Conformational Perturbation Due to an Extra Adenosine in a Self -Complementary

Oligodeoxynucleotide Duplex

SIDDHARTHA ROY,* Clinical Pharmacology Branch, National Cancer Institute; VLADIMIR SKLENAR,? Laboratory of Chemical Physics, NIDDK; ETTORE APPELLA, Laboratory of Cell Biology,

National Cancer Institute; and JACK S. COHEN, Clinical Pharmacology Branch, National Cancer Institute, National Institutes

of Health, Bethesdu, Maryland 20892

Synopsis

The conformation of an oligodeoxynucleotide pentadecamer, d(CGCGAAA’I””ACGCG), self- complementary except for an additional adenosine nucleotide at position 11, has been investigated with nmr spectroscopy. This oligomer was found to exist predominantly in the duplex form in solution rather than in the hairpin loop form, as observed previously for an analogous 13-mer sequence. Nuclear Overhauser effects indicate that the B-form conformation is disrupted by the extra A base, which forms a wedge within the duplex, producing a bent junction and an overall S-like structure. A downfield-shifted phosphate resonance was assigned using a two-dimensional 1 H-“I P correlation method, and was found to be P12-13. This and other results indicate that the extra A causes conformational distortions at some distance along the duplex structure.

INTRODUCTION

The subtlety of conformational preferences of oligodeoxynucleotides with minor alterations in base sequence, such as nonpaired insertions or deletions, is only now becoming appreciated. We have studied a series of oligomers with sequences derived from the self-complementary dodecamer d(CGCGAAT- TCGCG), crystallized by Dickerson and Drew.’S2 In a previous article we have shown that the sequence d(CGCGAATTACGCG), with an extra adenosine at position 9, a t room temperature and millimolar concentration, exists almost exclusively in the single-stranded hairpin loop conformation, with a four base-pair stem and a five base loop3 It was not possible for the duplex form to exist predominantly in solution under accessible conditions. Attempts to crystallize this oligomer did not yield suitable crystals for structure de- termination. A sequence isomer, d(CGCAGAATTCGCG), has been shown by nrnr to exist in a duplex form in solution, and the conclusion was drawn that the extra adenosine stacks inside the duple^.^ That sequence has been crystal- lized, and preliminary x-ray diffraction measurements have been r e p ~ r t e d . ~ Recently Hare et a1.6 have described two-dimensional nuclear Overhauser effect (2D NOE) and distance geometry analysis of a similar sequence, d(CGCAGAGCTCGCG), in which the nonpaired A4 stacks within the duplex.

*Current address: Department of Biophysics, Bose Institute, Calcutta, India. ‘On leave from the Institute of Scientific Instruments, Czechoslovak Academy of Sciences,

CS-61264, Brno, Czechoslovakia.

Biopolymers, Vol. 26, 2041-2052 (1987) B 1987 John Wiley & Sons, Inc. CCC OOO6-3525/87/122041-12$04.00

2042 ROY ET AL.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

o o m o m o o m 0 0

G-C-G-C,A, T-T-T-A-A-A - G-C-G-C

A-A\A C-G-C-G' \ m o o 0 T

I

G-C-G-C, T' A-T' Fig. 1. Representation of the interconversion of duplex and loop forms of I.

We now report our results on the pentadecamer, d(CGCGAAATT- TACGCG) (I), a sequence that has the potential to form a seven-membered loop (Fig. 1). It has been reported previously that the most stable loop size for oligodeoxynucleotides is 4-5 bases? It was anticipated that this factor and the increase of two extra base pairs in the central AT region, on going from the tridecamer to the pentadecamer, would stabilize the duplex form.8 Nuclear magnetic resonance solution studies described in this article show that this sequence I does indeed prefer the duplex form more than the sequence d(CGCGAATTACGCG).

Two-dimensional nmr techniques have been used extensively to characterize conformations of oligodeoxynucleotides,g particularly those in the duplex form. We have used NOE experiments to obtain spatial connectivities of protons in order to draw conclusions about the conformation of the duplex form of the oligonucleotide I. Crystallization of I yielded good crystals that diffract a t least to 3.4 A resolution." Thus, it may soon become possible to compare conformational properties of the pentadecamer I in solution, sug- gested by these nmr studies, and in the solid state.

