structure prevents addition of a third strand. However, if breaks are present in one or the other of the two strands, flexibility is increased and the addition of a third strand is facilitated. Another approach to the preparation of poly G poly C has been described (173, 174). Poly C was used as template for RNA polymerase (Micrococcus lysodeikticus) in the presence of G T P to give a product, apparently double-stranded, containing 1G :1C. Other than that the polyG formed appears to consist of short strands, the properties of this complex resemble those of the nonenzymatic preparation. The alkaline dissociation followed by neutralization yields a complex distinguishable from the original, possibly due to formation of aggregates containing 2G:lC (173). The optical rotatory dispersion of the above preparation of poly G poly C has been described (149). Somewhat surprisingly, a sharp transition occurs a t 90°C in 0.1 M Tris, with irreversible changes in magnitude. Similar changes were also observed in the UV absorption a t 90°C. In view of these results, serious doubts must be cast on the integrity of the material used, particularly since the same authors state that poly G is highly insoluble in aqueous solution, a statement that is as surprising as it is incorrect. ORD studies with the complex obtained by interaction between the two homopolynucleotides confirm an extreme stability and the complex exists in a range of pH between 2.0 and 12.2 (139). Other studies have shown that, a t pH 2.5, poly G-poly C is fully protonated (two protons

-

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MASSOULIB AND w.

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per base pair) yet nevertheless has a T, of approximately 8OoC ( 1 4 0 ) . Hence acidification does not destroy the complex, in contrast with the alkaline dissociation. This can readily be explained in terms of the base pairing characteristics. As might be expected, thermal dissociation of poly Gepoly C a t pH 2.5 is irreversible. At this pH both poly G and poly C possess structures much more stable than that of poly G-poly C. Complexes of poly G with poly BrC (or with poly iodo C) a t p H 2.5 are even more stable than is poly G-poly C. The complex of polydC and polydG can be obtained from dCTP and dGTP by means of DNA polymerase. The product generally contains 5 0 4 0 % of dG (that is, an excess) and shows a biphasic melting curve, the first step of which corresponds to the melting of polydC ( 1 4 6 ) . Alkaline dissociation is reversible with respect to ultraviolet absorption, but the viscosity does not return to the original value (175). It may be noted that poly dC.poly dG is markedly less stable than the corresponding ribonucleotide complex. Circular dichroic spectra (81, 84) of poly dCapoly dG are inverted by comparison with those of DNA or poly A-poly U. This has led to the perhaps unjustified suggestion that the structure is a left-handed double helix rather than right-handed (176). It would be of interest to have dichroic spectra of poly I (structured form).

B. Complexes between Poly A and Poly U The interaction of poly A and poly U to form helical complexes has been the most extensively studied of this type of reaction. The polymers can be prepared readily, and they react spontaneously in saline solution (177). It was thought a t first that only one kind of association could arise, a double-helical structure in which adenine and uracil would be paired like adenine and thymine are in DNA, and indeed it was shown that the complex between poly A and poly U has the crystallographic properties of a helix, the dimensions of which are very close to those of DNA (178-180). Moreover, the viscosity of the solution increases durdecreascs ing formation of the complex and the UV absorption at, , ,A while the optical rotatory power increases (177, 181). The hydrodynamic and optical properties of the complex resemble those of DNA. The stoichiometry of the association was studied by the continuous variation method (39, 40). An equivalent number of A and U residues entered the complex, which was therefore termed poly A-poly U. The method of continuous variation also showed that a second complex, containing two U residues for one A, was found when an appropriate mixture of polyA and polyU was allowed to react for a longer time, The reaction is rapid in the presence of divalent cations, or in a high enough

107

SYNTHETIC POLYNUCLEOTIDES

(>0.1) ionic strength, but it occurs even with low salt concentration (0.005 M Na’). It is thought that the two uracils are bound to the adenine in polyA-2polyU and not to each other. One of them is presumably in an orthodox Watson-Crick pairing, while the other is hydrogen-bonded through the remaining hydrogen of the 6-amino group and N7 of the adenine (98). This can be achieved in two ways, as is discussed below. Since the properties of poly A-poly U are of importance for an estimation of the contribution of one kind of base pair to the corresponding properties of DNA, the two complexes poly A poly U and poly A - 2 poly U received intensive study. Considerable confusion about the equilibrium of the complexes arose as a result of a coincidence in certain optical properties of the two species (182). The hypochromicity a t 259 mp does not distinguish between the following possibilities :

-

(1) A lA/lU mixture first gives rise to poly A*polyU which a t equilibrium rearranges itself into poly A.2 poly U liberating one half of the poly A. This process would lead to the same complex, poly A-2 poly U, a t equilibrium in all mixtures, since i t is the stable form of the 1A/2U mixture (40, 183). (2) Poly A-polyU is the stable complex formed in a lA/lU mixture. Such was generally assumed to be the case. The second case has now been proved by a variety of techniques such as NMR (116) and infrared spectroscopy (99) using concentrated solutions, and, in dilute solution M in polymer phosphate), by ultraviolet absorption a t wavelengths other than 259 mp, either a t discreet wavelengths (41, 42, 187) or by considering whole difference spectra (28, 187), by the use of formaldehyde to measure noncomplexed poly A (184, 187), and by the stimulation of ethidium bromide fluorescence by double- (but not single- or triple-) stranded structures (186). It is particularly interesting to consider ultraviolet absorption a t two “selective” wavelengths, X1 and Xz. X,I a wavelength close to 280 mp, is such that poly A * poly U shows no hypo or hyperchromicity; i t is not distinguished from its unreacted components, and poly A * 2 poly U is the only complex shown by the continuous variation technique a t this wavelength. At Xz, which is close to 283 mp, only poly A * poly U can be seen; i t is hyperchromic a t this wavelength (41, 42) (Fig. 1 ) . By considering simultaneously two “mixing curves” a t X1 and A?, one can determine the amount of poly A * poly U and poly A 2 poly U in any mixture (41). This leads to a rather odd conclusion regarding the evolution of a lA/lU mixture (186). It is true that poly A * poly U is formed rapidly, but in high enough salt concentration the formation of

-

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w. GUSCHLBAUER

poly A * 2 poly U is also rapid enough to take place during the association of the two strands of poly A and poly U. One can visualize this in the following manner: if the strands are not yet perfectly matched, there must be some free ends of A and some free ends of U. The latter will then tend to engage themselves in three-stranded segments-perhaps by folding back on the two-stranded structure. As a result of this, 1 or 2 hours after mixing, in 0.2 M Na+ solution, the totality of U residues has entered the complexes, but approximately 10% of them are in poly A 2 poly U. A slower rearrangement takes place, eliminating this, and eventually one finds only poly A poly U. This requires that the two poly U strands be antiparallel. Alternatively, the slow reaction involves poly A * poly 2 U formed by addition of a second strand of poly U to poly A * poly U. In this case, the poly U strands can be parallel or antiparallel.

-

-

C. Thermal Dissociation of Poly A Poly U and Poly A 2 Poly U (41,@1 187) By following the variation of ultraviolet absorption a t 259 mp, one can demonstrate the cooperative dissociation of poly A * poly U or poly A * 2 poly U a t the dissociation temperature, Tm.But simple dissociation, leading directly to free poly A and poly U, is not the only transition observed. For instance, a t low salt concentration (< 0.1 M Na+) and in the absence of divalent cations, poly A 2 poly U is dissociated in two steps:

-

poly A 2 poly U + poly A poly U 9

+ poly U + poly A + 2 poly U

It is necessary therefore to define several kinds of dissociation temperatures. The first one will be noted T m ( S - z ) , and the second one Tm(z-l), the figures designating the number of strands associated below and above the T,. I n low salt concentration, poly A poly U melts a t Tm(z-l), and between T,(3-2) and Tm(z-l)there is a temperature range where this complex is the only stable one. The absorption spectra of the lA/lU mixture in the two states, poly A * 2 poly U below Tm(s-z) and poly A poly U poly U between T,(3-z) and Tmcz-l,, intersect with that of the dissociated polymers (above Tmc2-1)) a t the selective wavelengths X1 and Xz. This is a simple way to demonstrate the existence of XI and Xz and to determine their values. Hence in low salt concentration one must consider two transition temperatures, T m ( 3 - 2 ) and Tnz(2-1,. Above 0.1 M (Na+), the corresponding transitions do not occur, and poly A * 2 poly U dissociates directly into the component polymers a t a temperature noted Tm(3-1).In contrast t o the situation in (Na+) less than 0.1 M , where the dissociation of poly A-poly U is direct and that of poly

-

-

+

109

SYNTHETIC POLYNUCLEOTIDES

-

A 2 poly U givcs poly A . poly U, we find in higher salt concentrations that poly A * poly U rearranges itself into poly A 2 poly U, which in turn dissociates a t Tm(3-1). This transformation is reversible and occurs This transition: at a transition temperature Tm(Z-3). 2[p0ly A * poly

-

U]

Tm(I-:)

poly A . 2 p01y U

-

+ poly A

was first demonstrated by infrared spectroscopy (100) and later by ultraviolet spectroscopy (41, 42). If the dissociation of poly A * poly U were direct, the absorption a t X1 would not vary during increase in temperature. One actually sees two equal and opposite variations a t Tm(2-3) and Tm(3-1).

70

30

10 log [No+]

FIG.7. Experimental phase diagram of interactions between poly A and poly U (41, 42, 187).

The variation of the four transition temperatures, Tm(3-2), Tm(2--l), and Tm(3--1), has been studied as a function of the salt concentration in neutral solution. It may be noted that the dissociation temperature of DNA varies linearly with the logarithm of the salt concentration. I n the same ivay, T m ( 2 - 1 ) , Tm(3--2),and Tm(3--1)increase with log (Na+) and their variation is perfectly linear, within the limits of experimental accuracy. On the contrary, Tm(2-3) decreases and this decrease is not linear. The stability diagram contains four lines, which do not intersect because, even though the transitions are cooperative, they are not perfectly sharp, and each T, defines a transition zone rather than a boundary. However, these four lines divide the plot into four regions (Fig. 7): Tm(2--3),

110

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A. M. MICHELSON, J . MASSOULI~ AND

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I corresponds to the situation found a t low temperature; both complexes exist, depending on the ratio of A and U residues. ( b ) Region 11, below 0.1 M (Na+), and a t temperatures intermediate is a domain of exclusive stability of between Tmc3-2, and Tm(P-l), poly A poly U, except perhaps a t high U/A ratios. (c) Region 111, above 0.1 M (Na+),and also a t intermediate temperatures, is a domain of exclusive stability of poly A 2 poly U. ( d ) I n region IV, the polymers are not associated.

(.a) Region

-

-

This diagram is valid in a certain range of pH around pH 7.

