Hairpin structures in synthetic oligodeoxynucleotides: sequence effects on the duplex-to-hairpin transition

Biochimie 71 (1989) 7 9 3 - 803 (~) S o c i r t 6 de C h i m i e b i o l o g i q u e / E l s e v i e r , Paris

793

Hairpin structures in synthetic oligodeoxynucleotidessequence effects on the duplex-to-hairpin transition Luigi Emilio X O D O 1., Giorgio M A N Z I N I l, Franco Q U A D R I F O G L I O 2, Gijs van der M A R E L 3 and J.H. van B O O M 3

1Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, 1-34127 Trieste, Italy; 2Institute of Biology, Faculty of Medicine, University of Udine, 1-33100 Udine, Italy; and 3Gorlaeus Laboratories, State University, P.O. Box 9502, 2300 RA Leiden, The Netherlands (Received 19-1-1989, accepted after revision 28-3-1989)

Summary-- We have synthesized and examined a number of fully and partly self-complementary palindromic oligodeoxynucleotides for their ability to assume in solution a unimolecular hairpin structure. The main results obtained by a combined optical and electrophoresis investigation show that: (i) D N A folding needs not be driven by mismatched base pairings over the dyad; fully self-complementary palindromic duplexes, comprising regular ( C G ) n D N A fragments, possess a considerable intrinsic propensity to make intramolecular base pairings; (ii) The duplex-hairpin interconversion is, in general, a slow process independent of the length and base composition of the palindrome; (iii) The palindromic sequences energetically least favored to form hairpin structures consist of C : G base pairs around the dyad axis and of T:A blocks in the arms of the inverted repeat; (/V) The base composition of the stem strongly influences the hairpin thermal stability. For instance, the substitution of one C:G with one A:T base pair in the stem helix of d(CG)7 diminishes the stability of the hairpin by 9oC. It is found that the stability of the stem helix, in hairpins of defined sequence and with the same loop length, decrt:ases in the order alt~rn~tina_("("~ h. . ..... .. .. . . - P. C .. . -.. .A~ t ~~ _J m ~l 1 f~- a-~t t.~. l.H. .a.u l l:__ ~, ~ - t ~ A~,l , i.e. ~,~ ill p o ly nucleotides. The thermo dy namic parameters for the hairpin-coil transition are reported. duplex-hairpin interconversion / oligodeoxynucleotides / electrophoresis / melting curves / thermodynamics

Introduction It is now well established that D N A is characterized by a high degree of polymorphism. Its double-stranded helix is no longer regarded as a static rodlike molecule, but as a chain with considerable conformational flexibility which may result in aberrant conformations (Z-DNA) [1, 2] as well as imperfect helices containing hairpins, bulges, loops, and so on. Since the control regions of the genome frequently contain inverted repeats, i.e. sequences of dyad symmetry (palindromes), it is likely that some biological processes require structural changes in the DNA. Extrusion of palindromic sequences in *Author to whom correspondence should be addressed.

cruciforms have been found near replication origins in prokaryotes [3] and in eukaryotic viruses [4-6]. Furthermore, cruciform structures are believed to be implied in transcription termination [7]. Recently it has been reported that proteins from human extracts [8] and from rat liver nuclei [9] bind specifically to artificial 4-way junctions and not to linear D N A of the same base composition. Since a cruciform is made up of 2 opposite hairpins, studies on the ability of oligodeoxynucleotid"s to form hairpin structures are important. Over the past few years a number of solution studies have been reported on hairpin formation from oligodeoxynucleotides [10-25]. Most of these studies have been

704

L.E. Xodo et al.

conducted on partly palindromic sequences which can m a k e hairpin structures with an alternating CG stem (see Table III). The most significant results so far obtained can be summarized as follows: (i) The presence of the loop does not seem significantly to reduce the conformational dynamics of the double helix stem. In fact, when the stem is Z-helicogenic, it assumes the left-handed conformation in the presence of a d d e d salt [16, 18, 22]; (ii) The overall hairpin stability is a function of loop size. Hairpins that can m a k e a two-residue loop are found to be particularly stable. This suggests that, contrary to the notion that highest stability is achieved when the loop comprises 4 or 5 residues, the occurrence of 2-residue miniloops is favored in B - D N A : a structural feature corroborated by N M R [12] and t h e r m o d y n a m i c [ 17] analyses; (iii) W h e n the stem is in the left-handed Z conformation it does not support a 2-residue loop [ 18]; (iv) The base composition of the loop region can also influence the overall hairpin stability, but to a minor extent [24]. In addition to this reseach line, the effects of topological stress on palindromic sequences have been investigated [26-29]. W h e n a palindrome is inserted in a negatively supercoiled plasmid it extrudes in a cruciform structure, with the process being accompanied by a reduction in superhelical density. In the light of these results the hairpin loops can be regarded as important components of D N A . As part of a program on hairpin loop structures in D N A , we present in this contribution some optical and electrophoretic results relative to the base sequence effects on the ability of a variety of palindromic or quasi-palindromic sequences to assume a unimolecular hairpin structure. The results reported here concern palindromic sequences with an increasing n u m b e r of A : T base pairs, located in the arms of the inverted repeat and with a central CG core.