MATERIALS AND METHODS

Sample Preparation

The oligodeoxyribonucleotides were synthesized on a Vega Coder 300 by the phosphoramidite method." After purification by reverse-phase high perfor- mance liquid chromatography,12 the oligomers were twice precipitated from aqueous solution by addition of 3 volumes of ethanol in the presence of 0.3M sodium acetate, and then dried in a Savant Speedvac concentrator. Con- centrations of oligonucleotides were determined spectrophotometrically a t 260

CONFORMATIONAL PERTURBATION 2043

nm, assuming extinction coefficients ( x for purines.

pmol) of 7 for pyrimidines and 14

NMR Methods

Proton nmr studies were performed on a Varian XL-400 nmr spectrometer at 400 MHz and on a modified Nicolet NT500 system at 500 MHz. Purified DNA samples were dissolved in D,O and 0.5 mL of appropriate buffers were added. The sample was then lyophilized. The lyophilized material was then relyophilized twice from D,O and finally dissolved in 0.5 mL of D,O. Temper- ature dependence of spectra was obtained by initially equilibrating the sample a t 4°C for 18 h, followed by preequilibration for a t least 30 min (15 min for temperatures above 35°C) after shifting to a higher temperature. The temper- ature settings on the spectrometer were calibrated using a thermocouple. The spectra were then recorded at each temperature. Relative proportions of each species were obtained by integrating the methyl region of the spectra. Van't Hoff enthalpy was obtained as described previou~ly.~ Two-dimensional NOE absorption mode spectra were obtained under standard conditions; in most cases spectra were recorded at 400 MHz or 500 MHz using mixing times of 300 and 100 ms, respectively. Proton 2D NOE spectra in H,O were obtained at 500 MHz with 100 ms mixing time using a recently described spin-echo water suppression technique in order to observe base imino and amino protons.13.14 The pulse scheme applied consists of the standard two nonselective pulses separated by the evolution period t,. During the mixing time a strong homogeneity spoiling pulse (duration 30 ms) is used t o defocus all transverse magnetization. Finally, a 1-1 echo sequence'* is used as a read pulse. The timing of the delays in the read pulse are adjusted to produce near-optimal excitation of both imino and amino protons in DNA.13 P r ~ t o n - ~ l P correlation spectra were also obtained in order to assign phos- phate signals. Proton-detected chemical shift correlation was applied with a constant time evolution period,15 and the phosphorus signals were identified using the assignments of the 3',4' and 5',5" protons from the 2D NOE experiment.

RESULTS Figure 2 shows proton nmr spectra of the methyl region of the pentade-

camer I as a function of temperature. A t 9°C only three peaks are observed in the methyl region at 1.32, 1.63, and 1.73 ppm. A t 25°C another set of three peaks can be seen at 1.6,1.76, and 1.88 ppm. At 40°C the peak at 1.88 ppm has grown considerably in size, whereas the other two peaks of the latter set merge with the bigger peaks at 1.63 and 1.73 ppm and can no longer be distinguished. The line width of the peak at 1.88 ppm is 3.2 Hz and that at 1.32 ppm is 6 Hz at 25°C.

Figure 3 shows the spectra of the aromatic region of oligomer I at 9°C. The sharper peaks at 7.11, 7.24, 7.62, and 7.76 ppm are probably from the H2 protons of the four adenosines present in the oligonucleotide. The rest of the proton resonances in this region come from the H6 protons of the pyrimidines and the H8 protons of the purines. Assignments shown in this figure are derived from the 2D NOE experiments described later.

2044 ROY ET AL.

C

B

A

I ' " " ' 1 m - z " " 1 f f " i ' ' I " 1 - - 1 9 1 c 4 I ? 1 c ( 3 F'FS1

Fig. 2. Proton nmr spectra at 400 MHz of the methyl region of oligomer I at three hfferent temperatures. (A) 9"C, (B) 25"C, and (C) 40°C. Oligonucleotide concentration was 4.2 mM. The sample was in 0.01M phosphate buffer, pH 7.0, containing 0.134 NaCl.