D. Stability of Poly A Poly U and Poly A 2 Poly U at Alkaline pH Poly A-2 polyU dissociates in two steps upon titration with alkali a t p H 10 and 10.4 (0.2 M Na+) (181).At alkaline pH, the uracil bases lose the protons involved in hydrogen bonding with adenine and therefore ionization and dissociation of the complexes are linked phenomena. The stability of poly A 2 poly U is diminished more than that of poly A . poly U. T m ( 2 - 3 ) is not changed in this p H range because the corresponding transition does not involve a variation of the number of free U residues. But decreases and, above a p H value that depends upon the salt concentration, the process of dissociation of the complexes goes and Tmcz-l), even a t high salt concentration (187, 188). through Tm(3-z)

E. Stability of Poly A Poly U and Poly A 2 Poly U at Acid pH (187, 188)

At acid pH, polyA forms a helical protonated structure, and this enters into competition with poly A poly U and poly A 2 poly U. I n the acid form, poly A reacts very slowly with poly U (161), so that dissociation of the complexes appears irreversible, and acid titration shows a hysteresis loop (189). The stability of the complexes can nevertheless be studied by first allowing association to take place a t pH 7, then bringing the p H down to a desired value. As a result of competition with the Tmcs-l), and Tmcz-l) decrease a t pH's such helical form of poly A, Tm(z-3), that the T,,, of helical polyA is sufficiently high. (It should be remembered that this T , increases as the pH decreases.) Tmcs-,) is not changed, because no polyA is liberated a t this transition. In low salt (0.03M Na+) the situation is rather complicated. Below pH 5.4, one finds only poly A * 2 poly U in all mixtures a t room temperature. Between p H 5.4 and pH 5, the dissociation of this complex is still in two steps, proceeding through poly A poly U. However, below pH 5, the poly A 2 poly

-

-

-

-

111

SYNTHETIC POLYNUCLEOTIDES

U dissociates directly, as shown by a comparison of the absorption variations that occur a t the different transitions.

F. Base Pairing in Poly A Poly U and Poly A 2 Poly U The existence of polyA * 2 poly U clearly demonstrates that adenine and uracil can be associated in at least two ways. B y studying the in-

crease of the carbonyl stretching frequencies of the uracil bases in poly A * poly U and poly A 2 poly U, Miles (98) has found that two different carbonyl groups must be involved in the hydrogen bonding present in the latter complex. His observations support the conclusion that poly A * polyU has a Watson-Crick type of bonding (190), while in poly A * 2 poly U, the second U is hydrogen-bonded through the C2 carbonyl to the 6-amino group of adenine. This differs from the bonding pattern originally suggested but is in accord with that observed in mixed base crystals (191). Both strands of poly U would be antiparallel to poly A if the same configuration of the glycosyl linkage is realized in all the chains. (Configuration here refers to rotation of the base about the glycosyl linkage.)

-

G. Thermodynamics of the Interaction between Poly A and Poly U One can give a quantitative explanation of the stability diagram presented in Fig. 7 (187) with the very simple formula,

AG

=

A(Tm- T )

where AG is the difference of free enthalpy between the separated polymers and the complex. Jf AH and A S were independent of temperature, A would be equal to AS, but it has been shown experimentally that this is not the case (162, 163, 192). By introducing the variation of T, with log (Na+) in the formulation of AG, AG can be expressed as a function of T and (Na+). This can be done for poly A * poly U and poly A 2 poly U, and involves two differentlA constants. The ratio of these constants can It is then be determined from the relationship that is realized a t Tm(2--3). possible to deduce the existence of the fourth T,, Tmc-3-2),and to calculate its value (Fig. 8). It can be seen that this very simple and even naive formulation is justified a posteriori: i t gives a rather good representation of the different equilibria a t the T,’s. A similar expression, in which p H is introduced, can also be written to define the equilibria among poly A poly U, poly A 2 poly U, the helical form of poly A, and the separated polymers a t acid pH. However,

-

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in this case it is not possible to give a quantitative prediction of the variation of the Tm’s. Experimental determinations of the heat of reaction of poly A and polyU show that AG increases with temperature (165). This variation is certainly due principally to changes in the properties of poly A. I n this polymer, the nucleotide residues show interactions that are noncooperatively disorganized by increase in temperature. This introduces a temperature-variable term in AG. Attempts to obtain a “true” AG by subtraction of this contribution have been made (4SJ 163),but i t is still doubtful if a reliable estimation has been obtained.

log [No*]

FIQ.8. Calculated phase diagram of interactions between poly A and poly U: 0,experimental values (187).

---, calculated;

The value AGA obtained for one A residue of poly A compared to the temperature of mid-variation in the properties of poly A seems to imply a rather high value for the corresponding ASA. The same order of magnitude as that of A8 for the formation of a rigid pair of bases is found. Moreover, AG for the reaction,

also varies somewhat with temperature (162) and the variation of AG for the reaction, poly A

+ p01y U

-+

poly A * p01y U

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SYNTHETIC POLYNUCLEOTIDES

with temperature is not the same a t different salt concentrations (1931, whereas the properties of polyA do not seem to depend upon the salt concentration, but only upon temperature (159). The simplified expressions used above are also in contradiction with the experimentally determined variation of AG with salt concentration. Instead of an increase in A G with increasing (Na'), the contrary is observed. Obviously, compensating factors are present and perhaps concomitant variations of AS and AG can explain this difficulty (194). However, the experimental decrease of A G when (Na') increases is difficult to understand. A priori, high ionic strength stabilizes the helical associations by shielding the negative charges of the phosphates and diminishing their repulsion. Thus poly A 2 poly U is more sensitive to variation of ionic strength than is poly A poly U. A tenfold variation of [Na'] corresponds to

-

=

21"

and

-

AT,(a-l, = 25"

By analyzing the dissociations of the complexes brought about by ionization of polyU a t alkaline pH, Warner et al. (181) were able to estimate indirectly AS and AG for the rupture of one pair or one triplet of bases.

H. Alternating Copolymers We here consider ribo- and deoxyribopolynucleotides containing alternating sequences, e.g., poly (rA-rU) , poly (dA-dU), and poly (dA-dT) . All these polymers possess an organized helical structure (195-197). This structure is like that of DNA, a double-stranded helix, and the two strands are antiparallel. The crystallographic properties of the lithium salt of poly (dA-dT) are identical to those of DNA in the B form. However the sodium salt differs from the known forms of DNA (198). Since each strand possesses a periodical self-complementary sequence, it can fold back over itself. Branched helices can thus be formed, and have been observed with the electron microscope (195). This particular behavior is probably the cause of several distinct properties of these alternated polymers. Instead of rising continuously during the thermal dissociation, the viscosity of a solution passes through a minimum a t a temperature slightly below the T, (T, being defined by changes in the ultraviolet absorption). Upon cooling, the absorption returns to the original value, but the viscosity does not. ( b ) The breadth of the thermal transition (again defined by the ultraviolet absorption) increases with the salt concentration. The reverse

(6)

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A. M . MICHELSON, J . M ASS O U LI ~AND

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-

variation is observed for poly A poly U, poly A 2 poly U and for DNA. In these cases, it may reflect the heterogeneity in length for poly A and poly U, or in composition for DNA. The breadth of the thermal dissociation of poly dG * poly dC does not vary with the ionic strength. I

I

I

I

log

I

"a+]

Fro. 9. Variation of T, with ionic strength of alternating copolymers containing A, U, T, and BrU. Symbol

Complex

References

@-a

poly r(A-BrU) poly d(A-BrU) poly r(A-U) poly A * poly U poly d(A-U) POIY d(A-T)

200 196 200 41, 42, 86 200 196, 200

c)-@

0-0

0-0 0-0

0-0

The T,'s of the dissociation of poly d(A-U) and poly r(A-U) increase linearly with log (Na') ; A T , is equal to 21" for a tenfold variation of (Na+), exactly as for Tm(z-l) in the case of polyA poly U. The effect of ionic strength (Figs. 9 and 10) is therefore the same on all these two-stranded helices containing A-U base pairs. It is also nearly the same for other two-stranded helices: poly dI * poly dC, 17°C; poly dG poly dC, 18OC; poly dI poly dBrC, 20°C (199). The polyr(A-U) helical form is 10°C more stable than poly A poly U (187); it is also

-

-

-

115

SYNTHETIC POLYNUCLEOTIDES

10°C more stable tlian the helical poly d(A-U), the T , of which coincides with Tm(2-1, of poly A poly U. An important difference may be noted between polyr(A-U) and poly d(A-U). The relative variation of absorption a t 260 nip is much

-

a-a x-x v-v A-A

+-+

rA . rU rA . rT dA . dT rA . dT dA . rU

41, 48, 86 21 8,222, 268,267 129, 266 266 266

larger for polyr(A-U) (70%) than for polyd(A-U) (45%) (600).The value for poly r (A-U) is not abnormal when compared to that for poly A poly U which is 61% [between 20 and 85°C (68)1. The differences between the deoxy and the ribo alternating polymers, +

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also observed between poly d (A-BrU) and poly r (A-BrU) (200), could arise from slightly different helical structures. It is unlikely that the unstructured chains should have very different optical properties, though this remains a possibility.

1. Association of Poly A and Poly I Polyinosinic acid is generally considered as equivalent to poly G. It does indeed associate with poly C, as one would expect, but it also associates with poly A. Both a two-stranded and a three-stranded complex can be formed, polyA * polyI and polyA * 2 poly I (201). In this respect, polyI behaves as an analog of poly U. However, the complexes between polyA and polyI dissociate a t lower temperatures than the corresponding A and U complexes (146). Poly A 2 poly I does not seem to be formed a t all a t low ionic strength (0.01 M Na+) (20201). Although this system has been but little investigated (202),i t seems from continuous variation curves that poly A 2 poly I in the conditions studied (0.05 M Na+, pH 6.8) is the equilibrium form even in a l A / l I mixture. No data are available yet to construct a stability diagram, similar to that described for poly A * poly U, and the effect of p H on the stability of the complexes is not known. The hydrogen-bonding pattern of poly A 2 poly I is probably similar to that of poly A 2 poly U (98). Here, however, the same acceptor and donor groups of inosine have to be used for both polyI strands. It follows that, if they have, as is most likely, the same configuration of the glycosyl linkage with respect to base rotation, these strands must be antiparallel.

