ultraviolet resonance raman bands of guanosine and adenosine residues useful for the determination...

JOURNAL OF RAMAN SPECTROSCOPY, VOL. 18, 221-227 (1987)

Ultraviolet Resonance Raman Bands of Guanosine and Adenosine Residues Useful for the Determination of Nucleic Acid Conformation

Yoshifumi Nishimura and Masamichi Tsuboi Faculty of Pharmaceutical Sciences, University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan

William L. Kubasek, Krzysztof Bajdor and Warner L. Peticolas Department of Chemistry, University of Oregon, Eugene, Oregon 97403, USA

The ultraviolet resonance Raman (UVRR) bands of several guanine- and adenine-containing mononucleotides were measured, and were compared with the classical and UVRR spectra of 9-ethylguanine, which contains no furanose ring, in order to assess the effect of sugar conformation on the UVRR spectrum. For several guanine mononucleotides containing different furanose rings some differences were found in the Raman bands normally attributed to the purine vibrations. A similar effect exists with different adenosine mononucleotides. The fact that these vibrations are allowed in the rigorous resonance Raman effect indicates that they are coupled to the electronic transitions of the base. Differences which are seen between cyclic nucleotides, ribonucleotides and deoxyribonucleotides probably arise from differences in the conformation of the attached furanose rings. This provides a method for the identification of conformationally sensitive vibrations of purine rings in nucleic acids.

INTRODUCTION

For a number of years it has been known that the Raman-active backbone vibrations of nucleic acids are useful in determining their c~nformation'-~ (for a review, see Ref. 5 ) . More recently it has been shown that several vibrations of the bases, particularly those of the purines, are sensitive to the nucleotide furanose conformation and to changes between the syn and anti

In particular, Nishimura et aL9 obtained the classical Raman spectra of a large number of mononucleotide and nucleoside crystals in which the molecular conformations were established by x-ray crys- tallography. The classical Raman effect has the advan- tage that one can obtain spectra from both crystalline measurements and in solution. However, the UVRR effect has the disadvantage that crystal samples will be photochemically damaged in the laser beam and hence crystals cannot be used as a primary standard for a structural reference in the UVRR spectra. By comparing the UVRR spectrum of a nucleotide in solution with the classical spectrum, however, conformational informa- tion can be obtained from the UVRR spectrum. The question naturally arises as to the nature of the vibrations which appear to come from the bases but which are sensitive to the backbone conformation. This sensitivity appears to require some coupling of the base vibrations with the displacement of the backbone atoms them- selves. The relative contributions of the base and the sugar-phosphate group to the vibrations cannot be obtained from classical Raman spectroscopy alone since the classical Raman spectral intensities are not directly coupled with low-lying electronic states of the bases. Hence it seemed worthwhile to make a comparison of the UVRR spectra of mononucleotides with varying types of furanose rings attached. Although the UVRR spectra of the bases have been reported from both their

0377-0486/87/030221-07 $05.00 0 1987 by John Wiley & Sons, Ltd.

260 nm10-12 and 210 nm absorption bands,'*-'' to date no detailed study has been made of the effect of different furanose groups on the UVRR bands of guanine and adenine. In this paper we report the detailed differences in the UVRR spectra of guanosine and adenosine nucleotides with varying types of furanose rings.

EXPERIMENTAL

The nucleotides were obtained from Sigma and Phar- macia and were used as received. The spectra were taken with a Quanta Ray YAG laser frequency doubled to 266 nm and shifted to 218 nm by stimulated Raman scattering from hydrogen gas. The scattered light was collected by a cassegrain lens and analyzed by a Spex 1401 monochromator and a solar blind Hamamatsu photomultiplier tube, as used in previous ~ t u d i e s . ' ~ - ' ~

RESULTS A N D DISCUSSION

9-Ethylguanine

In order to assess the effect of vibrational coupling of the furanose ring with the purine ring on the Raman spectrum of predominantly purine ring vibrations it would be desirable to have a 'standard' spectrum of the guanine residue which is free from ribose but which has a carbon atom attached to the nitrogen at 9-position (N-9). The UVRR spectra of 9-ethylguanine taken with 266 and 218nm excitation that is shown in the upper half of Fig. 1 may meet such a requirement. For guanine itself, which has a hydrogen attached to N-9, a reliable set of force constants has been obtained and used in a normal coordinate analysis of the guanine re~idue.'~"'

Received 21 April 1986 Accepted 16 May 1986

222 Y. NISHIMURA E T A L .

9EtG

=,"In g 266nm exc.