Materials and methods

Oligodeoxynucleotides The oligomers used for this study, reported in Table I and numbered from I to XIII, were synthesized according to a modified phosphotriester method as previously reported [30, 31]. Purification was performed by standard gel permeation chromatography using a G-50 Scphadex resin and eluting with a solu-

tion of 0.005 M tetraethylammonium bicarbonate. Sample purity was confirmed by reverse phase highperformance liquid chromatography (HPLC) and 20% polyacrylamide gel electrophoresis.

Buffers

Due to the very high thermal stability exhibited by CG-rich oligomers in the hairpin structures, most of the UV measurements were made in 0.5 mM NaCI, 0.5 mM Tris.HCl, 0.1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.4 (buffer A). In such low ionic strength buffer all the biphasic transitions were well resolved so that it was possible to perform a reliable thermodynamic analysis. The buffer ability of such a low concentration solution has been proved to be sufficient to avoid significant variations of the pH with increases in temperature (pH 7.4 at 25oC, pH 6.6 at 80oC). The electrophoretic experiments were performed in 1.0 mM NaCI, 0.5 mM EDTA, 50 mM EDTA, 50 mM Tris-HCl, pH 7.4 (buffer B).

Melting experiments The denaturation curves for oligomers I - X I I I were obtained with a Cary 219 or a DMS 80 (Varian) spectrophotometer interfaced with a PC system. The temperature was increased at a rate of 0. l o C / m i n through a Haake PG 20 temperature programmer, connected with a Haake water circulating bath equipped with a refrigerator. The melting curves were monitored at 270 nm and the oligomer concentration was determined by UV absorption at 260 nm in denaturing conditions (T>90oC), assuming as extinction coefficients 7000 M -1 cm -l for pyrimidines and 14 000 M -~ em -1 for purines. The strand concentration used for the melting experiments was in the order of 10-80/~M.

Electrophoresis PAGE experiments were carried out on gels (10 or 20 × 15 × 0.15 cm) obtained by polymerizing a solution containing 20% acrylamide, 3.3% bisacrylamide, 0.07% ammonium persulfate in buffer B. The stacking gels contained only 5% acrylamide. The gels were run at controlled temperature by means of a thermostated apparatus connected with a water circulating cryostat. Samples were prepared, before loading, in solutions containing 20% sucrose. In general electrophoresis was carried out at 30-50 mA for about 6 - 1 2 h. The applied voltage was in the order of 10-15 V / c m . Bromphenol blue was used as a marker and after the electrophoresis was stopped the bands were stained with a solution of "stains all" dye dissolved in 1:1 water-formamide. Electrophoretic bands appeared within 15 min and were immediately photographed.

Circular dichroism C D spectra were obtained with a Jasco 500 A spectropolarimeter equipped with a thermostatable cuvette holder which allows m e a s u r e m e n t s

Duplex-to-hairpin transition in oligodeoxynucleotides at controlled temperature. The dichrograph was connected to a Jasco DP 500 N data processor. Spectra are presented as Ae = (~L-ER) in units of M-~ cm -1. The spectra were obtained from sample solutions at a strand concentration of about 10/xM.

Table !. Palindromic and quasi-palindromic sequences studied for their ability to form a hairpin structure. d(TATATACCCCCTATATA) d(CCCCCC'IqqTFGGGGGG) d(CGCGCGqqTITCGCGCG) d(CGCGCGAAAAACGCGCG)

l,

1I. III. IV.

Results VI. VII. VIII. IX. X. XI. XII.

d(CGCGCGTACGCGCG) d(CGCGCGATCGCGCG) d(CGCGCGCGCGCGCG) d(CGCACGCGCGTGCG) d(CATACGCGCGTATG) d(TATATACGTATATA) d(ACACACGCGTGTGT) d(ACACACATGTGTGT)

XIII.

d(ATACGCGCGTAT)

V°

Thermal denaturation The denaturation behavior of oligomers I I I - V I was previously studied and reported elsewhere [17]. We briefly recall here that all 4 D N A fragments exhibit a two-step melting profile, under a rather wide range of nucleotide concentrations and ionic strengths (comprising buffer A). On the basis of spectroscopic and electrophoretic measurements we characterized the low-temperature (transition 1) and the hightemperature (transition 2) transformations as a duplex-hairpin and hairpin-coil, respectively (Fig. 1). This conformational picture should be kept in mind in interpreting the thermal behavmr of the other samples of Table I.

3'

795

moved down. In the case of X, where the presence of C:G is limited to the central dinucleotide, a broad transition, which probably overlaps duplex-hairpin and hairpin-coil transformations, is observed. In 0.1 M NaCI V, VI and VII maintain a two-step melting profile; by contrast both IX and X in this medium exhibit monophasic and more cooperative melting profiles with midpoint temperatures dependent on the nucleotide concentration (Fig. 2b). " ......

