A circular dichroism study of modified nucleosides

40

Biochimica et Biophvsica Acta, 801 (1984) 40-47

Elsevier BBA21797

A CIRCULAR D I C H R O I S M STUDY OF M O D I F I E D NUCLEOSIDES ANDRZEJ GALAT a,., PAWEL SERAFINOWSKI b and JACEK KOPUT b Institute of Biology, College of Pedagogics, Zolnierska 14, 10-561 Olsztyn and ~ Department of Chemistry, Adam Mickiewicz Unioersity, Grunwaldzka 6, 60-780 Poznah (Poland)

(Received January 17th, 1984)

Key words: Circular dichroism; Nucleoside structure," Uridine derivatwe

The conformation of 5-methoxycarbonylmethyluridine and 5-methoxycarbonylmethyl-2-thiouridine was studied by means of circular dichroism in various solvents. In order to calculate the accurate spectral parameters of the Cotton effects, the circular dichroism spectra were resolved into component Gaussian functions which simultaneously fit the adsorption spectra. On the basis of circular dichroism and proton magnetic resonance spectra, these nucleosides were found to occur in the fl-configuration with the 3 E - g g . a n t i conformation preferred. Due to the fact that the long-wavelength Cotton effect of mcmSs2U is not masked by the Cotton effects of the other nucleic acid monomers, the molecular parameters of this band may be useful for the conformational analysis of tRNA segments.

Introduction Transfer R N A s contain a variety of modified nucleosides [1-6]. In each t R N A , the p r o p o r t i o n of modified nucleosides to the, four that are normally found in R N A is considerable. To date, more than fifty modified nucleosides have been isolated and synthesized, and their conformations elaborated by a variety of physical and chemical methods [7-10]. A number of t R N A s contain thiolated nucleosides, which are known to exhibit different Cotton effects in comparison to those of unmodified nucleosides. This is due to the fact that the nucleosides which contain sulfur atoms in their structures exhibit at least one Cotton effect positioned above X = 300 nm, which is not masked by the Cotton effects of the four normal nucleosides [12-14]. * To whom correspondenceshould be addressed. Abbreviations: DBM, Debye-Bohrmagneton units; DMSO-d6, deuterated dimethylsulfoxide; mcmSU, 5-methoxycarbonylmethyluridine; mcmSs2U, 5-methoxycarbonylmethyl-2-thiouridine. 0304-4165/84/$03.00 © 1984 ElsevierScience Publishers B.V.

Limited access to a sufficient amount of thiolated nucleosides restricts the thorough study of the conformation and optical properties of this class of compounds. To date, only a few papers were devoted to the circular dichroism study of such nucleosides [7-10]. In this paper we present a study on the c o n f o r m a t i o n of 5-methoxycarbonylmethyluridine and 5-methoxycarbonylmethyl-2-thiouridine in various solvent, by using circular dichroism and 1 H - N M R spectroscopies. The CD data presented in this paper may be useful for the conformational analysis of t R N A s which contain these modified nucleosides, especially mcmSsZU. It has been shown, for example, that mcmSU occurs in the wobble position of t R N A Arg from brewer's yeast [15], whereas mcmSs2U occurs at the same position in tRNAL2y~ and tRNAG3lu from baker's yeast [16,17].

Materials and methods 5-Methoxycarbonylmethyluridine and 5-methoxycarbonylmethyl-2-thiouridine were synthesized

41 e m p l o y i n g t h e m e t h o d s o f F i s s e k i s a n d S w e e t [18]

photometrically, using the molar absorption coeffi-

and

S t r e h l k e [19], r e s p e c t i v e l y .