CONFORMATIONAL PERTURBATION 2045

I l l I I V 1 % ’ I ! ” , , , ‘ W T 1 ” I ’ ‘ I ’ I n ’ ’ 5 ; 8 - 1 9 , 9 , ‘ ’ 3 7 t ’ 4 7 L i’r 8

Fig. 3. Proton nmr spectra a t 400 MHz of the aromatic region of ohgomer I at 9°C. Solution conditions are same as in fig. 1. Assignments of the aromatic peaks are shown. Unlabeled sharper peaks are adenosine C2H protons.

Figure 4 shows the 2D NOE plot of the aromatic region of the oligomer I. In this region, only NOEs between base protons can be seen. An essentially identical result was obtained at 500 MHz with a shorter mixing time of 100 ms. Due to relatively large interbase distances, these NOEs are typically small. Four cytidine H6 protons have been identified by 2D correlated spectroscopy experiments at 7.33, 7.42, 7.54, and 7.70 ppm (data not shown).16

Figure 5 shows the region of methyl/H2’,H2’’ to aromatic cross-peaks in the 2D NOE plot of oligomer I. Each methyl group exhibits two large NOE cross-peaks. According to distance estimates for B-DNA, one of the NOEs is for the H6 proton on the same base and the other is for the H6 proton of the adjacent base on the 5‘ side. Clearly, the H6 proton of thymidine 10 has no methyl group on the adjacent base on the 3‘ side and should show an NOE for only one methyl group. On inspection one can thus immediately make the assignment of T10, and by connectivity shown in the figure assign T9 and T8. Thus, the H8 proton of adenosine 7 can be assigned to 8.21 ppm (because this is the only f a r downfield peak having a strong NOE for the T8 methyl peak). Thus one can assign the C6H protons of T8, T9, and T10 to 7.2,7.52, and 7.33 ppm, respectively. So the remaining aromatic proton peaks are purine H8 resonances.

In Fig. 4 one can observe a cytidine H6-purine H8-cytidine H6-purine H8-purine H8 connectivity (dashed line starting from the peak labeled “Cl”). Only Cl-G2-C3-G4-A5 can give rise to such a connectivity and hence the assignments are straightforward. Another cytidine H6-purine H8-cytidine

2046 ROY ET AL.

” b* 1 - 1

i. L ? ? - \

Fig. 4. The aromatic region of the 2D NOE plot of ligomer I at 9°C. The mixing time was 300 ms. The cross-peaks are the interaromatic NOEs. Solution conditions are same as in Fig. 2.

H6-purine H8 connectivity (solid line) must arise from C12-Gl3-Cl4-Gl5 and was assigned accordingly. The dotted line shows the connectivity of T9-TlO- A l l and the rest of the dashed line shows A5-A6-A7-T8 connectivity. There are four adenosine H2 protons at 7.76, 7.24, 7.11, and 7.62 ppm, which are connected by NOEs in that order (connectivity is not shown in the figure).

It may be noted that the A l l H8 proton resonance is connected to the H6 proton of T10 by a weak NOE, indicating that A l l stacks within the duplex (see Fig. 4). Similarly, the presence of an NOE between the H2 proton of A l l and A5 (one of the NOEs between the H2 protons described above) indicates stacking within the duplex of All. In fact, the cross-peak between Al l and A5 is only about half the intensity of the cross-peaks between A5-A6 and A6-A7 observed in the 500-MHz spectrum with 100-ms mixing time, suggesting that A l l is close to A5 and stacked in the helix.

Figure 6 shows the base-H1’ region of the 2D NOE plot of oligomer I at 500 MHz. The connectivities shown are fully consistent with the assignments derived from the NOE connectivities in the aromatic region. The results confirmed the interaction A l l H8-Tl0 Hl’, thus indicating stacking of the nonpaired All , although the cross-peak in this case was quite weak. Interest- ingly, interstrand NOE cross-peaks were also detected between A7 H2-T9 H1’ and A6 H2-T8 Hl’. The stacking of A l l in the helix is further confirmed by the observed NOE interaction between A l l H8 and C12 H5.