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Fig. 1. Conformations accessible to the DNA inverted repeat sequences of Table 1.

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Figure 2a shows the experimental absorbance versus temperature profiles in buffer A for V I I - X . Although VII can make a stable duplex, comprising 14 Watson-Crick C:G base pairs, it does show a two-step melting profile. If, in the two arms of this inverted repeat DNA fragment, one C and one G are substituted with one A and one T, respectively, as in VIII, the midpoint of both transitions 1 and 2 is reduced considerably (Table II). When more C:G base pairs are substituted, as in IX and X, the TMS are further

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Fig. 2. Panel A, Normalized absorbance (270 nm) versus temperature profiles for d(CGCGCGCGCGCGCG)(VII), d(CGCACGCGCGTGCG) (VIII), d(CATACGCGCGTATG) (IX), and d(TATATACGTA'I.ATA)(X) in bufferA. Panel B, Normalized absorbance (270 nm) for d(TATATACGTATATA) and d(CATACGCGCGTATG)in buffer A with the addition of salt (0.1 M NaCI).

796

L . E . X o d o et al.

Table !1. Thermodynamic parameters for the hairpin-coil conformational transitions in oligodeoxynucleotides.

Sample

Duplex-hairpin Conc. /.tM/strand

Hairpin-coil

TI/2 (oC)

T1/2 (oC)

AH (kcal/mol)

AS (e.u.)

35 57 57 55

116 172 167 161

d(TATATACCCCCTATATA)( a~ d(CCCCCC IJTI" IGGGGGG) d(CGCGCGrI-ITfCGCGCG) d(CGCGCGAAAAACGCGCG)

77 19 15 17

32 32

28 59 69 68

d(CGCGCGTACGCGCG)(b) d(CGCGCGATCGCGCG)(b) d(CGCGCGCGCGCGCG)(b) d(CGCACGCGCGTGCG)(b) d(CATACGCGCGTATG) d(TATATACGTATATA) d(ACACACGCGTGTGT) d(ACACACATGTGTGT)

15 20 24 21 28 10 30 19

38 38 49 42 37 notobserved 31 29

81 81 78 68 45 24 56 57

54 54 48 44 32 48 49

152 152 137 129 101 146 148

d(ATACGCGCGTAT)

10

30

39

32

102

(a)Data obtained in the presence of 0.1 M NaCI. (b~T~/2for hairpin-coil transition if, the average of T~,2 obtained from several individual melting curves, at different nucleotide concentrations. Data obtained in 0.5 mM Tris-HCl, 0.5 mM NaCI, 0.1 mM EDTA, pH = 7.4.

The 12-mer XIII behaves similarly to the 14rner IX, exhibiting biphasic profiles in buffer A, ,,uh~r~ac in fl 1 ~.I.VJL KIoF'! ;I,-,k . . . . . . U l l l y I . . ,'1 l l l U l l U .... vAvu~ z.tz v.l ..L~IKI.~.....,I Ilk ,..~IIU'I#'V phasic and more cooperative profile (not si,,own). Sequences XI and XII, based on the A C / G T repeating unit, are perfect palindromes, thus potentially capable of forming a monomeric hairpin structure. Their thermal profiles shown in Figure 3a suggest, indeed, that 2 conformational transitions occur in these D N A fragments and that transition 2 (hairpin-coil) is not significantly affected by the central loop generating sequence - A C G C G T - against - A C A T G T - . We observed that the fully palindromic sequences so far investigated, which exhibit a biphasic melting profile, share a common feature, i.e. upon cooling, transition 2 (hairpin-coil) is fully reversible, whereas transition 1 (duplexhairpin) shows a pronounced hysteresis. The initial absorbance value is restored only after many hours. Consequently, the 7"I/2 for the duplexhairpin transition of Table II are obtained in conditions of non-equilibrium and may convey some kinetic (but not thermodynamic) information. Figure 3b shows the melting profiles for I

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Fig. 3. Panel A, Normalized absorbance (270 nm) versus temperature profiles for d(ACACACGCGTGTGT) (XI) and d(ACACACATGTGTGT) (XII) in buffer A. Panel B, melting curves (270 nm) in buffer A for d(TATATACCCCCTATATA) (I) (with added salt, 0.1 M NaCi) and for d(CCCCCCIqTIq'GGGGGG) (II).