dents estimated at the long-wavelength ultraviolet

Ultraviolet absorption spectra, melting points and e l e m e n t a l a n a l y s i s o f t h e s e n u c l e o s i d e s a g r e e d well with those from chemically synthesized nucleos i d e s [18,19] a n d t h o s e f r o m n u c l e o s i t e s i s o l a t e d f r o m n a t u r a l s o u r c e s [20,21]. A c e t o n i t r i l e , d i o x a n e and methanol were supplied by Uvasol Merck,

a b s o r p t i o n m a x i m a , as g i v e n i n T a b l e I. T h e circular dichroism curves were obtained with a Jobin Yvon Mark III dichrometer. The absorbance of each sample was adjusted to be within the range

w a t e r w a s d e i o n i z e d , a n d 0.1 M HC1 s o l u t i o n w a s p r e p a r e d b y d i s s o l v i n g t h e s t o c k s o l u t i o n o f HC1

m a d e w i t h t h e u s e o f silica c u v e t t e s w i t h p a t h l e n g t h v a r y i n g f r o m 1 t o 10 m m . T h e s p e c t r a w e r e measured at least two times, and the results were averaged. The base line was recorded for each spectrum. The circular dichroism values were c a l c u l a t e d f r o m t h e r e l a t i o n s h i p A e = hs/lc, w h e r e h is t h e m a g n i t u d e o f t h e c i r c u l a r d i c h r o i s m b a n d i n m m , s is t h e s e n s i t i v i t y o f t h e i n s t r u m e n t i n A E / m m , I is t h e p a t h l e n g t h o f t h e c u v e t t e i n c m ,

Vorbri~ggen and

i n 1000 m l o f d e i o n i z e d w a t e r , a s r e c o m m e n d e d

0.5-1.5 measured violet absorption

by

the producers, POCh-Gliwice. DMSO-d 6 and TMS were suppiled by Merck, Ultraviolet absorption spectra were obtained with a Cary 118C and a Zeiss-Jena spectrophot o m e t e r . T h e c o n c e n t r a t i o n s o f all s a m p l e s u s e d i n the CD measurements were determined spectro-

at the long-wavelength maxima. Measurements

ultrawere

TABLE I CIRCULAR DICHROISM AND ULTRAVIOLET ABSORPTION PARAMETERS OF 5-METHOXYCARBONYLMETHYL-2THIOURIDINE AND 5-METHOXYCARBONYLMETHYLURIDINE IN VARIOUS SOLVENTS The accuracy of the wavelength position is not larger than :t: 1 nm over the entire spectrum. The uncertainties of the A ~ values given are as follows: ±0.2 M - l . c m -1 in the range 350-250 nm, and ±0.4 M - l . c m -1 in the range 250-200 nm. The uncertainties of the e values given are as follows: ±200 M - l . c m -1 in the range 350-250 nm, and 5=500 M - l - c m -1 in the range 250-200 nm. * Too high absorption of the solvent. Solvent

5-Methoxycarbonylmethyl-2-thiouridine

5-Methoxycarbonylmethyluridine

CD

CD

~. (nm)

H20

0.1NHC!

Methanol

Acetonitdle

Dioxane

Absorption Ae

322 286 270 221

- 7.8 12.2 12.7 -6.5

322 282 271 222

- 7.7 11.9 12.9 -6.1

330 289 269 220

- 8.6 11.6 13.5 -6.7

327 291 271 220

- 9.4 11.5 10.5 - 5.8

327 291 270 220

- 10.6

11.5 10.5 - 5.8

Absorption

A (nm)

e

~, (nm)

Ae

A (nm)

e

280

15200

9500

14300

2.4 - 1.4 -2.1

265

222

273 241 217

207

9100

277

15800

9400

14400

2.4 - 1.4 - 1.8

265

220

272 240 216

207

9000

280

15400

9300

14200

2.6 - 1.6 - 1.6

265

220

272 242 218

208

8900

290 270 220

11800 11600 13800

275 245 220

2.6 - 1.6 -- 1.6

265

9600

209

8900

293 270 220

13100 8900 13500

275 245 221

2.6 - 1.4 - 1.5

265

9600

*

42

and c is the concentration in m o l / l . The circular dichroism, Ae,is expressed in M - 1 . cm-1. The t HN M R spectra were measured with a Tesla BS 487A spectrometer in glass tubes, using TMS as the internal standard.