-

J. Associations Involving Hypoxanthine and Cytosine In neutral saline solutions, poly I and poly C associate into a doublestranded helical complex, poly I * poly C (2Q3).No triple-stranded structure has been detected. Poly I poly C has a hydrogen-bonding pattern homologous to that of G and C, but it cannot be considered entirely analogous to poly G * poly C since the T, (60.2"C in 0.15 Na') is much

-

lower (146). This T, increases linearly with log (Na+) (204, 206) (Fig. 5). The acid titration of poly I poly C is complex (206). Poly C forms a semiprotonated helical structure in acid solution but even a t pH's such that this poly (C, C') melts higher than poly C * poly I, the complex is not dissociated. In contrast with the effect of decrease in p H on poly A poly U, the T, of poly I poly C rises and the complex is protonated. In an ionic strength of 0.05 M Na+, the titration presents two cooperative steps. The first, a t pH 4.9, is reversible; the second, a t p H 3.7, shows hysteresis and the reverse titration takes place a t p H 4. Analysis of the complex peak, isolated from various mixtures of poly I

-

-

117

SYNTHETIC POLYNUCLEOTIDES

and polyC by sucrose gradient centrifugation, shows that the ratio of I to C is always 1. Poly (C, C + ) ,the acid foEm of poly C, does not react appreciably with poly I a t pH 6. At neutral pH, the T,, of poly I * poly C (ionic strength 0.05 M ) is 5O"C, a t pH 4.6 (where half of the cytosines are protonated) it is 53"C, and a t pH 3 (cytosines fully protonated) it is 62°C (no titration of poly I occurs in this p H range). It would be interesting to know whether

FIQ.11. Possible types of base pairing between inosine and cytidine. The neutral form is that found in poly I * poly C at neutral pH. Forms A-C represent three possible configurations involving protonated cytidine residues. The solid circles represent the position of the carbon atom (Cl') of the ribose residue [after Giannoni and Rich (906)l. the increase of T, with decreasing p H occurs as a regular variation or, as seems possible, in more or less abrupt steps, a t the pH's of protonation. Three hydrogen-bonding schemes have been considered for poly I polyC+ (Fig. 11). One of them, A, has been eliminated because the slow rate of reaction of formaldehyde with poly I poly C+ implies that the amino groups must be involved in hydrogen bonding. I n C, the cytosine is rotated about the glycosyl linkage as compared to the neutral poly I poly C pairing. This means either an inversion of the direction of the C strand, which is impossible without dissociation of p o ly I * poly

-

-

-

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A. M . MICHELSON, J. MASSOULI~ AND

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C and therefore formation of poly (C, C+), or else a rotation of the base in the double helix which also presents difficulties. This leaves model B for poly I poly C+. The structure a t pH 4.6 is well-defined, different from both the fully protonated and the neutral forms, as indicated by the cooperativeness with which it loses or gains one proton for two cytosines. The assumption has been made that it is an incompletely protonated polyI poly C. It must be an original structure in which two kinds of base pairs coexist, perhaps alternating. A completely different possibility is that the bases are arranged in triplets, poly I * poly C * poly C+ (Fig. 12). A structure of this type would explain why poly (C, C+) and polyI can associate a t pH 4.6. The authors do not seem to have checked the stoichiometry of the complex in this state, but only at

-

-

FIG. 12. Possible type of base interaction between poly I, poly C, and poly C'.

pH 3. However, they report a higher degree of association a t pH 4.6 than at neutral pH or pH 3 and they suggest that this may arise from the combination of individual molecules of protonated poly C combined with both polyI and polyc. However, a random process does not account for the cooperative nature of the transition. Poly dI polydC has a lower thermal stability (205) than poly I * poly C; the T, is 14°C lower (in 0.1 M Na+). The relative variation of absorption that takes place a t 246 mp during dissociation is also less than for poly I poly C, 52% compared to 67%. In this series we now find, besides the double-stranded structure poly dC poly dI, a triple-stranded one, 2 poly dI * poly dC (207). It is difficult to establish a stability diagram in this case because two more structures complicate the picture: the acid form of poly dC (145) and the helical structure of polydI. The acid form of polydC a t pH 6.9 still melts around 45°C in 0.41 M Na+ (or 60°C in 0.1 M Na') but can be eliminnted by raising the pH above 7.5. The helical structure of poly dI melts lower than poly d I poly dC, but the difference of the T,'s

-

-

-

119

SYNTHETIC POLTNUCLEOTIDES

diminishes as the salt concentration rises. The triple-stranded 2 poly dI * poly dC can be found only in high salt concentration, that is, in a limited range of temperature and ionic strength. Increased thermal stability of the complex formed between polydI and poly BrC allows an easier study of 2 poly dI * poly BrC (kW7). I n 0.41 M Na+ a t 42°C (above the dissociation of poly dI), the continuous variation method demonstrates the existence of both poly dI poly dBrC and 2 poly dI * poly dBrC, according to the ratios of the polymer residues. At 20°C the reaction of polydI with poly dBrC proceeds only to the formation of poly d I * poly dBrC. At low salt concentrations, 2 poly dI * poly dBrC dissociates in two steps: 2 poly dI poly dBrC -P 9

poly dI poly dBrC +

+ poly dI + 2 poly dI + poly dBrC

(the first dissociation takes place a t temperatures lower than 20°C, below 0.1 M Na'). Above a certain salt concentration (0.35M Na+), however, the dissociation of the three-stranded complex is direct: 2 poly dI . poly dBrC + 2 poly dI

+ poly dBrC

We thus find a set of equilibria similar to those for the associations of poly A and poly U, and it appears that i t might be completed by a fourth transition, a rearrangement of poly d I * poly dBrC above 0.35 M Na+: 2(poly dI .poly dBrC) -+ 2 poly dI poly dBrC

+ poly dBrC

The hydrogen bonding in such three-stranded complexes is unknown but it probably involves a triangular scheme in which each base is hydrogenbonded to the other two, unlike those proposed for poly A * 2 poly U and poly A 2 poly I . It is not unlikely that a triple-stranded complex, 2 poly I * poly C, is possible in the ribopolynucleotides a t high enough salt concentrations.

VI. Role of Sugar Phosphate Backbone A. The Phosphate Residues We examine here the effects of the sugar phosphate "backbone" on the stability of helical structures. The negative charges of the phosphate residues in general tend to repel each other, and the repulsions may be minimized in certain helical structures. In most associations of polynucleotides, the negative charges carried by the two or three strands result in a repulsion that decreases the stability of the structure. Exceptions to this are the acid helical forms of polyA and poly C. In poly A, the proton carried by N1 is not involved in hydrogen bonding

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between bases, but hydrogen bonding between one phosphate and the amino group of an adenine in the opposite strand has been postulated. The positive charges of the bases give an attractive force with the phosphate and stabilize the association of the two strands. Hence the T , of the acid form of poly A decreases as the salt concentration increases. The variation is much more complex for poly (C, c')for which the T , is a decreasing function of (Na+) above about pH 4 and an increasing one below. Opposite variations occur on each side of the pK of cytosine because only half of the bases must be titrated in the helix. The effect of salt concentration on the T , of polyI poly C+ has not been investigated. It may be expected that the T,,, of poly I * poly C' will be higher a t low ionic strength. I n most neutral complexes, shielding of the negative charges increases the stability; the effect is greater in triple-stranded helices. Divalent cations such as Mg++,a t low concentrations, also increase the stabilities of most neutral complexes. I n the presence of excess Mg++, T,(s-l) of poly A-2polyU is nearly independent of Na+ but decreases (187)above 0.01 M Mg++.Concomitantly, the breadth of the transition increases very markedly: one fourth of the optical density change takes place in a 0.2"C interval with no Mg++in 0.5 M Na+, and in a 12.5"C interval with 0.02 M Mg".

-

B. TheSugar We have already noted many differences between ribo- and deoxyribonucleotide structures : The helical semiprotonated forms of poly dC and poly dBrC are more stable than those of poly C and poly BrC (199). The T,, of poly (dA-dU) is 10°C lower than that of poly (rA-rU) (200). The T, of poly dI-poly dC is 14°C lower than that of poly I.poly C (in 0.1 M Na+) (205). Poly dG poly dC is dissociated a t considerably lower temperatures than poly G poly C ($9, 176). The T,'s of poly d T and poly dU are lower than those of the corresponding polyribonucleotides (147, 2008, 209). In cases where DNA-DNA and RNA-RNA helices of identical sequence and composition have been compared, the T,'s of the latter are about 8°C higher (200) (if thymine did not replace uracil in DNA, the difference would be larger).

-

-

Apart from the differences in stability, in the few cases examined, hypochromicity is larger in the ribose series. These differences are per-

121

SYNTHETIC POLYNUCLEOTIDES

haps partly a consequence of RNA helices being in the A form while DNA exists in solution in the B form. Evidence for this lies in the observation that neither RNA helices nor hybrid helices bind actinomycin D, which is bound only by DNA in the B form and not in the A form (210, 211).

Hybrid RNA-DNA helices melt lower (4OC) than the corresponding DNA-DNA helices (130,131, 212). Few polynucleotide hybrids have been extensively studied. Poly dG poly C has been found in a CsCl density gradient (128). It melts higher (83°C in 0.0013 M Na’) than poly dG * poly d C but lower than poly G * poly C. Two hybrids, poly dG poly C and poly G poly dC, have been synthesized with the enzyme “nucleic acid hybrid polymerase” (213).Their Tmlsare not very different, and both are about 20°C higher than that of poly dG poly dC. However, the stoichiometry of all these hybrids has not been wellestablished, and their study was not very extensive. The order is thus

-

-

-

poly G * p01y C

> poly G . poly dC > poly dG * poly C > poly dG . poly dC

A thorough examination of all four combinations of poly I and poly C has been carried out (206).The order of stability is as follows: poly I * poly C

> p01y I

*

poly dC

> p01y dI

poly dC

> poly dI

*

poly C

The stability of a hybrid structure is therefore not necessarily intermediate between those of the two corresponding nonhybrid associations. The crystallographic characteristics of all these complexes differ and do not resemble those of RNA and DNA helices. Postulates concerning A and B forms may be very oversimplified or even wrong, but it remains that, depending on the nature of the sugar phosphate backbone, geometrically different double helices can be formed, always using the same base-pairing pattern. It is difficult to make detailed explanations of the role of the sugar.2 However, the large differences in the stability and the optical properties of homologous deoxy, ribo, and hybrid complexes illustrate the importance of this part of the polymer in the formation of helices. It is sometimes overlooked, but one must expect the contribution to be decisive, since the sugar phosphate backbone has direct interactions with the surrounding aqueous medium (117). It may be relevant to the transcription of information from DNA to messenger RNA that a DNA-DNA helix is more stable than a hybrid. This may ensure that the process can take place, leaving the DNA un* Recent comprehensive ORD studies of ribo and deoxyribopolynucleotides

have provided plausible explanations for many of the stability differences (261).

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changed while producing free RNA. This point has received support recently, using the various complexes of polyI and poly C as templates (205, 214). The remaining double helix is the more stable one, regardless of the template used.

C. Right- or left-Handed Helices, Parallelism, and Antiparallelism I n all cases where crystallographic studies have been made, polynucleotide helices have been found to be right-handed: DNA (2161, poly (dA-dT) (198), polyI poly C (216), poly A poly U (190), the acid forms of poly A ( 1 5 7 ) ,and poly C (168). Like DNA, poly (dA-dT), poly A * poly U, poly I * poly C, and poly A polyI all possess antiparallel structures, whereas, in the acid forms of poly A and poly C, the strands are parallel. Can a generalization be made for all homopolynucleotides? It is reasonable to assume that the two strands in such a structure must be equivalent ( 2 ) .This implies that each base pair is symmetrical, with an axis of symmetry. The helical 'axis will therefore also be a dyad axis in this type of structure built by association of two identical strands. It follows that the strands must always be parallel. With the same assumption that the strands will be absolutely equivalent, three-stranded homostructures must have a threefold axis of rotation. Such structures must also be parallel. (One can also reason as follows: a t least two strands must be parallel. If the third one is to be equivalent to the other two, it will be parallel to them.) It may be noted that, with respect to formation of organized secondary structure in homopolynucleotides, the rate will be independent of polymer concentration if an antiparallel structure is involved (folding of a single strand). In the case of parallel strands in a double (or triple) helix, the rate of formation of secondary structure will be concentrationdependent.