797

Duplex-to-hairpin transition in oligodeoxynucleotides

and II in buffer A (the low thermal stability of I imposed the addition of salt, 0.1 M NaC1). Both sequences exhibit a one-step transition whose ?'1/2 are constant with the nucleotide concentration, suggesting that the conformation adopted in solution by I and II is a monomeric looped structure. Table II collects all the data relative to the transitions.

abcdefghi

Electrophoresis

Polyacrylamide gel electrophoresis is a suitable technique to distinguish between DNA conformations differing in size. Since the duplex-tohairpin interconversion in short palindromic sequences is, in general, a slow rate process, it is possible to detect the secondary structure adopted in solution by sequences I - X I I I through gel electrophoresis. In a previous paper we resolved by 20% polyacrylamide gel electrophoresis (PAGE) the duplex and hairpin forms of I I I - V I [17]. Following the same procedure described in [17] we analyzed by electrophoresis all the samples of Table I and observed that already in buffer B, the hairpin form is detectable in most sequences I - X I I I , except for X, IX, and XIII. Some gels are herewith described. Figure 4 shows the mobilities of X, XI, XII, and XIII, at 15oC, which are compared to those of a 7-mer duplex (lane i) and a 14-mer duplex " . . . . g)." .i. nc . . . . 1. .,+-mer . . . . . . A m~grates with a sin gl e Unuc band corresponding to a duplex (lane e). When the sample is heated at 90oC before loading, to overcome the kinetic barrier to hairpin formation, the same electrophoretic profile is obtained (lane J0. This suggests that the duplex-hairpin interconversion does not occur in X, with the duplex reconstitution being a slow process with respect to the electrophoretic time scale. This is corroborated by the fact that by running the gel at 35°C, i.e. at a temperature where the expected hairpin form should be strongly favored, a unique band corresponding to the duplex was observed. Oligomers XI and XII run in lanes c and a. Two bands are present for each sample: the slow moving band corresponds to a duplex; the fast moving and much less intense band to a hairpin. By heating the sample before loading more duplex is converted into hairpin, and this is observed in lanes d and b, where the bands corresponding to the hairpins are now much more intense. These experiments conclusively establish the presence of a duplex-to-hairpin

•

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i

Fig. 4. 20% polyacrylamide gel electrophoresis in buffer B of d(ACACACGCGTGTGT) (XI) (lane a), d(ACACACATGTGTGT) (XII) (lane e), d(TATATACGTATATA) (X) (lane e), d(ATACGCGCGTAT) (XIII) (lane h), and d(CGCGCGATCGCGCG) (VI) lane g). Samples XI, XII, and X heated to 100°C just before loading run in lanes b, d, g.til~.i i 9 l V O l . J ~ l . , t l V ~ . , l ~ .

ILF

g3tligJ

15oC.

interconversion in XI and XII, as was hinted by their melting behavior. Lane h contains the 12mer XIII which migrates as a linear duplex, as expected by its melting behavior at this ionic strength. Figure 5 shows the electrophoretic results for VII and VIII. The mobilities of the reference sequences, 8-mer and 10-mer duplexes, are shown in lanes a and b. The 14-mers VII (lane d) and VIII (lane 3") migrate as linear duplexes, under the gel conditions. However, when these DNA fragments are heated prior to loading, the hairpin structures of VII and VIII appear in the gel (lanes c and e). This is consistent with the biphasic denaturation profiles observed in buffer A for these DNA fragments. As for the 17-mers I and II, analogously to III and IV [17], they migrate essentially as monomolecular hairpin structures, under a wide range of nucleotide concentrations, ionic strengths, and temperatures (not shown).

L.E. Xodo et ai.

798

a

b

c

d

e

where Eh and Ec are the extinction coefficients of the hairpin and coil forms, respectively, Cr is the total strand concentration in solution. Introducing in Eq. 2 the expression of f obtained by Eq. 1, the temperature dependence of the absorption becomes: A(T) = E~ CT/(I+Kh) + Eh CT/(I+Kh-') (3)

f

Eq. 3 was used to fit [33] the experimental melting curves by means of a nonlinear least-square program (algorithm of Marquardt, [34]), leaving as adjustable parameters eh, e~, AH, and AS. However, since the pre- and post-transition regions showed, in general, a temperature dependence, the extinction coefficients of the hairpin and the coil were assumed to vary linearly as Eh = mh(T-T1) + ~1 and E~ = m~(TT2) E2.

W Fig, 5. 20% polyacrylamide gel electrophoresis in buffer B of d(CCTATAGG) (lane a), d(CCq ATATAGG) (lane b), d(CGCGCGCGCGCGCG) (VII) (lane d), and d(CGCACGCGCGTCG) (VIII) (lane f). Samples VII and VIII heated to 100°C just before loading run in lanes c and e. respectively. PAGE was carried out at l0 V / cm and 5°C.

Thermodynamic analysis

Duplex to coil

Single strand-to-hairpin loop We used the melting profiles to study the the~nodynamics of intramolecular hairpin loop formation from the coil. On the basis of a twostate model, which is valid for short oligodeoxynucleotides [32], the reaction can be written as: single stranded coil ~ hairpin loop, with equilibrium constant, Kh, equal t o l l (l--f), wherefis the hairpin fraction at a given temperature. Kh is a function of the thermodynamic parameters according to: Kh = f / 0 - - f ) = e x p ( - A n / R T + A S / R ) , (1) where AH and AS are the enthalpy and entropy changes of the reaction. The absorbance of the sample solution as a function of temperature is given by: A(T) = Ehf CT + Ec (I--f) CT

This method, based on the lower and upper baselines subtraction, accurately reproduces the melting curves (Figure 6). The relative error, defined as the absolute value of (Ac - Ae) / A e , where Ac and Ae are the computed and experimental absorbance values at a given temperature, is around 0.5%. The uncertainties on the AH and AS parameters were assessed to be + 3 - 5 % , i.e. of the same magnitude as found by other authors for similar van t'Hoff analyses [35]. At the semitransition point, f=0.5, the free energy is AG=0, thus the standard entropy is given by AS = AH/T1/2. Values for AH and AS are reported in Table II.