less than 0.01 of absorbance; M is the number of bands. The oscillatory strength ( f ) , and the dipole strength ( D ) were calculated from the following relationships:

Resolution of the ultraoiolet absorption and CD spectra. With the aid of the method of Meiron [22],

f = 4.32.10

we were able to fit the ultraviolet absorption and C D spectra into the sum of component bands, Gaussian in wavenumber. The absorbance of a spectrum composed of M individual bands can be written as the following sum:

9fe(~)

d~

D = 9.18.10 3fe(x) dlnZ

dimensionless (debye)2, D 2

The rotatory strength ( R ) was calculated using the well-known relationship:

M

R = 0.248fa~(x) d In X

k--I

Debye-Bohr magnetons ( D B M units)

where B(~) is the background value, and A , ( ~ ) is the absorbance value of the k th band at wavenumber ~. Absorption and C D bands were described with the Gaussian function in wavenumber, ~, as:

The anisotropy factor g was calculated from the relationship: p4R I g= T

AG(v) = xl-exp[-(~-

Bohr m a g n e t o n s / D e b y e ( B M / D units)

x2)2/x~]

The parameters x~, x 2 and x 3 are directly related with the spectroscopic parameters of the band: xa is the absorbance at band maximum, x 2 is the position of absorption maximum, and x 3 is the exponential half-bandwidth. The background was described by the exponential fuction: B(~) = a - e x p [ b ( ~ - ~,)]

where ~t is the fixed wavenumber referring to the short wavelength end of the spectrum, a is the background value at ~g, and b is the parameter decribing the curvature of the background. The convergence between the measured and calculated band envelope was achieved by minimizing the error function: N

+ = E (A~bs-Ar'c) 2 i--1

where A °bs and A talc refer to the observed and calculated absorbances, and N is the number of ordinate measurements. Usually ten iterations were enough to reach the standard error:

S= ( ~ / [ N - ( 3 M + 2)]} 1/2

Results and Discussion

5-Methoxycarbonylmethyl-2-thiouridine The CD spectra of mcmSsZU in various solvents are shown in Fig. 1A. The complex CD curves were resolved into four Gaussian bands with considerable ambiguity as to band energies and intensities. Their molecular parameters are listed in Table II. In the near-ultraviolet region of the CD spectra, a negative Cotton effect occurs, the maximum of which varies within the range 322-330 nm when solvent polarity is altered. The observed C D at these wavelength changes from A e----- --7.7 M - 1 . cm -1 in aqueous solution to A e = - - 1 0 . 6 M -1 c m - a in dioxane (see Table I). Although the maximum of this Cotton effect changes within the range 322-330 nm, its rotatory strength remains almost constant (see Table II). This seeming discrepancy between the observed CD and rotatory strength values is due to a considerable overlapping of the bands positioned at about h = 320 nm and ~ = 297 nm (see Fig. 2). It shows that the rotatory strength values are more adequate for the quantitative characterization of the overlapped Cotton effects than their Ae values. The 320-nm

43

A 12

/, \

a 4

22O

,~-4 -a

~,y;,/ x~

0

34o

_IL

Fig. 1. Circular dichroism spectra of 5-methoxycarbonylmethyl-2-thiouridine (A) and 5-methoxycarbonylmethyluridine (B) obtained in: aqueous solution ( ); 0.1 M HC1 (. . . . . -); methanol ( - - - - - - ) ; acetonitrile ( . . . . . ); dioxane (. . . . . ).