-

-

VI I. Reversibility Most of the associations studied are formed spontaneously when two polynucleotides are mixed in saline solution, and it follows that, after dissociation by elevation of temperature or by a change of pH, reassociation occurs when conditions are brought back to the original. However, hysteresis phenomena are found when polynucleotide complexes are titrated (189, 206) and, in certain conditions, thermal dissociations appear to be irreversible. Hysteresis loops have been observed in the acid dissociation of poly A poly U, poly A * 2 poly U and poly I poly C, and similar observations would be possible for poly A poly I and poly A * 2 poly I (but not poly G poly C because protonation does

-

-

-

SYNTHETIC POLYNUCLEOTIDES

123

not lead to dissociation) ( 2 9 ) . In all these cases, acidification allows the formation of a new structure, the protonated forms of polyA or poly C. When the complex is dissociated, the poly A (or poly C) strands associate, and in this form they do not react easily with complementary polynucleotides. Thus the original association is reformed a t a higher pH than the pH at which it was broken. There are no hysteresis loops in the alkaline titration of the same complexes, since alkaline, stable, ordered structures do not exist for these polynucleotides. Apparently irreversible thermal dissociations can also be observed when polynucleotides are first allowed to associate under favorable conditions, then transferred to different conditions (of salt concentration or pH) and heated. For example, preformed poly dI poly dC has a T , of 51°C a t pH 6.1 in 0.286 M Na'. The acid form of poly dC in the same solution melts a t 67"C, and polydI, also associated a t low temperature, melts a t 32°C. The thermal dissociation of poly d I * poly dC is irreversible and leads, when the solution is cooled, to a mixture of the separate ordered structures of poly dC and poly dI ( 1 4 5 ) . Another example lies in the thermal dissociation of poly G * poly C a t pH 2.5 (after preparation of the complex at pH 7). Recooling does not lead to reformation of the complex

-

(29)

f

Competition with protonated ordered structures is not the only case in which irreversible transformations, indicative of frozen equilibria, can be obtained. Thus poly dI and poly dBrC seem to give only polydI poly dBrC a t 20°C in 0.41 M Na+. But a t 42"C, as poly d I is dissociated, the reaction proceeds and 2 poly dI * poly dBrC is formed. This complex, which melts at 8loC, is then stable when brought down to 20°C (207).

-

VIII. Displacement Reactions The question of competition between two different associations is of interest. Such a competition arises when one polynucleotide (K) is able to associate with two different complementary polynucleotides (L and M). If the association K L is more stable than K M, we can expect L to displace M from its less stable complex. It is widely assumed that the higher the T,, the more stable the complex, and the T, has even been considered as a measure of absolute stability. If this is correct, we can predict which of two polynucleotides will displace the other. As far as neutral complexes are concerned, many examples have been found in which the complex of higher T, is the end product of a displacement, and no example to the contrary has been reported. But i t is not necessarily true for polynucleotide associations of very different types. For example, the reaction between poly A and poly U a t acid pH, though slow, goes in the opposite direction (187, 188). Poly A is displaced from

-

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A. M. MICHELSON, J . M A S S O U L I ~AND ~

w. GUSCHLBAUER

the association with itself even though this has a higher T, than poly A * poly U. A few examples of displacement reactions between neutral helical structures follow.

+

poly A . 2 poly I 2 poly C T, = 39°C (0.11 M Na+, pH 7)

-+

2(p0ly I * poly C) T, = 58°C

+ POIY A

This reaction (202) has been demonstrated by various methods such as ultraviolet absorption, infrared spectroscopy, and sucrose gradient sedimentation. Another example is (146): poly dI poly dC T,,,= 43°C (0.1 M Na+)

+ poly dBrC -+

poly dI . poly dBrC T, = 70°C

+ poly dC

Here poly dC may be in a structured form (T,,, = 48OC a t p H 7, in 0.1 M Na+),although no corresponding transition can be detected in the dissociation profiles. I n these two cases, therefore, it may be argued that the driving force in the reactions is the tendency to accommodate the maximum number of bases into rigid helical structures. However, a complete set of displacement reactions between the polydeoxy, polyribo, and hybrid associations of poly I and poly C has been reported (206).

+ poly dC poly I . poly dC + poly C T, = 52.3"C poly dI poly C + p01y I T, 35.4"C p01y dI poly dC + poly I

poly dI . poly C T, = 35.4"C

*

=

T,

= 46.1"C (0.1 M Na+, 30°C)

-+

-+

-+

poly dI . poly dC T, = 46.1"C

+ poly C

+ poly dC p l y I poly C + p01y dI T, = 60.2"C poly I . p01y dC + poly dI p l y I * poly C T, = 60.2"C *

T,

=

52.3"C

These reactions were demonstrated by examination of the melting profile a t given intervals of time. The fluorescence of ethidium bromide in the presence of double-stranded structures also provides a useful technique in certain cases ( 2 0 6 ~ ) . Moreover, a helix-helix rearrangement has also been shown by the same authors (206): poly dI p01y C 1

+ poly I

*

poly dC + poly I . poly C

+ poly dI

*

poly dC

Another example of reaction between two multistranded secondary structures lies in the association of poly A and poly X a t p H 5 (221).

125

SYNTHETIC POLYNUCLEOTIDES

Another series of displacements occurs when poly C and its halogenated analogs compete for association with poly I or poly G (217). In 0.15 M Na+, pH 7, the T, values are the following: poly I . poly c poly I poly BrC poly I . p01y IC

60.2"C 89.2"C 91.3"C

According to expectation, poly BrC and poly IC displace poly C. Equilibrium is reached in 1 hour a t 20°C. This system offers an opportunity to compare the stabilities of two structures, the Tm's of which are nearly equal. Such a comparison is of interest in view of the use that can be made of T, as a measure of stability. If one can write AG = A ( T , - T) , a direct comparison of AG values is possible for different complexes, provided the A constants are equal. The values are, of course, different for two- and three-stranded helices, but it may be supposed that, for similar double-stranded structures, A will be the same. Poly I poly BrC and poly I * poly I C constitute a most favorable case. If the A constants are really equal, polyI * poly I C should have a slightly lower free energy than poly I poly BrC, a t any temperature. This is confirmed by a slow displacement of poly BrC by poly I C ; half of the poly BrC is displaced, in dilute polymer solution ( l k 4 M in nucleotides), in 1 month a t 20°C. Conversely, poly BrC does not displace poly IC. Some expected displacement reactions have not been found to occur a t measurable rates, even though the T , differences must be large in both cases (217):

-

poly G poly C poly I * poly C

+ poly BrC

+ p01y G

-f,

-f,

+

poly G . poly BrC poly C] p01y G * poly C plyI

+

However the reverse displacements do not occur either, and it has been argued that large activation energies linked to the breakage of G-C or G-G bonds prevent these systems from reaching equilibrium.

IX. Polynucleotide Analogs A wide variety of polynucleotides containing analogs of the purines and pyrimidines occurring in nucleic acids has been examined (217~). Included in this group are also homopolyniers of methylated nucleotides, which occur in rather minor proportions as natural constituents. When hydrogen-bonding possibilities are blocked, as for example in poly N1-methyluridylate (86, 218) poly N1-methylinosinate, poly Nl,N7dimethylinosinate, and poly N1-methyladenylate ('7) , then complex formation with the appropriate polynucleotide (poly A, poly C,or poly U)

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GUSCHLBAUER

does not occur. Nevertheless, double- (or multi-) stranded secondary structures may be possible; for example, poly NlMeA readily gives an “acid” form corresponding to that of poly A. This is in accord with the postulated structure for poly A a t acidic pH involving the 6-amino group and N7 for hydrogen bond formation. Alkylation of the 6-amino group in adenosine produces an effect that depends on the alkyl group. Thus polyA treated with formaldehyde to give essentially the 6-hydroxymethyl derivative does not give an “acid” form or complex with poly U (219), nor does a copolymer of adenylate and 6-methylaminoadenylate (some 70% of the latter) ( 7 ) . However, the homopolymer of “6-methyladenylate” (6 MeA) apparently interacts with poly U (but only to give the 1:1 complex) ; the thermal transition of this complex is very low (near 15°C) (220). I n the case of poly 6-hydroxyethyladenylate,no interaction occurs with either poly U or poly BrU (use of the latter would be expected to increase stability of a possible complex by some 20°C) (83). Poly 6-hydroxyethyl adenylate (HEA) does not give a doublestranded “acid” form and, in accord with the lack of complex formation with poly U, no interaction occurs with poly I. Stacking of the bases in poly HEA a t neutral p H is nevertheless demonstrated by dichroic studies; hence a rather powerful steric effect of the alkyl group must be postulated to explain the lack of interaction with polyU and poly I. Nevertheless it may be noted that a very stable complex is formed with polyxanthylate (221). Here the geometry must be such that steric hindrance by the hydroxyethyl group is eliminated. [Similarly, formaldehyde treatment of polyA does not prevent hydrogen bond formation with poly X (221).] Methylation a t N7 in purine nucleosides (guanosine, inosine) also reduces structural stability (7). Thus no secondary structure can be observed in poly N7-methylinosinate a t pH 7 (at this p H the base is zwitterionic) ; the thermal stability of secondary structure in poly N7methylguanylate acid is markedly less than that of poly G, though this is partly the effect of liberation of the free purine on heating. Similarly, the double helical structures formed by these polymers with polyC (or poly BrC) are dissociated under milder conditions than those containing the nonmethylated polymers. It may be noted that in these complexes (and also in structured poly 7-methylguanylate) the purine base is protonated a t pH 7 and hence carries a positive charge. The complexes are thus similar to poly G * poly C or poly G a t pH 2.5, where a reduction of stability also occurs despite possible interactions with the negative phosphate groups. In addition, the alkaline titration of poly 7MeI * poly C is noncooperative, suggesting that formation of the zwitterion of

127

SYNTHETIC POLYNUCLEOTIDES

7-methylinosine on removal of a proton does not lead to a disruptive influence on the rest of the hydrogen-bonded base pairs (7). Methylation a t C5 in pyrimidine polynucleotides leads to increased stability in both the homopolynucleotide secondary structure and in the heterologous complex (85, 222). For example, poly 5-methyluridylate (poly ribothymidylate) has a T , some 30°C higher than that of poly U, and the complex with poly A is also more stable than poly A poly U by about 20°C (85, 218). A wide variety of analogs of polyuridylate have now been described (85, 223). These include homopolynucleotides containing 5-fluoro-, 5-chloro, 5-bromo-, s-iodo-, and 5-hydroxyuracil. With respect to the secondary structure of homopolynucleotide a t low temperatures, stability is increased by substitution by bromine or iodine and decreased by fluorine, chlorine, and hydroxyl. However, when the analogous complexes with poly A are considered, although decreased stability occurs with poly 5-hydroxyuridylate, a marked increase in stability is found not only with poly 5-bromo- (or iodo-) uridylate but also with poly 5-chlorouridylate (Fig. 13).The case of poly 5-fluorouridylate is somewhat special since the pK of the polymer is 8.1 (in 0.15 M NaCl). At pH 7 poly A 2 poly FU is about 10°C less stable than poly A 2 poly U, but at p H 6 the Tmlsare virtually identical (224). Polypseudouridylate may also be regarded as an analog of polyuridylate (225).Here the substituent a t C5 is the sugar phosphate chain itself. P o l y q has a remarkably stable secondary structure with a T , some 55°C higher than that of poly U. Both double- and triple-helical complexes are obtained with poly A. Absorbance-temperature profiles of poly A 2 poly q are biphasic, the second dissociation occurring a t a higher temperature than poly A * poly U in the absence of magnesium ions. Since magnesium ions have no effect on the T, of poly A poly @, the thermal stabilities of poly A * poly U and poly A * poly Q are identical in solvents containing magnesium ions (1W2M ) . An anomalous alkaline titration behavior is observed with both poly A * 2 poly q and the structured form of poly q acid. Considerable noncooperative loss of protons can occur before there is a cooperative rupture of secondary structure (85, 225). Halogen-substituted poly C’s have also been studied. Like poly C, both poly BrC and poly iodoC show no indication of a rigid secondary structure a t pH 7 (226). Contrary to an earlier report ( 8 5 ) , polyBrC readily forms an “acid” structure, comparable with th a t of poly C, but is much less stable. Similar decrease in stability of the “acid form” has been reported previously for poly dBrC compared with poly dC, both of which are nevertheless more stable than the corresponding polyribo-