(2)

The melting temperatures for X, IX, and XIII in 0.1 M NaCl are concentration dependent; thus they have been analyzed according to the bimolecular reaction: 2 single-stranded coils "=" duplex. According to the procedure described above the temperature dependence of the absorbanee becomes now:

A(T)=edCr/2 + (ec--ed/2) (Kd/4) [(l+8Cr/Kd)l/2-1]

(4)

where ed and ec are the extinction coefficients of duplex and coil; Cr the total strand concentration; and Kd the equilibrium constant for the process. Treating Ed and ec as linear functions of temperature, Eq. 4 was fitted to the experimental melting curves by using the Marquard algorithm with 4 adjustable parameters (AH, AS, md, and me). The values found are:

Duplex-to-hairpin transition in oligodeoxynucleotides AH exp (kcal / mol) d ( T A T A T A C G T A T A T A ) 10"7(+_ 3) d ( C A T A C G C G C G T A T G ) 10U (_+ 5) d(ATACGCGCGTAT) 82 (± 3)

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1

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AH cal [36] (kcal / moi) 95 112 !00

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~ H cal [37] (kcal / mol) 104 111 95

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799

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T(°O )

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Fig. 6. Best ~ tsolid fine) for the hairpin-to-coil trar~ition of d ( A C A C A C A T G T G T G T ) in buffer A. Eq. 3 (see Thermodynamic analysis) was used for tl~e fitting. Best fits are: AH = 49.4 (-+ 4), AS = 149 (+- 13), T112 = 57.

-2 -4 -6 -8 -10 220

The AH values are consistent with a duplexcoil transition. The experimental AH values have been compared with calculated AH, obtained according to available empirical stacking energies [36, 37]. The discrepancies between experimental and calculated values are in the order of 10% for IX and X, whereas for XIII the discrepancies are slightly higher. This can be attributed to inherent limits of an all-or-more model, and / or the presence of a small quantity of hairpin.

Circular dichroism All the D N A fragments of Table I, except II, exhibit, at low ionic strength, a CD spectrum consistent with B-form DNA. Sequence II adopts in solution a monomeric hairpin structure with a d ( C C C C C C ) : d ( G G G G G G ) stem. Fig. 7 shows tl:~e CD spectra of II and III at 25°C and of II at 90°C. Sequence III forms a hairpin of the same size and base composition as II does, but with an alternating-CG stem. The spectra of the two oligomers are quite different: whereas that of III is consistent with B-form DNA, that of II deviates significantly from it. A close inspection reveals: (i) A reduced negative band for II. The positive ellipticity (Ae=4.7 M -1 cm -l) of II is

I

I

I

l

260 300 ~,(nm)

Fig. 7. Circular dichroism spectra for d(CCCCCCqTFITGGGGG) (II) ( . . . . . ) and d(CGCGCGT1TITCGCGCG) (II!) ( - - ) in 0.1 M NaCl The dotted line is the CD spectrum of II at 95oC. The inset shows a plot of the ellipticity values at 265 nm for II as a function of temperature• about one and a half times more intense than the negative one (Zle= - 3 . 2 M-~ cm-l), whereas the contrary holds for III, i.e. the positive band (AE=3.7 M -1 cm -l) is half the negative ( A e = - 7 . 6 M -1 cm-l); (ii) A blue shift of the positive ellipticity of II, which is centered at 263 nm. In III as well as other samples of Table I, the positive band occurs around 280 rim. The above CD spectra do not reflect only one pure conformation since contributions from both the stem helix and the single-stranded loop are present. This makes the overall spectra rather complex and not clearly partaking of one of the canonical conformations. The CD spectral features of II resemble those of polydG:polydC and may hint at a stem helix in the A-form DNA. However, we are cautious on this point since Raman spectra on polydG:polyC in solution are rather intriguing due to the presence of diagnostic frequences of both A- and B-conformations

SO0

L.E. Xodo et al.

in different proportions depending on the sample concentration [38]. In addition, it is known that the presence of a G cluster in oligomers results in anomalous CD spectra [39]. On raising the temperature the CD spectrum of II changes, eventually assuming the form of the coil conformation at'90°C. When the change in ellipticity at 267 nm is plotted as a function of temperature, a denaturation curve for II is obtained (see inset), whose T1/2 is found to be 59oC, in accord with UV melting experiments. Note that the melting temperature of III is 69oC, indicating that an alternating-CG stem confers more stability on the hairpin structure than a homo-CG stern does, in line with the relative thermal stabilities of poly(dGdC):poly(dGdC) and poly(dG):poly(dC) [40]. Discussion