CD band has no counterpart in the absorption spectra, which suggests that this transition must be electrically forbidden but magnetically allowed. Thus, the pronounced Cotton effect at X = 320 nm is due to the n ~ ~r * transition, which takes place at the C=S group [11,24]. It was shown that both the intensity and position of this Cotton effect were not affected by the change in pH within the range 8-1. A similar effect has been noted for 5-methyl-2-thiouridine [11]. The Cotton effects below X = 300 nm were found to show different spectral properties. For example, a decrease in solvent polarity involves a considerable enhancement of both Ae and R of the Cotton effect positioned at about X = 298 nm, although its position is only slightly affected. The rotatory strength changes from 0.040 DBM in aqueous solution to 0.254 DBM in dioxane. In contrast, the rotatory strength of the Cotton effect positioned within the range ~ = 269-275 nrn de-

TABLE II MOLECULAR PARAMETERS OF 5-METHOXYCARBONYLMETHYL-2-THIORIDINE IN VARIOUS SOLVENTS The oscillatory strength ( f ) is a dimensionless quantity; the dipole strength ( D ) is expressed in units D 2; the rotatory strength ( R ) is expressed in DBM units; the anisotropy factor ( f ) is expressed in BM/D units. * Due to the low signal to noise ratio below h = 210 nm, it was difficult to estimate reliable values o f f , D and R for the band at about ~ = 220 rim. Solvent

Ultraviolet absorption

Circular dichroism

h(nm)

f

D

h(nm)

R

g

297 277 221

0.028 0.359 0.257

1.79 21.33 12.09

322 296 275 222

- 0.230 0.040 0.506 - 0.108

0.089 0.096 0.036

298 277 221

0.032 0.364 0.293

2.03 19.89 11.43

323 294 271 223

-0.215 0.110 0.412 - 0.117

0.217 0.083 0.041

298 277 221

0.034 0.316 0.230

2.15 18.60 10.32

331 296 271 220

- 0.228 0.141 0.439 - 0.123

0.263 0.094 0.048

299 274

0.116 0.232

7.35 13.48

328 295 269

- 0.241 0.203 0.326

0.110 0.097

297 271

0.134 0.196

8.53 11.53

328 298 269

- 0.235 0.254 0.302

0.119 0.116

H 20

0.1 M HCI

Methanol

Acetonitrile *

Dioxan¢ *

44

A

.'-"- '

. -';

300 \ 260 nm 280 -" " "".\.,,

320

3,10

~51)

260

320

~0

L 360

4

-8

-12

16

12 'Q 6

/

'\'~

4 2 200

220

240

2BO

300

~.,nm

Fig. 2. Resolution of circular dichroism data (A) of mcmSs2U into component Gaussian functions which simultaneously fit the absorption data (B); both the CD and absorption spectra were measured in aqueous solution.

creases simultaneously with the diminution in solvent polarity. It is worth to note that during the change in solvent polarity, the sum of the rotatory strengths of these two C o t t o n effects remains almost constant, R - - 0 . 5 4 5 + 0.025 DBM. Such dipole strengths and rotatory strengths as for these two b a n d s characterize the electronic transitions, which are simultaneously electric dipole-allowed and magnetic dipole-allowed. The b a n d at about X = 275 n m arises from the lowest energy ~r --~ ~r * transition of the pyrimidine framework [25-27], and has B2u s y m m e t r y [28]. The b a n d at about X = 298 n m appears to be due to the intramolecular charge transfer n --* ~r * transition that is usually found in the near-ultraviolet absorption and C D spectra of thiolated nucleosides and enones [23,24]. Such a transition is strongly dependent on solvent polarity, and usually exhibits considerable dipole and rotatory strength. The anisotropy fac-

tor for this b a n d is comparable with that of a thiolated enone [23], for which it was calculated to be g = 0.128, c o m p a r e d to g = 0.110 for mcmSs2U (both measurements were done in acetonitrile). It could be concluded, therefore, that both the rotatory strength and dipole strength of the n --* ~r * intramolecular charge-transfer b a n d increase with the decrease in solvent polarity, mostly due to the weakening interaction between the n o n b o n d e d electrons at the sulfur atom and the solvent. All these effects indicate a considerable influence of the solvent on the n and ~r electrons of the pyrimidine framework. The rotatory strength and position of the Cotton effect at X = 222 nm remain almost constant when solvent polarity is varied. The dipole strength, and rotatory strength, were shown to be relatively large, and it may thus be presumed that the next ~r ~ 7r * transition takes place in this spectral region. The chemical shifts of the protons of mcmSs2U dissolved in D M S O - d 6 are listed in Table III. The position and coupling constant for the H - I ' indicate that this ribonucleoside occurs in an anti c o n f o r m a t i o n [29]. A more thorough study of the high-resolution I H - N M R spectra of mcmSs2U has proved that, due to the steric repulsion between the bulk 2-thiocarbonyl group and the substituents on the C-2', the 3E-gg-anti c o n f o r m a t i o n is preferred [30]. A similar conclusion was derived by Hillen et al. [31], who established that mcmSsZU in the crystalline state indeed occurs in the 3E-gg-anti conformation.