-

-

-

-

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nucleotides. Poly iodoC shows rather more complex behavior since thermal dissociation of the acid form is biphasic. Spectral properties indicate two ordered “acid” structures, both of which are protonated. (226). Introduction of the bromine (or iodine) atom again increases the stability of hetero complexes. Thus both poly I poly BrC and poly I poly I C possess Tm’s some 30°C higher than that of polyI * poly C

-

-2

-I

0

log “a*]

FIG.13. Variation of T, with ionic strength of triple-stranded complexes of A and T, U, ClU, BrU, and IU. Symbol

0-0

0-0 V-V

v-v x-x

+-+

A- A

Complex

References

rA . rU rU rA rBrU. rBrU rA rClU .rClU dA . rU .rU dA * dT * dT dA rU . d T rA rIU .rIU

41, 48, 86

+

86, 818 86, 818 266 266 266 86

(85). Similar increase in stability is also seen in the corresponding complexes with poly G (29). As is generally the case, increase in thermal stability is accompanied by an increase in the pH necessary t o cause dissociation of such complexes. This apparent inversion of the effect of substitution a t C5 by bromine depending on whether a homologous or a heterologous complex is involved is readily explained. The acid form of poly C requires addition of a proton for each pair of bases. Since substitution a t C5 by bromine (or iodine) reduces the basicity of the pyrimi-

129

SYNTHETIC POLYNUCLEOTIDES

dine (as shown by pK values of the nucleosides), uptake of protons is rendered less facile and hence leads to an apparent decrease in stability at slightly acidic pH. At neutral pH, the normal stabilizing effect of increased dipole interactions is apparent. That other factors are involved is shown by the fact that a similar inversion occurs if ribo- and deoxyribopolycytidylates are compared. Here the “acid” homologous complex formed by poly C is less stable than that of poly dC, whereas poly I * poly C is more stable than poly I poly dC. The stabilizing effect of substitution by bromine a t C5 of pyrimidines has been fully documented in the case of polydeoxyribonucleotides. Poly (dA-dBrU) has a T, 8°C higher than that of poly (dA-dU) (198) whereas the difference between poly A * 2 poly U and the corresponding polyA 2 poly BrU is some 36”C, that is 18°C per substitution since two strands of the polyU analog are involved (85). However, poly dI poly dBrC has a T, some 26°C higher than poly dI * poly dC (199), that is, the same difference as that between poly I poly C and poly I poly BrC in the ribose series. It thus appears that vertical interactions play a marked role in the stability of helical complexes. This is also seen in a comparison of the T,’s of poly (A-U) and poly A poly U since it may reasonably be assumed that the strengths of the hydrogen bonds (in both cases between A and U ) are the same. It has been suggested that subsitution by bromine increases hydrogen bond strength, but this seems insufficient as an explanation, since the effect should be independent of sequence, and this is clearly not the case. The explanation quoted by Inman and Baldwin (199) for an alleged differential effect of bromine substitution a t C5 in cytosine or in uracil cannot be accepted. First, there is no validity in the statement that bromine is ortho to the donor group in one case and meta in the other since both systems are similar (Br-C-C-N-H). Second, the direct comparison of an alternating copolymer with a complex formed from two homopolymers is not valid, as we have seen above. Third, if homologous structures are considered, poly BrU is more stable than poly U while poly BrC (acid form) is less stable than poly C. Another nucleotide analog that bears a resemblance to pseudouridylic acid is 3-isoadenylic acid ( 4 ) , in which the glycosyl linkage is a t N3 instead of N7. Polyisoadenylate readily interacts with poly U or polyI to give 1:l complexes, which are extremely stable in comparison with the corresponding structures containing poly A. Unlike poly A, however, poly isoA does not give a double-helical structured “acid form.” The hypochromicity of the polymer and the effect of increase in temperature a t p H 7 suggest that, like poly A, the bases are stacked to a high degree, thus giving a single-stranded helical conformation. While the greater

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basicity of 3-isoadenosine (pK = 5.5) compared with adenosine (pK = 3.45) may promote stronger hydrogen bonding, it is more likely that the increased stability of the complexes is a result of increased interplanar interactions, with perhaps an increase in organization of solvent molecules also playing a role ( 4 ) . In contrast with poly A * poly U, the complex poly isoA poly U shows no hyperchromic effect in the region of 290 mp. This is perhaps another demonstration that the assignment of particular transitions ( T + T” or n+ x ” ) to specific wave bands is a little premature a t present. Like poly I and poly G, the homopolymer of xanthylic acid possesses a helical secondary structure (221). The structural stability of poly X is somewhat greater than that of poly I, but markedly less than that of poly G. In contrast with polyI and poly G, polyX does not interact with poly C (or poly BrC) under a wide variety of experimental conditions. However complexes are readily formed with poly A, polyI, and poly U, and also with a wide variety of analogs of poly A, including poly 3-isoadenylate7 poly N1-methylA, and poly N6-methyl (or hydroxymethyl, or hydroxyethyl) A. Certain peculiarities are shown by these complexes. Thus the stability of poly A * 2 poly X is increased on lowering the pH (removal of negative charges from ionized xanthine residues) whereas that of poly U poly X is decreased (2221). Several other extremely interesting polynucleotide analogs have been prepared, but details of their physical properties are not as yet available (227). e

-

X. Theory and Practice of Helix-Coil Transitions Ever since the proposal of the DNA model by Crick and Watson (1954) (215), the topological, mechanical, and thermodynamical prob-

lems of this structure have interested physical chemists. Numerous suggestions have been presented (228-233) for the untwisting and or “unzippering” of a double-stranded helix of the type of DNA, particularly in view of mechanisms of replication of DNA. These investigations essentially show the mechanical feasibility of the sequential opening mechanism for a double helix. No studies of the considerably more complicated case of a triple helix have been reported. On the other hand, considerable energy has been invested, particularly in recent years, in studies of the energetical aspects of the helixrandom coil transition. Statistical mechanics was originally used to describe this transition in polypeptides (i.e., the single-stranded a! helix) (234, 2%). This treatment has been extended to the DNA double helix and to polynucleotides by Hill and others (236-939). Zimm (6373 also investigated the effect of chain ends on the stability and coopera-

SYNTHETIC YOLYNUCLEOTIDES

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tiveness of the transition. He introduced a parameter, uo (in later papers 1 , ’ ~ related )~ to the free energy, C, of stacking the base pairs on top of each other: UO = exp (-e/kT) Zimm demonstrated that the sharpness of the transition depended on the value of the stacking parameter, u0. Comparing experimental values for the melting of DNA (in 1959) which indicated a breadth of the melting transition of about 2”C, Zimm estimated 0.1 < uo < 0.01 and therefore 3 kcal < L < 1.5 kcal. Since the melting curves become steeper (more cooperative j with more homogeneous and longer DNA chains, he was inclined to prefer the higher value. In practice, it has been found that this value still seems too small. I n a more refined treatment of poly (dA-dT) and poly dI * poly dC, Crothers and Zimm (239), using the partition function of Lifson and Zimm (S40), evaluated u0 between le3 and for these two cases. They used in their studies the measured value of AH = 7 keal/mole of Rawitscher et at. (163) for the stacking energy in poly A. This gives a breadth of the thermal transition of less than l0C, quite in agreement with the best recent observations on polynucleotides and DNA. This means that the transition of a completely and perfectly aligned long helix is much more cooperative than Zimm had evaluated from the earlier DNA data. Similarly, statistical thermodynamical treatments have been used by Applequist and Damle (941) to investigate the effect of chain length on polynucleotide transitions and by Magee et al. (242, 243) to study homo- and copolymeric binding. These calculations all predict a decrease with decreasing degree of polymerization and a broadening of the of T,,, melting profile (244-246a). This broadening is not always seen (246). Ionic strength has a very marked effect upon the stability of the helix. Schildkraut and Lifson (247) have studied this in detail, and developed a generalized theory of electrostatic repulsion based on the Debye-Huckel treatment.

XI. Factors Governing Structure All homopolynucleotides derived from natural, or almost natural, nucleotides form multistranded secondary structures under appropriate conditions. In the case of the “basic” (or 6-amino) nucleosides (A and C) , slightly acidic conditions are necessary, whereas the “acidic” (or 6-keto) polymers (G, U, I, X ) all give defined secondary structures a t pH 7, albeit with vastly different stabilities. Disregarding complexes containing substituted bases where substitution merely increases or decreases the stability without change in specificity, the family of inter-

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actions so far experimentally observed with homopolynucleotides can be summarized as follows: A A

+

c

-

C (H+)

+

U

G

+

-

-

+

(H+)

+

I +

+

+

-

-

-

X

+

u

+

-

G

-

+

-

I

+

+

-

-

+

+

x

+

-

+

-

+

+

+

-

-

While certain interactions cannot be demonstrated (for example, of A with G ) , this may be simply a consequence of the greater stability of secondary structure in poly G compared with poly A poly G ; that is, G-G interactions (in a total sense) are greater than those for A-G, whereas, in the case of poly I, the 1-1interactions are weaker than those present in poly A poly I. However, if interaction between G and G is excluded by some means such as spatial distribution, the possibility arises that A-G base pair formation could occur. This argument, also applicable to poly U poly G (compare with the known poly U poly X) cannot be extended to the absence of poly U poly C, and one can only assume that the stability of such a structure is extremely low. General rules governing stability of helical complexes have usually been analeptic in character. Despite the wealth of information now available, prediction is still hazardous. For example, although the alternating poly (A-U) is some 10°C more stable than polyA poly U, it cannot be assumed that alternating poly (G-C), when available, will likewise be more stable than poly G poly C. This involves virtually the simplest comparison possible:

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-

It is quite likely that vertical G-G and C-C interactions are in total stronger than G-C interactions (248),whereas A-U (or T) interactions are greater than those of A-A plus U-U (or T-T), thus leading to an

inversion of the relative stabilities. The fact that the same enzyme produces poly dC poly dG but alternating poly (dA-dT) suggests that this might be the case, if one assumes that the enzyme catalyzes formation of the most stable possibility under admittedly abnormal conditions.