Linear duplexes and hairpin loops The duplex-to-hairpin conversion in synthetic oligodeoxynucleotides has been previously reported. Table III shows some sequences studied over the past few years. Apart from the homologous series of Haasnoot et al. [10] and the recent hairpin system of Senior et al. [24], all the sequences so far investigated form hairpin structures with a CG alternating stem, supporting a variety of loops. Consequently, the question of whether short inverted repeats of any sequence can form hairpin structures has not yet met a complete experimental answer. The most unambiguous experimental evidence for the coexistence in solution of two ordered structures

for a palindromic DNA fragment, namely the duplex and the hairpin, is obtained by gel electrophoresis. This is possible since the duplexhairpin interconversion is, in general, a very slow process, also with respect to the electrophoretic time scale. We found that the duplexhairpin interconversion is slow in all the fully palindromic sequences of Table II and even in very short palindromes: for instance, the mismatched d(meCGmeCGTGmeCG) octamer studied by Orbons et al. (Table III) migrates in a 20% P A G E and 0.1 M NaCI with two bands corresponding to the duplex and hairpin forms (not shown). The parameter that most affects the duplex-hairpin interconversion is temperature, although we found, as already noted [11], that the oligomer concentration and, in particular, the ionic strength also significantly influence this transition. Fragments I - I V are quasi-palindromic sequences since the two copies of the corresponding inverted repeat stretch are not contiguous. Such sequences are found to strongly favor the intramolecular base pairing, resulting in the formation of hairpin looped structures. In fact the alternative conformation would be the dimeric duplex with 5 mismatches or a bulge at the center, a rather unstable structure which is observed only at low temperature and high nucleotidc concentration. Electrophoretic experiments on I - I V reveal that at 5oC, these samples migrate mainly as monomeric hairpin structures (the duplex is present in traces). This feature holds for palindromes forming hairpins with a stem of different base composition: 100% AT-alternating in I, 100% CG-alternating in III and IV, CG-homo in

Table !!i. DNA fragments of dyad symmetry studied in solution and reported in the literature.

Sequence

Comment

Reference

ATCCTAT,TAGGAT

duplex-hairpin n = 0 - 3 most hairpin n>3 Z-hairpin PAGE, thermodynamics duplex / hairpin kinet. NMR study NMR study thermodynamics duplex-hairpin 3-residue loop duplex-hairpin kinet. two-residue loop hairpin X=C, G, A, T

10

CGCGCGTsCGCGCG CGCGCGTA(ATICGCGCG CGCGCG ~ C G C G C G CGCGTIS~CGCG CGCGAATI'CGCG CGCGTATACGCG CGCGATTCGCG CGCGCGAATI'ACGCGCG m5CGm5CGTGm5CG CGAACGX4CGTTCG

16 17.18 19 14 15 11 21 20 13 12 24

Duplex-to-hairpin transition in oligodeoxynucleotide~ II. These types of sequences are therefore suitable systems for studying looped structures in DNA by means of techniques requiring high nucleotide concentrations. With reference to the fully self-complementary and palindromic DNA fragments of Table I, they cover a broad range of sequence patterns. At very low ionic strength (buffer A) the duplexto-hairpin interconversion is experimentally observed in all these DNA sequences. On the contrary, in 0.1 M NaCI the melting curves indicate that the folding is precluded to IX, X, and XIII, as found by electrophoresis in buffer B. Hence, the propensity to form unimolecular hairpin structures in short self-complementary palindromes is dominated by sequence and ba~:e composition effects. It appears that sequences containing a central CG tract around the axis and some amounts of AT in the arms of the inverted repeat, like IX, X, and XIII, are those less favored for forming hairpin structures. We interpret this behavior by observing that the duplex and hairpin conformations in these types of sequences show different amounts of A:T base pairs in the corresponding helices (see Table IV). It follows that as the AT content increases in the stem region, the hairpin structure becomes less stable with respect to the competing duplex. This effect becomes more significant as ionic strength increases since it favors the duplex over the hairpin [11]. Table IV. Percentage of T:A base pairs in the dou-

ble helix of the duplex and hairpin forms relative to oligomers VII, VIII, IX, and X. Sequence

CGCGCGCGCGCGCG CGCACGCGCGTGCG CATACGCGCGTATG TATATACGTATATA

PercentageofTA Duplex

Hairpin

0 14.2 42.8 85.7

0 16.6 50 100

Hairpin stability The data in Table II for the hairpin-coil transition reveal that: (i) The hairpin stability is strongly influenced by the base composition of the stem helix. The hairpins formed by V-VII have a d(CGCGCG): d(CGCGCG) stem and they exhibit, as expected, similar thermal stabilities. The substitution of one C:G of the stem with one A:T, as in VIII,