CONFORMATIONAL PERTURBATION 2047

-~ _________ SUGAR H2',2" METHYL

T I 0 T 9 T 8

T---T--r---T- i-7-7 rn

3.0 2.5 2.0 1.5 1-13 FPP Fig. 5. The aromatic-methyl/HY-H2" region of the 2D NOE plot of oligomer I at 500 MHz.

Conditions are same as in Fig. 4, but with a mixing time of 100 ms. T8, T9, and T10 labels in the figure refers to the methyl groups of the corresponding thymidine residues.

Figure 7 shows the 2D NOE spectra a t 500 MHz of sequence I at 9°C in H,O using the new spin-echo sequence for water ~ u p p r e s s i o n . ~ ~ ~ ~ ~ All the 7 imino protons are observed. The imino protons and consequently the adeno- sine H2 proton signals have been unambiguously assigned. For the assignment of the T imino protons, the connectivities between T and AH2 and the mutual connectivities between AH2 protons measured in D,O have been used. Each T imino proton has a strong NOE for the AH2 of the same base pair and a weaker interaction with the AH2 in the ( n - 1) residue. G imino protons have been identified by the connectivities G-C amino-C H5-C H6 in the same base pair. The cross-peak intensities of the G imino protons and the two C amino protons are almost the same, which suggests relatively rapid 180" flips about C-N bonds. These fast flips relay the magnetization to the C H5 protons, and the observed cross-peaks with these protons can be used for C amino and consequently G imino assignments.13 These connectivities are not shown in Fig. 7. No NOE between the A l l H2 and T10 imino protons was detected, and only a very weak interaction (observed a t a lower level contour plot) between the A5 H2 and G4 imino protons was found. The latter interaction was quite significant in the regular B-DNA structure of the Dickerson d~decamer, '~ and this finding suggests a change in the stacking geometry in the G4-A5 step, as compared to the regular B-DNA conformation. The comparison of the intensi- ties of the exchange cross-peaks of imino protons with water shows that the G4 imino proton has a faster exchange rate than the C3 and even G2 imino

2048

AROMATIC pu H8 PY H6

18

T10,C3

C 14

19 c12

c1

iG4 G13 G2,15

A7,6

ROY ET AL.

0 - -

A5 A1 1

SUGAR H1'

G . 2 6.0 5 . B 5.6 S .'t s.z PPm

Fig. 6. The aromatic-HI' region of the 2D NOE plot of oligomer I at 500 MHz. Conditions are same as in Fig. 5. Aromatic H1' connectivities are shown by dashed lines.

hydrogens. This probably implies a more open structure of the double helix due to distortion by the extra A l l .

31P-nmr spectra of I showed that a single signal is shifted downfield relative to the rest of the spectrum. This signal has been assigned using the proton- detected 2D correlation experiment with a constant evolution time (Fig. 8). The correlation of the shifted 31P signal with sugar H3', H4', and H5',5'' signals are clearly resolved. The assignment of the H3' and H4' signals in the 2D NOE spectrum allows us to assign the 31P resonance to the phosphorus of C12. This phosphorus has a scalar interaction with its own H3' and with the H4' and H5',5" of the adjacent G13 residue on the 5' side. The assignment of the H3' and H4' was done using the 1'-2',2"-3' and 1'-3'-4' connectivities.

CONFORMATIONAL PERTURBATION 2049

I I ' " ' I 7 ' l ' ~ ~ " ' " " ' ' ' ~ 1 ~ '

1 Y 13 PPfl 8 7 6 5 PPM

Fig. 7. Part of the 2D NOE absorption mode spectrum of oligomer I measured in 10% D,O using a mixing time of 100 ms at 9"C, displaying the interactions between imino, amino, and aromatic protons. The sample conditions were the same as in Fig. 2. The one-dimensional nmr spectrum obtained with the spin-echo water suppression technique is shown on the top of the 2D matrix.