5-Methoxycarbonylmethyluridine The C D spectra of mcm~U are shown in Fig. lB. There is a small change in the C D spectra of mcmSU when going from aqueous solution to organic solvents. Therefore, for clarity in the presentation of the figure, only the C D curves measured in aqueous and dioxane solutions are shown. F o r the same reason, the molecular parameters are also constant, and the averaged values of f , D, and R are given. In the C D spectra of mcmSU, three distinct C o t t o n effects occur, the spectral parameters of which are given in Table I. A decrease in solvent polarity involves a slight red shift of the C o t t o n effect positioned at X - - 2 7 3 nm, whereas its C D

45 TABLE III CHEMICAL SHIFTS ppm OF 5-METHOXYCARBONYLMETHYLURIDINE THIOURIDINE

AND 5-METHOXYCARBONYLMETHYL-2-

½co 3

The PMR spectra were recorded in DMSO-d6; s, singlet; d, doublet; b, broad. The protons f, g, and h were grouped as a multiplet within the range 4.65-4.40 ppm. Compound mcmSU mcmSs2U

Protons (see Scheme) a

b

c

d

e

3.55 s 3.66 s

3.30 s 3.35 s

7.75 s 7.80 s

11.35 b 11.35 b

5.17 d J = 5 Hz 6.46 d J = 2 Hz

value r e m a i n s a l m o s t constant. This long-wavelength C o t t o n effect is d u e to the lowest energy ~r---, 7r * transition. F o l l o w i n g the t e r m i n o l o g y of C l a r k a n d T i n o c o [28], this transition should fall into B2u s y m m e t r y . The ultraviolet a b s o r p t i o n b a n d exhibits the relatively large d i p o l e strength D = 11.50 D 2 ( f = 0.200), which is a characteristic f e a t u r e for all electric d i p o l e - a l l o w e d electronic excitations whereas the r o t a t o r y strength of this transition is characteristic for a w e a k l y m a g n e t i c d i p o l e - a l l o w e d electronic excitation. The C D b a n d was, therefore, relatively weak, a n d its average r o t a t o r y strength was e s t i m a t e d to be R = 0.055 D B M for all solvents used. T h e 240-nm C D b a n d c o r r e s p o n d s to a d e e p m i n i m u m in the ultraviolet a b s o r p t i o n s p e c t r a (see Fig. 3). T h e averaged r o t a tory strength o f this C o t t o n effect was e s t i m a t e d to b e R = - 0.025 ± 0.05 D B M , d e p e n d e n t on solvent p o l a r i t y . It m a y b e a s s u m e d that this C o t t o n effect is d u e to an n---, ~r * t r a n s t i o n [32]. T h e C o t t o n effect p o s i t i o n e d at ) , - - 2 1 7 n m ( R = - 0 . 0 5 0 D B M ) c o r r e s p o n d s to a weak a b s o r p t i o n b a n d at ~, -- 215 n m ( D = 0.87 D 2, f = 0.20), whereas at ~ 204 n m a p r o n o u n c e d a b s o r p t i o n b a n d occurs ( D = 13.50 D 2, f = 0.310); the d a t a were calcul a t e d from the a b s o r p t i o n a n d C D s p e c t r a of aqueous solution o f mcmSU (see also Fig. 3). These