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SYNTHETIC POLYNUCLEOTIDES

133

However, several major factors govcrning structural stability of polynucleotide complexes can be defined. Clearly, direct interplanar interactions (London, van der Waals forces) (%’&a) play an important role and can be modified by substitution of the bases. I n addition “hydrophobic” forces, defined as organization of solvent molecules in the vicinity of the helix (249),exert a considerable control, and can be affected by base substitutions (including methyl) and also by the nature of the sugar. Finally, hydrogen bonds certainly control specificity of interactions and contribute t o stability. This contribution is by no means overwhelming and the concept that the three hydrogen bonds possible in poly G poly C are responsible for the greater stability than that of poly A poly U, containing two hydrogen bonds, is unwarranted. Despite the failure of efforts to obtain interactions involving only one hydrogen bond (e.g., poly A poly N1-methyl-5-bromouridylate), such a possibility cannot be excluded, since steric or disruptive effects on solvation by the N1-methyl group may be important. One hydrogen bond may well be insufficient to create the planarity induced by two or more hydrogen bonds, but one can nevertheless visualize possible structures. The presence of considerable stacking of bases in single-stranded polynucleotides and even oligonucleotides indicated by earlier studies (1) has now been extensively confirmed (70, 82, 169, 161). I n this connection, mention must be made of the definitive ORD studies on oligonucleotides by Tinoco and his co-workers (69, 70, 260, 251). The effect of chain length (251a) has also been studied by various techniques and it is now clear that interplanar interactions contribute significantly to single-stranded secondary and tertiary structures. However, this concept has been much misunderstood as leading to virtually rigid single-helical structures which is certainly not the case, either in polyA or in tRNA. The contradiction between hydrodynamic and optical results can be resolved readily if account is taken of the time of stacking, and the fluctuation of this stacking along a chain. Again, stacking of bases in a single strand represents not the lack of freedom as in a DNA double helix, but rather a restriction of rotation or separation of the bases, which are nevertheless in movement even when stacked. Nevertheless, this kind of stacking interaction is undoubtedly important with polymerizing enzymes such as DNA polymerase and RNA polymerase, where hydrogen bonds provide specificity, but interplanar interactions between the terminal nucleotide in the chain and the approaching nucleoside triphosphate provide some of the binding energy. Among other approaches indicating base stacking can be mentioned the transfer of excitation energy between the neighboring nucleotide bases determined by studies of the luminescence of various oligonucleotides. Here again, the order of

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+

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sequence, that is, direction of the chain, plays an important role (252, 253). The question of flexibility of double helices has received little attention recently, apart from studies of the polarization of fluorescence of acriflavine conjugates of polyribonucleotides (254). Some loss of rigidity precedes ultraviolet absorption effects accompanying thermal dissociation of double- and triple-stranded structures. Flexibility in a double helix is possibly quite important for the formation of certain triple helices. For example poly G and poly C give only a double helix whereas degraded polymer (either G or C) gives rise to 2 p o l y G poly C; that is, a third strand can be added only if a certain number of breaks are present in one strand of the double helix. Similar reasoning may explain why polyG (probably double helix) is much less viscous than oligo G, which is multistranded and can apparently aggregate to the point of precipitation. Yet another indication of the fact that hydrogen bonds alone play a relatively slight role in the stability of double helices is the observation that the Tm’sof complexes of a series of oligouridylates with polyA are markedly less than those of an analogous series of oligo A’s complexed with poly U. It is probably too naive to attempt explanations of the biological effects of various nucleotide analogs in terms of the physical behavior of homopolynucleotide analogs, despite certain marked effects. Explanations of the mutational effect of incorporation of bromouracil as a result of increased tautomerization are equally suspect despite the beauty of simplicity. It may well be that such an analog modifies the behavior of an adjacent base by increase in the interplanar interaction. I n this respect it may be noted that the observed energy transfer is much more marked in A +-BrU than in A + U. This may well lead to a different electron distribution giving increased tautomerization and even a change in chemical reactivity of the neighboring base in addition to the modified behavior of the analog compared with the natural base. Another overlooked possible cause of biological effect may be the slowing down of reading, thus increasing the possibility of error due to misreading of a tautomer. If replication, transcription, and translation use somewhat different time scales (even if very different from the time of tautomeriaation), one could visualize an analog (e.g., FU) behaving abnormally in one case and normally in another.

REFERENCES 1 . A. M. Michelson, “The Chemistry of Nucleosides and Nucleotides.” Academic

Press, Few York, 1963; R. F. Steiner and R. F. Beers, “Polynucleotides.” Elsevier, Amsterdam, 1961; A Rich and D. W. Green, Ann. Rev. Biochem. 30,

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93 (1961); J. R. Fresco and D. B. Strauss, Am. Scientist 50, 158 (1962); B. H. Zimm and N. R. Kallenbach, Ann. R ev . Phys. Chem. 13, 171 (1962); I. Tinoco and D. N. Holcomb, ibid. 15, 371 (1964). 2. M. Grunberg-Manago, This series, 1, 93 (1963). 3. A. M. Michelson, J. Dondon, and M . Grunberg-Manago, Biochim. Biophys. Acta 55, 529 (1962). 4. A. M. Michelson, C. Monny, R. A. Laursen, and N. J. Leonard, Biochim. Biophys. Acta 119,258 (1966). 5. M. N. Thang, M. Graffe, and M. Grunberg-Manago, Biochim. Biophys. Acta 108, 125 (1965). 6. J. Weill, N. Befort, B. Rether, and J. P. Ebel, Biochem. Biophys. Res. Commun. 15, 447 (1964). 7. A. M. Michelson and F. Pochon, Biochim. Biophys. Acta 114, 469 (1966). 8. L. Grossmann, Proc. Natl. Acad. Sci. U S . 50, 657 (1963). 9. J. H. Philips, D. M. Brown, R. Admann, and L. Grossmann, J . Mol. Biol. 12, 816 (1965).

10. W. E. Fletcher, J. M. Gulland, D. 0. Jordan, and H. E. Dibben, J . Chem. SOC. p. 30 (1944). 11. A. M. Michelson, unpublished results (1965). 12. C. M. Radding and A. Kornberg, J . Biol. Chem. 327, 2877 (1962). 13. H. K. Schachman, J. Adler, C. M. Radding, I. Lehmann, and A. Kornberg, J . Biol. Chem. 235, 3242 (1960). 14. F. J. Bollum, E. Groeniger, and H. Yoneda, Proc. Natl. Acad. Sci. U.S. 51, 853 (1964).

15. C. Byrd, E. Ohtsuka, M. W. Moon, and H. G. Khorana, Proc. Natl. Acad. Sci.

U.S. 53, 79 (1965). 16. P. J. Flory, “Principles of Polymer Chemistry.” Cornell Univ. Press, Ithaca, New York, 1953. 17. T. Svedberg and K. 0. Pedersen, “The Ultracentrifuge.” Oxford Univ. Press, London and New York, 1940.

18. H. K. Schachman, “Ultracentrifugation in Biochemistry.” Academic Press, New York, 1960. 10. C. Tanford, “Physical Chemistry of Macromolecules.” Wiley, New York, 1961. 20. R. L. Baldwin and K. E. Van Holde, Fortschr. Hochpolymer-Forsch. 1, 451 (1960). $1. H. K. Schachman, in “Methods in Enzymology” (S. P. Colowiclr and N. 0. Kaplan, eds.), Vol. IV, p. 32. Academic Press, New York, 1957. 22. A. Peterlin, J . Coll. Sci. 10, 587 (1955). 23. A. Peterlin, Makromol. Chem. 18-19, 254 (1956). 24, H. G. Elias, Angew. Chem. 73,209 (1961). 26. T. Lindahl, D. D. Henley, and J. R. Fresco, J . Am. Chem. SOC.87, 4961 (1965). 25a. E. F. Cassassa and H. Eisenberg, Advan. Protein Chem. 19, 287 (1964). 26. R. Thomas, Biochim. Biophys. Acta 14, 231 (1954). 27. G. Felsenfeld and G. Sandeen, J. Mol. Biol. 5, 587 (1962). $8. J. MassouliB, W. Guschlbauer, L. C. Klotz, and J. R. Fresco, Compt. Rend. h a d . Sci., 260, 1285 (1965). 29. F. Pochon and A. M. Michelson, Proc. Natl. Acad. Sc. U.S. 53, 1425 (1965). 30. J. R. Fresco, L. C. Klotz, and E. G. Richards, Cold Spring Harbor Symp. Biol. 28, 83 (1963). 31. G. Felsenfeld and S. Z. Hirschman, J . Mol. Biol. 13, 407 (1965).

136

A. M. MICHELSON, J . M A S SO U LI ~AND

w.

GUSCHLBAUER

3.9. H. R. Mahler, B. Kline, and B. D. Mehrotra, J. MoZ. Boil. 9, 801 (1964). 33. G. Felsenfeld and G. L. Cantoni, Proc. Natl. Acad. Sci. U S . 51, 818 (1964). 34. W. Guschlbauer and A. M. Michelson, in preparation (1966) 36. W. Guschlbauer, Biophysik 3, 156 (1966). 36. W. Guschlbauer, Nature 209, 258 (1966). 37. P. Job, Ann. Chim. 9, 113 (1928). 38. G. Felsenfeld, Biochim. Biophys. Acta 29, 133 (1958). 39. G. Felsenfeld, D. R. Davies, and A. Rich, J. Am. Chem. Soc. 79, 2023 (1957). 40. G. Felsenfeld and A. Rich, Biochim. Biophys. Acta 26, 457 (1957). 41. J. Massoulie, R. Blake, L. C. Klotz, and J. R. Fresco, Compt. Rend. Acad. Sci.

259, 3105 (1964). 42. C. L. Stevens and G. Felsenfeld, Biopolymers 2, 293 (1964). 43. D. Shugar and J. J. Fox, Biochim. Biophys. Acta 9, 199 (1952). &. J. J. Fox and D. Shugar, Biochim. Biophys. Acta 9, 369 (1952). 46. D. Voet, W. B. Gratzer, R. A. Cox, and P. Doty, Biopolymers 1, 193 (1963). 46. G. H. Beaven, E. R. Holiday, and E. A. Johnson, In “The Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. 1, p. 493. Academic Press, New York, 1955.

47. T. V. Venkstern and A. A. Bayev, “Absorption Spectra of Nucleotides and

Oligonucleotides.” Nauka Press, Moscow, 1965. S. E. Kerr and K. Seraidarian, J . Biol. Chem. 159, 211 (1945). 49. R. F. Steiner, J. Biol. Chem. 236, 3037 (1961). 60. J. C. Reid and A. W. Pratt, Biochem. Biophys. Res. Commun. 3, 377 (1960). 61. S. K. Vadenko, S. G. Komzolova, and D. G. Knorre, Biokhimyia 27, 142 @.

(1962).

62. S. Lee, D. McMullen, G. L. Brown, and A. R. Stokes, Biochem. J . 94, 314 ( 1965).

W. Guschlbauer, E. G. Richards, K. Beurling, A. Adams, and J. R. Fresco, Biochemistry 4, 964 (1965). 64. A. W. Pratt, J. N. Toal, G. W. Rushizky, and H. A. Sober, Biochemistry 3,

63.

1831 (1964).

66.

W. Kauzmann, “Quantum Chemistry,” Chapters 15 and 16. Academic Press,

New York, 1957. 66. J. B. Biot, Mem. Acad. Sci., Toulouse 2, 41 (1817). 67. P. Drude, “Lehrbuch der Optik.” Hirzel, Leipzig, 1906.