DISCUSSION

We have studied the pentadecamer sequence, d(CGCGAAATTTACGCG) (I), as a continuation of our study of the loop-forming abilities of self- complementary or near self-complementary sequences. Previously we had studied the tridecamer, d(CGCGAATTACGCG), and the heptadecamer, d(CGCGCGAATTACGCGCG), and concluded that at 37°C they are predomi- nantly in a single-stranded hairpin loop c~nformation.~ Similarly, as shown in Fig. 2, two sets of thymine methyl resonances clearly indicate the presence of two conformations for I in a temperature-dependent equilibrium exchanging slowly on the nmr time scale. Our results indicate that this is a duplex-hairpin loop equilibrium (Fig. 1) on the following grounds:

The pentadecamer I exists predominantly as the duplex form a t 9"C, a t an oligonucleotide concentration of 4.2 m M in 0.01M phosphate buffer (pH 7.0, containing 0.1M NaC1). Two-dimensional NOE spectra in H,O with water suppression (Fig. 7), which include the imino-proton signals a t 9"C, clearly indicate that the correct number of AT and GC base pairs are formed as expected for a duplex.

We have also obtained the Van't Hoff enthalpy of this transition and derived a value of 40 kcal/mole. Clearly, the higher temperature form is not a single-stranded random coil. The Van't Hoff enthalpy of such a melting would probably be in the range of 100 kcal/mole.8,'7 For the tridecamer sequence, d(CGCGAATTACGCG), the Van't Hoff enthalpy of the duplex to hairpin loop transition is 30 k~al /mole,~ where only the AT base pairs are disrupted in the loop form. Two extra AT base pairs are expected to be disrupted in the pentadecamer I compared to the similar duplex to hairpin transition in the tridecamer, d(CGCGAATTACGCG). Hence the pentadecamer I is expected to have a somewhat higher Van't Hoff enthalpy for the duplex to hairpin loop

2050 ROY ET AL.

P12-13 + G13

613

I I " ' l " ' l " ' l " ' / " ' I " ' l ' T

-7.0 -2.2 - 2 . 3 -2 .6 - 2 . 8 -3.0 -3.7 -3.9 PPM

P-31 CHEMICAL SHIFT

Fig. 8. Two-dimensional absorption mode H-31P correlation spectrum of oligomer I obtained using constant time evolution period with the effective t , acquisition times ranging from - 25 to 25 ms. Proton and 31P chemical shifts are reported relative to TSP and phosphate buffer, respectively. The spectrum results from a 2 X 25 X 512 data matrix; total measuring time was 12 h. The resolution enhanced one-dimensional 31P-nmr spectrum is shown along the 31P frequency axis.

transition than for the tridecamer. A value of 40 kcal/mole is thus consistent with a duplex to hairpin loop transition for the pentadecamer I.

Although we have not structurally characterized the high-temperature form, the chemical shifts of the methyl groups and the slow rate of intercon- version (on the nmr time scale), taken with the arguments presented above, makes the likelihood that this is a duplex-hairpin loop equilibrium a reason- able one.

We have assigned all the aromatic protons of the oligomer I in the duplex form. Assignments in the duplex state are straightforward and consistent, based on intraaromatic and aromatic-HI' connectivities. hoimportant conclu- sion one can draw is that the H2 proton of A l l is within 4 A of the H2 proton of A5. Also, the A l l H8 proton is connected by an NOE to the H6 proton of T10. Thus one can conclude that A l l is stacked within the duplex above the A5-TlO base pair.

CONFORMATIONAL PERTURBATION 2051

A

5’\ /3’

3’’ ‘5’

Fig. 9. Two possible structures of oligomer formed by uniform separation of the base pairs expanding only one strand between the base wedge.

B 3 B 5’

P

3’ 3‘

Fig. 9. Two possible structures of oligomer I near the extra adenosine 11. (A) The structure formed by uniform separation of the base pairs G4-Cl2 and A5-TlO. (B) The structure formed by expanding only one strand between the base pairs G4-Cl2 and A5-Tl0, with A l l acting as a wedge.

3‘ I near the extra adenosine 11. (A) The structure G4-Cl2 and A5-TlO. (B) The structure formed by pairs G4-Cl2 and A5-Tl0, with A l l acting as a

We have been unable to observe a significant and unambiguous NOE connecting the base protons of A l l and C12. But there is a clear, unambigu- ous connectivity between C12 H6 and A l l Hl‘. This probably is a reflection of distortion (but not disruption) of the double helix. Another interesting point is that the NOE connectivities between the base protons of G4 H8 and A5 H8, as well as of A5 H8 and G4 Hl’, are also clear and substantial.