218 nrn exc

218nrn exc

Figure 1. Raman spectra of 9-ethylguanine in 2 - 3 m ~ aqueous solution a t pH 7, and of guanosine in 1 mM aqueous solution at pH 7, taken with 266 and 218 nm excitation. In the upper part of the figure the classical Raman spectrum of guanine is shown, together with i ts infrared spectrum, for comparison. To each Raman line a proposed assignment of the vibrational mode is given. The in-plane vibrational modes of guanine are numbered according to Nishimura and co-workers' normal coordinate Some of the modes (with asterisks) are shown in Fig. 2. An out-of-plane vibrational mode of guanine is indicated as 'out pl.'

Although the guanine molecule will have a slightly different set of normal modes of vibration than the 9-substituted guanines because of the difference in the mass of the atom attached at the 9-position, we may still use the normal coordinate analysis of guanine to under- stand the UVRR spectrum of 9-ethylguanine.

The 1700-1600 cm-l region of the UVRR spectra of 9-ethylguanine seems to have two UVRR-active normal vibrations, whose modes are similar to the mode 6 (C=O stretching) and mode 7 (C-NH, stretching) of guanine.

Figure 2. Vibrational modes of the in-plane normal vibrations of guanine as derived from the force constants calculated by an ab initio MO method.l6.l7 The numbering of the modes was made by Nishimura et aL16 and is used in Fig. 1 for assigning vibrational modes to the observed Raman lines of guanine.

(See Fig. 1 and note the Raman bands of crystalline guanine listed at the top for comparison. The atomic displacements for some of the guanine vibrations are shown in Fig. 2.) The Raman scattering intensity corresponding to these vibrations is more pronounced with the 218 nm excitation than with the 266 nm excitation. The weak 1602 cm-' line of 9-ethylguanine may correspond to the 1604 cm-' line of dGMP. This vibration is considered to involve the NH, scissoring motion, because Fodor et aLi3 found that it disappeared on hydrogen-deuterium exchange. It must also involve the C-NH, bond stretching and some skeletal ring stretching motion since it is active in the UVRR spectrum in addition to the classical spectrum. The six Raman lines of 9-ethylguanine in the 1600-1300 cm-' region are impressive in their intensities in the UVRR spectra. Those at 1576, 1489, and 1306cm-' are fairly strong with 266 nm excitation, whereas they are absent or weak with 218 nm excitation. On the other hand, the 1369 cm-' Raman band is very strong with 218 nm excitation, whereas it is weak with 266 nm excitation. The 1560 and 1415cmp' lines are both very weak, but are clearly observed in the UVRR spectra, so they cannot be ignored. These six lines may correspond to the modes 8, 10, 11, 12, 13 and 15 of guanine (see Fig. 2). Guanine has the N-9-H bending vibrations (mode 14) at about 1400cm-' and this motion may be coupled with the ring-stretching vibrations, whose frequencies are close to 1400cm-'. Such a coupling should be absent in 9- ethylguanine, and therefore the ring-stretching vibrational modes in 9-ethylguanine may be very different from those in guanine itself in this vibrational frequency range.

The excitation profiles of these Raman intensities should be explained in terms of the vibrational modes and the excited state geometry. It is not impossible to construct a self-consistent explanation for each excited state on the basis of our present data using the bond order changes which may be calculated in going from the ground state to the particular excited ~ ta te . '~ ' ' ' "~ For example, according to quantum mechanical calculations carried out in several laboratories the C-4=C-5 bond should be greatly elongated on going from the ground state to the first allowed excited electronic state of guanine, but not so much in going to the second allowed state. 12,18,19 Therefore, Raman lines with the modes hav- ing the C-4=C-5 stretching motion (modes 10 and 13,

DETERMINATION OF NUCLEIC ACID CONFORMATION 223

for example) should be enhanced in the longer wavelength UV excitation (266 nm, for example), but not in the shorter wavelength UV excitation (218nm, for example). A detailed discussion of this point should perhaps be deferred until both the normal modes of 9-ethylguanine and its excited state geometries are well established. It seems likely that we shall come to the conclusion that most of these normal modes are not localized in any particular bond in the ring and also that any of the geometrical changes observed in going from the ground state to an excited electronic state are also not localized in any particular bond.