68. C. Djerassi, “Optical Rotatory Dispersion.” McGraw-Hill, New York, 1960. 69. W. Moffitt, J . Chem. Phys. 25, 467 (1956). 69a. W. Moffitt, Proc. Natl. Acad. Sci. U S . 42, 736 (1956). 60. W. Moffitt and J. T. Yang, Proc. Natl. Acad. Sci. U S . 42, 596 (1956). 61. W. Moffit, D. D. Fitts, and J. G. Kirkwood, Proc. Natl. Acad. Sci. US.43, 723 (1957). 62. S. F. Mason, Nature 199, 139 (1963). 63. I. Tinoco, J. Am. Chem. Soc. 82, 4785 (1960). 64. I. Tinoco, J. Chem. Ph.ys. 34, 1067 (1961). 66. J. R. Fresco, Tetrahedron 13, 185 (1961). 66. J. T. Yang and T. Samejima, J . Am. Chem. Soc. 85, 4039 (1963). 67. T. Samejima and J. T. Yang, Biochemistry 3, 613 (1964). 68. T. Samejima and J. T. Yang, J. Biol. Chem. 240,2094 (1965). 69. D. N. Holcomb and I. Tinoco, Biopolymers 3, 121 (1965). 70. C. R. Cantor and I. Tinoco, J. MoZ. B i d . 13, 65 (1965).

SYNTHETIC POLYNUCLEOTIDES

137

71. P. S. Sarin and P. C. Zamecnik, Biochem. Biophys. Res. Commun. 19, 198 (1965). 72. P. S. Sarin and P. C. Zamecnik, Biochem. Biophys. Res. Commun. 20, 400 ( 1965). 79. M. R. Lamborg and P. C. Zamecnik, Biochem. Biophys. Res. Commun. 20, 328 (1965). 74. J. Brahms, J . Am. Chem. SOC.85, 3298 (1963). 75. M. Grosjean and M. Legrand, Compt. Rend. Acad. Sci. 251, 2150 (1960). 76. G. Holzwarth, Rev. Sci. Znst. 36, 59 (1965). 77. J. Brahms and G. Spach, Nature 200, 72 (1963). 78. J. Brahms, Nature 202, 797 (1964). 79. J. Brahms, W. F. H. M. Mommaerts, and C . Sadron, Compt. Rend. Acad. Sci. 258, 2203 (1964). 80. W. F. H. M. Mommaerts, J. Brahms, J. H. Weill, and J. P. Ebel, Compt. Rend. Acad. Sci. 258, 2687 (1964). 81. J. Brahms and W. F. H. M. Mommaerts, J . Mol. BioZ. 10, 73 (1964). 82. J. Brahms, J . Mol. Biol. 11, 785 (1965). 83. K. E. Van Holde, J. Brahms, and A. M. Michelson, J . Mol. Biol. 12, 726 (1965). 84. J. Brahms, A. M. Michelson, and K. E. Van Holde, J. MoZ. Biol. 15, 467 (1966). 85. J. MaasouliB, A. M. Michelson, and F. Pochon, Biochim. Biophys. Acta 114, 16 (1966). 86. L. J. Bellamy, “The Infrared Spectra of Complex Molecules.” Methuen, London, 1958. 87. C. L. Angell, J. Chem. SOC.p. 504 (1961). 88. Y. Kyogoku, T. Shimanouchi, M. Tsuboi, and I. Watanabe, Nature 195, 160 (1962). 89. M. Tsuboi, K. Yoshimasa, and T. Shimanouchi, Biochim. Biophys. Acta 55, 1 (1962). 90. M. Tsuboi, K. Matsuo, T. Shimanouchi, and Y. Kyogoku, Spectrochim. Acta 19, 1617 (1963). 91. H. T. Miles, Biochim. Biophys. Acta 22, 247 (1956). 92. H. T. Miles, Biochim. Biophys. Acta 27, 46 (1958). 93. H. T. Miles, Biochim. Biophys. Acta 30, 324 (1958). 94. H. T. Miles, Biochim. Biophys. Acta 35, 274, (1959). 96. H. T . Miles, Nature 181, 1814 (1959). 96. H. T. Miles, Biochim. Biophys. Acta 45, 196 (1960). 97. H. T. Miles, Proc. NatZ. Acad. Sci. U S . 47, 791 (1961). 98. H. T. Miles, Proc. Natl. Acud. Sci. US.51, 1104 (1964). 99. H. T. Miles and J. Frazier, Biochem. Biophys. Res. Commun. 14, 21 (1964). 100. H. T. Miles and J. Frazier, Biochim. Biophys. Res. Commun. 14, 129 (1964). 101. R. Langridge and M. Sundarlingham, This series. To be published. 102. F. H. C. Crick and J. C. Kendrew, Advan. Protein Chem. 12, 133 (1957). 103. J. C. Kendrew, Progr. Biophys. Chem. 4, 244 (1954). 104. V. Luzzati, This series, 1, 347 (1963). 105. V. Luzzati, Acta Cryst. 13, 939 (1960). 106. 0. Kratky, Progr. Biophys. MoZ. BioZ. 13, 105 (1963). 107. G. Porod, Fortschr. Hochpolymer-Forsch. 2, 363 (1961). 108. J. Witz, Ph.D. Thesis, Strasbourg (1965). 109. A. R. Stokes, Progr. Biophys. 5, 140 (1955). 110. E. P. Geiduschek and A. Holtzer, Adv. Biol. Med. Phys. 6, 431 (1958).

138

A. M . MICHELSON, J.

MASSOULIE AND w.

GUSCHLBAUER

A. Guinier, Compt. Rend. Acad. Sci. 204, 1115 (1937). J. Witz and V. Luzzati, J. Mol. Biol. 11,620 (1965). A. Peterlin, J . Polymer Sci.47, 403 (1960). C. D. Jardetzky and 0. Jardetzky, J. Am. Chem. SOC. 82, 222 (1960). M. P. Schweitzer, S. I. Ohan, G. K. Helmkamp, and P. 0. P. Ts'O, J. Am. Chem. SOC.86,696 (1964). 116. 0. Jardetsky, Biopolymer Symp. 1, 501 (1964). 117. C. C. McDonald, W. D. Phillips, and S. Penman, Science 144, 1234 (1964). 118. C. C. McDonald, W. D. Phillips, and J. Penswick, Biopolymers 3, 609 (1965). 119. L. A. Blumenfeld, Mem. Acud. Roy. Belge, Classe sci. 33, 93 (1961). 119a. R.0.Rahn, R. G. Shulmann, and J. W. Longworth, Proc. Natl. Acad. Sci. U.S.

111. 112. 113. 114. 115.

53, 893 (1965).

120. C. E. Hall, Proc. 4th Intern. Congr. Biochem. 9, 90. Pergamon Press, Oxford,

1959.

121. M. Beer and E. Moundranakis, Proc. Natl. Acad. Sci. U S . 48, 409 (1962). 12%'.P. J. Highton and M. Beer, J. Mol. Biol. 7, 70 (1963). 123. E. N. Moundrianakis and M. Beer, Proc. Natl. Acad. Sci. U.S. 53, 564 (1965). 124. E. N. Moundrianakis and M. Beer, Biochim. Biophys. Acta 95, 22 (1965). 126. M. N. Meselson, F. W. Stahl, and J. Vinograd, Proc. Natl. Acad. Sci. U S . 43,

581 (1957).

126. N . Sueoka, J. Marmur, and P. Doty, Nature 183, 1429 (1959). 187. C. L. Schildkraut, J. Marmur, and P. Doty, J. M o l . Biol. 4, 430 (1962). 128. C. L. Schildkraut, J. Marmur, J. R. Fresco, and P. Doty, J. Biol. Chem. 234PC4

(1961).

129. J. Marmur and P. Doty, J. Mol. Biol. 5, 109 (1962). 130. R. L. Sinsheimer and M. Lawrence, J. Mol. Biol. 8, 289 (1964). 131. M. Chamberlin and P. Berg, J. Mol. Biol. 8, 297 (1964). 13%'.M. Gellert, M. N. Lipsett, and D. R. Davies, Proc. Natl. Acad. Sci. U S . 48,

2013 (1962).

133. J. Iball, C. H. Morgan, and H. R. Wilson, Nature 199, 688 (1963). 134. I. Bang, Beitr. Chem. Physiol. Pathol. 4, 331 ; 5, 317 (1904). 136. P.K. Sarkar and J. T. Yang, Biochem. Biophys. Res. Commun. 20, 346 (1965). 136. A. M. Michelson, Nature 182, 1502 (1958). 137. M. N.Lipsett, J. Biol. Chem. 139, 1250 (1964). 138. J. R. Fresco and J. MassouliB, J. Am. Chem. SOC.85, 1352 (1963). 139. T. L. V . Ulbricht, R. J. Swan, and A. M. Michelson, Chem. Commun. 63 ( 1966).

140. A. M . Michelson and C. Monny, In preparation (1966). 141. P. K. Sarkar and J. T. Yang, Biochemistry 4, 1238 (1965). 142. M. Chamberlin and P. Berg, Federation Proc. 21, 385 (1962). 143. C.M. Radding, J. Josae, and A. Kornberg, J. Biol. Chem. 237, 2869 (1962). 144. A. Rich, Biochim. Biophys. Acta 29, 502 (1958). 145. R.B. Inman, J. Mol. Biol. 9,624 (1964). 146'. P. Doty, H. Boedtker, J. R. Fresco, R. Haselkorn, and M. Litt, Proc. Natl.

Acad. Sci. U S . 45,482 (1959). l4Y. M . N . Lipsett, Proc. Natl. Acad. Sci. U S . 46, 445 (1960). 148. E. G.Richards, C. P. Flessel, and J. R. Fresco, Biopolymers 1, 431 (1963). 149. P. K. Sarkar and J. T. Yang, J. Biol. Chem. 240,2088 (1965). 150. A. M. Michelson and C. Monny, Proc. Natl. Acad. Sci. U S . In press (1966). 151. J. R. Fresco and P. Doty, J. Am. Chem. SOC.79, 3928 (1957). 162. J. R. Fresco and E. Klemperer, Ann. N.Y. Acad. Sci. 81, 730 (1959).