It has been hypothesized that in order to accommodate the extra base the phosphate A5pG4 may be in an unusual conformation, and bases G4 and A5 may be separated by more than the standard interbase pair di~tances.~ Such a structure is shown in Fig. 9(A). Evidence presented here indicates that G4 and A5 are stacked close enough to have NOE connectivities similar to that observed in B-DNA. Since one has to expand the space between T10 and C12 to physically accommodate A l l stacked into the duplex, keeping close proxim- ity to G4 and A5 leads to A5-Tl0 and G4-Cl2 base pairs forming a bent structure. This conformation is represented in Fig. 9(B), with A l l acting as a wedge. Thus, one may hypothesize that CG sections are stacked on AAAT- TTA/ATTTAAA sections forming this wedgelike bent junction. The struc- tural consequences of this would lead to an overall S-like conformation for the oligomer, with bends at each A l l wedgelike junction. It will be very interest- ing to see if the x-ray structure now being determined” in any way supports this structural proposal. The structure determined recently for the 13-mer d(CGCAGAAGCTCGCG) by 2D NOE and distance geometry methods is quite similar to the “bent” structure proposed here.

We have also observed that one phosphate signal is shifted downfield from the main bulk of phosphate resonances in the 31P-nmr spectrum of the pentadecamer I. Pate1 et al.4 also reported a similar downfield-shifted phos- phorus resonance in the sequence with an extra adenosine that they studied, and concluded that it must arise from the phosphodiester opposite the unpaired A base inserted into the helix, in this case G4pA5 (Fig. 9A). Using the 2D 1H-3’P correlation technique, we have assigned this downfield-shifted resonance to the phosphodiester between C12 and G13. This is somewhat surprising, but indicates that the presence of the extra A causes distortion along the duplex, resulting in an unusual conformation at a distant phosphate.

ROY ET AL.

There is evidence from hydrogen-exchange rates" as well as our other results that such distortions in base pairs far from the extrahelical base are indeed present, and any realistic structure must also account for these distortions.

In conclusion, we have presented evidence that the extra A l l in the sequence I stacks within the duplex, forming a bent junction.

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4. Patel, D. J., Kozlowski, S. A., Marky, L. A., Rice, J. A., Broka, C., Itakura, K. & Breslauer,

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T. J. J., Hilbers, C. W., van der Marel, G. A. & van Boom, J. H. (1983) J . Biomol. Struct. Dynum. 1, 115-129.

8. Miller, M., Kirchhoff, W., Schwarz, F., Appella, E., Chiu, Y. Y. H., Cohen, J. S. & Sussman, d. I,. (198X) Nucleic Ac& Res. 15, 3877-3890.

9. Wemmer, D. E. & Reid, B. R. (1985) Ann. Rev. Phys. Chem. 35, 105-137.

H. & Cohen, J. S. (1986) Biochemistry 25, 7417-7423.

K. (1982) Biochemistry 21, 445-451.

Bid. 188, 111-113.

10. Miller, M., Wlodawer, A., Apella, E. and Sussman, J. L. (1986) J . Mol. Biol., in press. 11. Adams, P., Kanka, K. S., Wiks, E. J., Holder, S. B. & Galupi, G. R. (1983) J . Am. Chem.

12. Zon, G. & Thompson, J. A. (1986) Biotechnigues 1, 22-32. 13. Sklenar, V., Brooks, B. R., Zon, G. & Bax, A. (1987) FEBS Lett. 216, 249-252. 14. Sklenar, V. & Bax, A. (1987) J . Mugn. Reson. in press. 15. Sklenar, V., Miyashiro, H., Zon, G., Miles, T. & Bax, G. (1986) FEBS Lett. 208, 94-98. 16. Borah, B., Roy, S., Zon, G. & Cohen, J. S. (1985) Biochem. Biophys. Res. Commun. 133,

17. Marky, L. A., Blumenfeld, K. S., Kozlowski, S. & Breslauer, K. J. (1983) Biopolymers 22,

18. Pardi, A., Morden, K. M., Patel, D. J. & Tinoco, I., Jr. (1982) Biochemistry 21, 6567-6574.

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380-388.

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Received November 11, 1986 Accepted June 3, 1987