d a t a indicate that at least two different electronic transitions take p l a c e in this spectral region, n a m e l y the ~r ~ ~r * transition ( E 1 s y m m e t r y ) at a b o u t X = 205 nm, a n d the n ---, ~r * transition posit i o n e d at a b o u t X = 217 nm. T h e C D spectra of m c m 5U a n d u r i d i n e are very alike [25-27,33]. This m a y suggest that the m e t h o x y c a r b o n y l m e t h y l g r o u p b o u n d to the C-5 a t o m does not p e r t u r b significantly the electronic levels of the p y r i m i d i n e ring, since the m e t h y l e n e g r o u p restricts the m e s o m e r i c effect. However, d u e to the bulkiness of this group, the syn-anti equil i b r i u m m a y be affected. F o r example, a n u m b e r o f m o l e c u l a r interactions can t a k e p l a c e b e t w e e n the m e t h o x y c a r b o n y l m e t h y l g r o u p a n d the O H - 5 ' a n d H - 3 ' in the 3E ribose ring puckering, a n d the O H - 5 ' a n d H - 2 ' in the 2E puckering. The effect of this steric h i n d r a n c e is explicitly seen in the C D spectra, since the change in solvent p o l a r i t y does n o t p r o d u c e a n y a l t e r a t i o n in Ae a n d R, while in the case of uridine, significant a l t e r a t i o n s of these values have been o b s e r v e d [33]. Thus, the syn-anti e q u i l i b r i u m in mcmSU is m o r e restricted in c o m p a r i s o n to that in uridine. T h e 1 H - N M R p e a k o f the H-1 a p p e a r e d at a b o u t 8---6.20 p p m as a d o u b l e t of which the c o u p l i n g c o n s t a n t was e s t i m a t e d to b e 4 Hz. T h e

46

A

~ ( t

I

l

t

-2

J

It

.';/"

I

~\

V

8 6

B

\ ~\ ~t

X ~O 2

200

~\\\\

220

240

260

280

300

320

~nm Fig. 3. Resolution of circular dichroism data (A) of mcmSU

into component Gaussian functions which simultaneously fit the absorption data (B); both the CD and absorption spectra were measured in aqueous solution.

Due to this steric hindrance, the syn-anti equilibrium is highly restricted. As has been shown in our previous paper [11], the change in solvent polarity apparently does not influence the syn-anti equilibrium. Therefore, one may assume that any alteration of the Cotton effects positioned above X = 300 nm, caused by solvents or salts added to aqueous solution of tRNA, will be due to the change in the conformation of the segment in which 2-thiopyrimidine is located. Hence, it is obvious that the change in R, or Ae, of the Cotton effects positioned above X = 300 nm will be primarily due to alterations of the stacking between the thiobase and its neighbouring nucleotides, since the syn-anti equilibrium in thionucleosides was shown not to be changed by solvents. The changes in the C D spectra of a t R N A containing 2thiophyrimidine should be easily recognized, since the characteristic Cotton effects of thionucleosides are not masked by the Cotton effects of the four normal nucleosides. 5-substituted pyrimidine nucleosides without sulfur at the C-2 are also more rigid than uridine or cytidine. This is due to the repulsion between the bulk substituent at the C-5 and the substituents in the ribose ring. For example, the methyl group at the C-5 in 5-methyluridine was shown not to disturb the syn-anti equilibrium [11 ], whereas the methoxycarbonylmethyl groups is sufficiently large to freeze this equilibrium in mcm 5U. References

~H-NMR signals of the protons H-2', H-3' H-4' were grouped as a multiplet at about 8 = p p m (see Table III). Such results indicate fl-configuration and anti conformation of ribonucleoside [29].

and 4.62 the the

Conclusions

A number of analyses of the C D and high-resolution 1 H - N M R spectra of 5-substituted 2thiopyrimidine nucleosides have revealed that, due to the repulsion between the bulk thiocarbonyl group (of which the C=S bond is relatively long (1.66 /k) and which has a large° Van der Waals radius of the sulfur atom (1.85 A)) and the substituents at the C2', these nucleosides occur exclusively in the 3E-gg-anti conformation [11,30,34].

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