SYNTHETIC POLYNUCLEOTIDES

139

Ts’O,G. K. Helmkamp, and C. Sander, Proc. Natl. Acad. Sci. U.S. 48, 686 (1962). 154. R. F. Steiner and R. F. Beers, Biochim. Biophys. Acta 32, 166 (1959). 155. J. Massoulik, Compt. Rend. Acad. Sci. 260, 5554 (1965). 156. J. R. Fresco, J. Mol. Biol. 1, 106 (1959). 157. A. Rich, D. R. Davies, F. H. C. Crick, and J. D. Watson, J. Mol. Biol. 10, 28 (1961). 158. V. Luzzati, A. Mathis, F. Masson, and J. Witz, J. Mol. Biol. 10, 28 (1964). 169. M. Leng and G. Felsenfeld, J. Mol. Biol. 15, 455 (1966). 160. M. M. Warshaw and I. Tinoco, J. Mol. Biol. 13, 54 (1966). 161. J. Massoulib and A. M. Michelson, Compt. Rend. Acad. Sci. 259, 2923 (1964). 162. P. D. Ross and R. L Scruggs, Biopolymers 3, 491 (1965). 163. M. A. Rawitscher, P. D. Ross, and J. M. Sturtevant, J. Am. Chxm. SOC.85, 1915 (1963). 164. A. M. Michelson, J. Chem. SOC.p. 3655 (1959). 166. P. 0.P. Ts’O, G. K. Helmkamp, and C. Sander, Biochim. Biophys. Acta 55, 584 (1962). 166. G. X. Helmkamp and P. 0. P. Ts’O, Biochim. Biophys. Acta 55, 601 (1962). 167. E. 0.Akinrimisi, C. Sander, and P. 0. P. Ts’O, Biochemistry 2, 340 (1963). 168. R.Landgridge and A. Rich, Nature 198, 725 (1963). 169. G. D.Fasman, C. Lindblow, and L. Grossman, Biochemistry 3, 1015 (1964). 170. J. R. Fresco, in “Informational Macromolecules” (H. J. Vogel, V. Bryson, and J. 0. Lampen, eds.), p. 121.Academic Press, New York, 1963. 171. M. N. Lipsett, Biochem. Biophys. Res. Commun. 11, 224 (1963). 172. M. N.Lipsett, J. Biol. Chem. 139, 1256 (1964). 173. R. Haselkorn and C. F. Fox, J. Mol. Biol. 13, 780 (1965). 17.4. L. Hirschbein and J. R. Fresco, Personal communication (1966). 176. C. M. Radding, J. Josse, and A. Kornberg, J. Biol. Chem. 237, 2869 (1962). 176. J. Brahms and C. Sadron, Personal communication (1W). 177. R. C.Warner, J. Biol. Chem. 229,711 (1957). 178. A. Rich and D. R. Davies, J. Am. Chem. SOC.78, 3548 (1956). 179. A. Rich, Rev. Mod. Phys. 31, 191 (1959). 180. A. Rich, in “The Chemical Basis of Heredity” (W. D. McElroy and B. Glass, eds.), p. 557. Johns Hopkins Press, Baltimore, Maryland, 1957. 181. R. C. Warner and E. Breslow, Proc. 4th Intern. Congr. Biochem., 9,157. Pergamon Press, Oxford, 1959. 182. R. F. Steiner and R. F. Beers, “Polynucleotides.” Elsevier, Amsterdam, 1961. 183. R. F.Steiner and R. F. Beers, Nature 181, 30 (1958). 184. J. Massoulib, Compt. Rend. Acad. Sci. 259, 3392 (1964). 186. J. B. Le Pecq and C. Paoletti, Compt. Rend. Acad. Sci. 260, 7033 (1965). 186. R. D. Blake and J. R. Fresco, J. MoZ. Biol. 19, 145 (1966). 187. J. Massoulik, Ph.D. Thesis, University of Paris (1966). 188. J. Massoulik, Biochim. Biophys. Acta In press (1966). 189. R. A. Cox, Biochim. Biophys. Acta 68, 401 (1963). 190. V.Sasiselcharan and P. B. Sigler, J. Mol. Biol. 12, 296 (1965). 191. A. E.V .Haschemeyer and H. Sobell, Proc. Nutl. Acad. Sci. U.S. 50, 873 (1963). 192. M. Saunders and P. D. Ross, Biochem. Biophys. Res. Commun. 3, 314 (1960). 193. R.F. Steiner and C. Kitzinger, Nature 194, 1172 (1962). 194. S. Glasstone, K . G. Laidler, and H. Eyring, “The Theory of Rate Processes.” McGraw-Hill, New York, 1941. 196. R. B. Inman and R. L. Baldwin, J. Mol. Biol. 5, 172 (1962). 163. P. 0. P.

140

A. M. MICHELSON, J . M AS SOU LI ~AND

w.

GUSCHLBAUER

196. R. B. Inman and R. L. Baldwin, J. Mol. Biol. 5, 185 (1962). 197. H. C. Spatz and R. L. Baldwin, J. Mol. Biol. 11, 213 (1965). 198. D. R. Davies and R. L. Baldwin, J. Mol. Biol. 6, 251 (1963). 199. R. B. Inman and R. L. Baldwin, J . Mol. Biol. 8, 52 (1964). 200. M. Chamberlin, R. L. Baldwin, and P. Berg, J. MoZ. Biol. 7, 334 (1963). 201. A. Rich, Nature 181,521 (1958). 202. P. B. Sigler, D. R. Davies, and H. T. Miles, J. MoZ. Biol. 5, 709 (1962). 203. D. R. Davies and A. Rich, J . Am. Chem. SOC.80, 1003 (1958). 20.4. R. L. Haselkorn, Ph.D. Thesis, Harvard University (1960). 206. M. J. Chamberlin and D. L. Patterson, J. Mol. Biol. 12, 410 (1965). 106a. J. B. Le Pecq and C. Paoletti, Compt. Rend. Acad. Sci. 261, 838 (1965). 206. G. Giannoni and A. Rich, Biopolymers 2, 399 (1964). ,207. R. B. Inman, J. Mol. BioZ. 10, 137 (1964). 208. F. J. Bollum, Personal communication (1966). 209. W. Szer, M. Swierkowski, and D. Shugar, Acta Biochim. Polon. (English Transl.) 10, 87 (1963). 210. R. Haselkorn, Science 143,682 (1964). 211. E. Reich, Science 143, 684 (1964). 212. E. P. Geiduschek, J. W. Moohr, and S. B. Weiss, Proc. Natl. Acad. Sci. U S . 48, 1078 (1962). 213. S. L. Huang and L. F. Cavalieri, Proc. Natl. Acad. Sci. U S . 51, 1022 (1964). 214. M. J. Chamberlin and D. L. Patterson, J. MoZ. BioZ. I n press (1966). 216. F. H. C. Crick and J. D. Watson, Proc. Roy. SOC.A223, 80 (1954). a16. D. R. Davies, Nature 186, 1030 (1960). 217. J. MamouliB and A. M. Michelson, Biochim. Biophys. Acta, In press (1966). $ 1 7 ~ A. . M. Michelson, Bull. SOC.Chim. BioZ. 47, 1553 (1965). 218. W. Szer and D. Shugar, Acta Biochim. Polon. (English Transl.) 10, 219 (1963). 219. R. F. Steiner and R. F. Beers, Biochim. Biophys. Acta 33, 470 (1959). 220. B. E. Gr&, W. J. Haslam, and C. B. Reese, J. MoZ. Biol. 10, 353 (1964). 221. A. M. Michelson and C. Monny, Biochim. Biophys. Acta. In press (1966). 222. W. Szer and D. Shugar, J. Mol. Biol. 17,174 (1966). 223. A. M. Michelson, J. Dondon, and M. Grunberg-Manago, Biochim. Biophys. Acta 55, 529 (1962). 924. M. Grunberg-Manago and A. M. Michelson, Biochim. Biophys. Acta 87, 593 226.

(1964).

F. Pochon, A. M. Michelson, M. Grunberg-Manago, W. E. Cohn, and L.

Dondon, Biochim. Biophys. Acta 80, 441 (1964). 226. A. M. Michelson and C. Monny, In preparation (1966). 227. E. Reich, D. C. Ward, A. Cerami, and P. Fromageot, In preparation (1966). 238. C. Levinthal and H. C. Crane, Proc. Natl. Acad. Sci. U.S. 42, 436 (1956). 229. M. Fixmann, J. Mol. Bid. 6,39 (1963). 230. W. Kuhn, Ezperientia 13, 301 (1957). 231. M. V. Wolkenstein, Dokl. Akad. Nauk. SSSR 130,889 (1960). 232. H. C. Longuet-Higgins and B. H. Zimm, J. Mol. Biol. 2, 1 (1960). 833. E. Freese, Cold Spring Harbor Symp. Quant. Biol. 23, 13 (1958). 23.4. J. H. Gibbs and E. A. Dimarzio, J . Chem. Phys. 28, 1247 (1958). 236. B. H. Zimm and J. K. Bragg, J. Chem. Phys. 28, 1246 (1958); B. H. Zimm and S. E. Rice, Mol. Phys. 3, 391 (1960). 236. T. Hill, J. Chem. Phys. 30, 383 (1959). $87. B. H. Zimm, J. Chem. Phys. 33, 1349 (1960).

SYNTHETIC POLYNUCLEOTIDES

Z38.

239. 240. 241. 242. 24% 244.

141

R. F. Steincr, J . Chem. Phys. 32, 215 (1960). D. M. Crothers and B. H. Zimm, J . M o l . Biol. 9, 1 (1964). S. Lifson and B. H. Zimm, Biopolymers 1, 15 (1963).

J. Applequist and V. Damle, J . Chem. Phys. 39, 2719 (1963). W. S. Magee, J. H. Gibbs, and B. H. Zimm, Biopolymers 1, 133 (1963). W. S. Magee, J. H. Gibbs, and G. F. Newell, J . Chem. Phys. 43, 2115 (1965). M. F. Singer, L. A. Heppel, G. W. Rushizky, and H. A. Sober, Biochim. Biophys. Acta 61, 474 (1962). 2.44~.P. Doty, J . Polymer Sci. 55, 1 (1961). 246. M. N. Lipsett, L. A. Heppel, and D. I?. Bradley, Biochim. Biophys. Acta 41,

175 (1960). 246a. M. N. Lipsett, L. A. Heppel, and D. F. Bradley, J. Biol. Chem. 236, 857 (1961). 246. A. M. Michelson, In preparation (1966). 2.47, C. L. Schildkraut and S. Lifson, Biopolymers 3, 195 (1965). 248. B. Pullmann, B. Claverie, and J. Caillet, Proc. Natl. Acad. Sci. U S . 55, 904 (1966). 248a. A. M. Michelson, Biochim. Biophys. Acta 55, 841 (1962). 249. W. Kauzmann, Advan. Protein Chem. 14, 1 (1959). 260. M. M. Warshaw and I. Tinoco, J . Mol. Biol. 13, 54 (1965). 261. C. R. Cantor, S. R. Jaskunas, and I. Tinoco, J . MoZ. Biol. I n press (1966). 261a. A. M. Michelson, T. L. V. Ulbricht, T. R. Emerson, and R. J. Swan, Nature 209, 873 (1966). 268. C . Helene, P. Douzou, and A. M. Michelson, Biochim. Biophys. Acta 109, 261 (1965). 263. C . Helene, P. Douzou, and A. M. Michelson, Proc. Natl. Acad. Sci. U.S. 55, 376 (1966). 864. D. B. S. Millar and R. F. Steiner, Biochim. Biophys. Acta 102, 571 (1965). 266. M. Riley, M. Maling, and M. Chamberlin, J . Mol. Biol. 20, 359 (1966). 266. W. Szer and D. Shugar, J . Mol. Biol. 5,580 (1962). 267. M. Swierkowski, W. Szer, and D. Shugar, Biochem. 2. 342, 429 (1965). 268. K. A. Hartmann and A. Rich, J . Am. Chem. SOC.87, 2033 (1985). 269. M. Chamberlin, Federation Proc. 24, 1446 (1965). 260. W. Szer, Biochem. Biophys. Res. Commun. 20, 182 (1965). 261. P. 0. P. Ts’o, S. A. Rapaport and F. J. Bollum, Biochemistry. In press.