801

produces the effect of considerably reducing the T~/2 of the hairpin-coil transition, from 78 ° to 68°C. When more C:G base pairs of the stem are replaced with T:A, the resulting hairpins are stable only in a narrow temperature range (Table II); (ii) At a given base composition also the sequence of the stem can influence the hairpin stability. This is clearly seen by considering oligomers II and III, whose hairpin structures are characterized by a h o m o and an alternating-CG stem, respectively. The melting curves show that III is more stable than II for some 10°C. Since the enthalpy changes for the two hairpins are found to be the same, i.e. 57 kcal/moi, the different stability appears to be mainly entropic in origin; (iii) The loop length rather markedly influences the stability of hairpins with the same stem, as can be seen in [10] and from a comparison of samples III and IV with V, VI, and VII (see also [17]); (iv The loop base composition influences the hairpin stability, but to a relatively minor extent with respect to the stem composition. Senior et al. have found that in the homologous hairpin series d(CGAACGX4CGTTCG) (X=C, T, A, G,) the stability decreases as T > C > G > A , with 4oC of difference between a T-loop and A-loop [24]. In our hairpins this effect is even lower. That the loop exerts a modest influence on the c t n h i l l t v n f t h o h n i r n i n ~ n f t h e t w o series I II. III, IV and V, VI, VII, and XII can also be inferred by considering that it parallels that found in the corresponding polynucleotides poly(dGdC)" poly(dGdC) > poly(dG)" poly(dC) > poly(dAdc): poly(dGdT) > poly(dAdT)"

poly(dAdT) [401. Duplex-hairpin energetics Since this conformational transition in fully palindromic sequences shows a pronounced hysteresis, the evaluation of the duplex-hairpin enthalpy change, based on the analysis of the melting profiles, is misleading. Therefore~ we estimated indirectly the AH for this conversion by using the experimental A/-/for hairpin denaturation and the empirical stacking energies reported in the literature [36, 37], considering that: AH (D--+H) = AH (D-+C) - 2all (H---,C). The calculated values for most of the sequences V - X I I I are found in the range of 25-40 kcal / tool. Similar values have been reported for

81)2

L.E. X o d o et al.

the duplex-to-hairpin transition ot d ( C G C G T A T A C G C G ) , 30 kcal-mol [21], d ( C G C G A A T T A C G C G ) , 26 k c a l / m o l [13], and of III, 23 k c a l / m o l [i6]. An insight into the mechanism of the duplexto-hairpin transformation was obtained by Tjump experiments [13, 19, 21]. We recently reported activation energy values of 87 and 96 k c a l / m o l for the duplex-to-hairpin transition of V and VI, respectively [19]. Roy et al., studying the same transition, found a value of 85 k c a l / mol for d ( C G C G C G A A T F A C G C G C G ) [13], whereas W e m m e r et al. found 55 k c a l / m o l for d ( C G C G T A T A C G C G ) [21]. These results are consistent with a duplex-to-hairpin transformation through a high energetic transition state, probably implying a nearly total duplex unwinding rather than a cruciform extrusion. As for the reverse reaction, hairpin-to-duplex, it was found to be so slow that we could not obtain useful relaxation spectra for estimating the relative activation energy. The energetics of this transition was recently studied in our laboratories with a 20-mer D N A fragment containing an inverted repeat whose termini are flanked by 4 bases that are not self-complementary within one strand, but are complementary with those of the opposite strand [41]. The two strands are d ( C C A T C G C G C G T G C G C G T A C C ) and d(GG T A C G C G C G T G C G C G A T G G ) . They assume the unimolecular hairpin structure and, more interestingly, their 1:1 complex exhibits a facile dunlex-hairnin i n t o r e n n v ~ r e i n n T&hl l;l~O gI I,I ~I U, l4I I; ~n ~ indicates that the presence of sticky dangling ends in the above 20-mer D N A fragment provides suitable loci for duplex formation. In the case of the "blunt end" palindromes of Table I, the duplex formation from the hairpins is extremely slow because these lack any structural feature that can facilitate their interstrand interaction. Thus part of the stem should be disrupted to promote the hairpins association, and this high energetic step ~s responsible for the slow rate of reaction. •

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Acknowledgments This work was supported by the Italian Research Council (CNR) and the Netherlands Organization for Advancement of Pure Research.

References 1 Pohl F.M. & Jovin T.M. (1972) J. Mol. Biol. 67, 375-396

2 Wang A.H.-J., Quiglcy G.J., Kolpak F.J., Crawford J.L., van Boom J.H., van der Mare! G.A. & Rich A. (1979) Nature 282,680-688 3 Stalker D.M., Thomas C.M. & Helinski D.R. (1980) Mol. Gen. Genet. 181, 8--12 4 Crews S., Ojala D., Posakony J., Nishiguchi J. & Attardi G. (1979) Nature 277,192--198 5 Tapper D.P. & Clayton D.A. (1981) J. Biol. Chem. 256, 5109 -5115 6 Hsu M. (1987) Virology 143,617-621 7 Rosenberg M. & Court D. (1979) Annu. Rev. Genet. 13,319-353 8 Elbourough K.M. & Wcst S.C. (1988) Nucl. Acids Res. 16, 3603-3616 9 Bianchi M. (1988) EMBO J. 7,843-849 10 Haasnoot C.A.G., Hilbers C.W., van der Marel G.A., van Boom J.H., Singh U.C., Pattabiraman N. & Kollman P.A. (lq~q6)J. Biomol. Struct. Dyn. 3,843-857 11 Marky L.A., Blumenfeld K.S., Kozlowski S. & Breslauer K.J. (1983) Biopolymers 22, 1247-1257 12 Orbons L.P.M., van der Marel G.A., van Boom J.H. & Altona C. (1986) Nucl. Acids Res. 14, 4187-4195 13 Roy S., Weinstein S., Borah B., Nickol J.. Appella E., Sussman J.L., Miller M., Shindo H. & Cohen J. (1986) Biochemistry 25, 7417-7423 14 Ikuta S., Chattopadhyaya R., Ito H., Dickerson R.E. & Kearns D.R. (1986) Biochemistry 25, 5341-5350 15 Hare D.R. & Reid B.R. (1986) Biochemistry 25, 5341-5350 16 Xodo L.E., Manzini G., Quadrifoglio F., van der Marel G.A. & van Boom J.H. (1986) Nucl. Acids Res. 14, 5389-5398 17 Xodo L.E., Manzini G., Quadrifoglio F., van der Marel G.A. & van Boom J.H. (1988a) Biochemistry 27,6321 - 6326 18 Xodo L.E., Manzini G., Quadrifoglio F., van der Marel & van Boom J.H. (1988b) Biochemistry 27, 6327-6331 19 Xodo L.E., Manzini G., Quadrifoglio F., van dcr Marei G.A. & van Boom J.H. (1988c) J. Biomol. Struct. Dyn. 6, 139-152 20 Summers F., Byrd R., Gallo K., Samson C.J., Zon G. & Egan W. (1985) Nucl. Acids Res, 13, 6375-6386 21 Wemmer D.E., Chou S.H. & Hare D.R. (1985) Nucl. Acids" Res. 13, 3755-3771 22 Germann M.W., Schoenwalder K.-H. & van de Sande J.H. (1985) Biochemistry 24, 5698-5702 23 Patei D.J., Kozlowski S.A., Ikuta S., Itakura K., Bhattand R. & Hare D.R. (1983) Cold Spring Harbor Symp. Quant. Biol. 47, 197-206 24 Senior M.M., Jones R.A. & Breslauer K. (1988) Proc. Natl. Acad. Sci. USA 85, 6242-6246 25 Gupta G., Sarma M.H. & Sarma R.H. (1988) Biochemistry 26, 7715-7725 26 Sinden R.R. & Pettijohn D.E. (1984) Biol. Chem. 259, 6593

Duplex-to-haiJpin transition in oligodeoxynucleotides 27 Courey A.J. & Wang J.C. (1983) (_'ell 33, 817-829 28 Gellert M,, O'Dea M.H. & Mizuuchi K, (1983) Proc. Natl. Acad. Sci. USA 80, 5545-5549 29 Lilley D.M.J. (1980) Proc. Natl. Acad. Sci. USA 77, 6468-6472 30 van der Marel G.A., van Boeckel C.A.A., Wille G. & van Boom J.H. (1981) Tetrahedron Lett. 22, 3887-3890 31 van Boom J.H., van de:: Marel G.H., Wille G., Hoyng C.F. (1982) in: Chemical and Enzymatic Synthesis of Gene Fragments (H. Gassen and H. Lang, eds.), pp. 53-70 32 Borer P.N., Dengler B. & Tinoco I. Jr. (1974) J. Mol. Biol. 86, 843-853 33 Albergo D.D., Marky L.A., Breslauer K.J. & Turner D.H. (1981) Biochemistry 20, 1409-1413 34 Bevington P.R. (1969) Data Reduction and Error Analysis for the Physical Sciences. McGraw-Hill,

h03

New York 35 Freier S.M., Aikema D., Sinclair A.. Neilson T. & Turner D.H. (1985) Biochemistry 24. 4533-4539 36 Goton O. & Tagashira Y. (I981) Biopolymers 20, 1033-1042 37 Breslauer K.J., Frank R., Blocker H. & Marky L.A. (1986) Proc. Natl. Acad. Sci. USA 83, 3746-3750 38 Benevides J.M., Wang A.H.J., Rich A., Kyogoky Y., van der Marel G.A., van Boom J.H. & Thomas G.J. Jr. (1986) Biochemistry 25, 41-50 39 Sato M., Ono A., Higuchi H. & Ueda T. (1986) Nucl. Acids Res. 14, 1405-1415 40 Klump H.H. & Loftier R. (1985) Hoppe-Seyler Biol. Chem. 366, 345-353 41 Xodo L.E., Manzini G., Quadrifoglio F.. Yathindra N., van der Marel G.A. & van Boom J.H. (1989) J. Mol. Biol. 205,777-781