This conclusion is in agreement with the recent calculations of Vergoten et uZ.,~' who performed a normal coordinate calculation on oligonucleotides containing guanine and found that the ring niodes are very delocal- ized and mix with furanose vibrations. In the 1300- 600 cm-' range, ten Raman lines of 9-ethylguanine are observed at frequencies of 1238 and/or 1220, 1188 and/or 1178, 1089, 1075, 1030, 855, 715 and 627 cm-'. Guanine itself has ten in-plane vibrations in this frequency range-the modes numbered 16, 17, 18, 19, 20,21,22,23,24 and 25 (shown in Ref. 17). One of these modes (mode 20) is assigned to the amino-rocking vibration, which should be inactive in the UVRR spectrum. 9-Ethylguanine should have two additional skeletal stretching vibrations in comparison with guanine because the N-9-H group in the latter is changed to N-9-ethyl in the former. By taking this into account, we can correlate each prominent peak in the UVRR spectrum of 9-ethylguanine with a guanine in-plane vibrational mode. In Fig. 2, only one mode (mode 23) of these ten modes is illustrated as an example. The Raman line assignable to this mode is found at 855 cm-' and this appears only with the 218nm excitation and not with the 266 nm excitation. This mode is mainly a ring deformation but also involves an appreciable amount of C-5-C-7 stretching motion. According to the quantum mechanical calculations of Nagata et a1.,I9 the C-5-N-7 bond order is lowered only when guanine is excited to its third lowest excited electronic state, which is probably the second lowest allowed state corresponding to the 210nm absorption band. However, the bond order changes of Hug et al. as reported by othersI2 shows a much smaller difference in the two excited electronic states. In this case, the calculations of Nagata et al. seem more in agreement with the experimental UVRR results.

Guanosine

The lower half of Fig. 1 shows the UVRR spectra of guanosine taken with 266 and 218 nm exciting light. In guanosine the ribose ring is attached to the guanine ring at the N-9 position. In the 1700-1350 cm-' region of the UVRR spectra of guanosine eight Raman bands are observed. These occur at 1685 (probably two overlap- ping lines make up this band), 1604, 1581, 1539, 1492, 1420 and 1367 cm-I. These are very similar to the lines at 1676 (probably a doublet), 1602, 1576, 1560, 1489, 1415 and 1369cm-' which are observed in 9-ethylguanine. Both the frequencies (except for 1539 versus 1560 cm-') and the relative intensities are similar if one compares the UVRR spectra of the two molecules taken with exciting light of the same frequency (see Fig. 1).

The 1323 cm-' line of guanosine, however, is located at a considerably higher frequency than the corresponding line at 1306 cm-' of 9-ethylguanine. In addition, the former is appreciably broader than the latter.

These facts may be interpreted as indicating that this mode of the guanine residue couples with a ribose vibration when the residue is linked with ribose. Its frequency therefore may depend on the torsional angle, chi, around the glycosidic bond.879 If the chi angle of guanosine in solution is fluctuating over a greater range, it is under- standable that the 1320cm-' band is observed to be broader. The same explanation would be possible for our observation of a very broad ring-breathing Raman band in the 700-600 cm-' region of the guanosine UVRR spectra. In between these two structure-sensitive guanosine lines679 there are several weaker UVRR lines. A few guanosine lines including those at 1211 and 1184 cm-' should be correlated with the four lines (1238, 1220, 1188 and 1178 cm-') of 9-ethylguanine and four modes (modes 16, 17, 18 and 19) of guanine. The three lines at 1089, 1082 and 1031 cm-' correspond probably to the 1089, 1075 and 1030 cm-' lines of 9-ethylguanine. The vibrational modes for these lines would be some proper linear combinations of modes 21 and 22 of guanine and some of the N-C and C-C stretching motions of the glycosidic bond. The 877cmP1 line of guanosine, which is only active with 218 nm excitation, must correspond to the 855 cm-' line of 9-ethylguanine, which is also active only with 218 nm excitation.

Guanosine-5'-phosphoric acids-RNA vs DNA

Two classical Raman spectra are shown at the top of Fig. 3. One of these is that of a crystal of the nickel salt of riboguanosine-5'-phosphoric acid with eight molecules of water, and the other of a crystal of the nickel salt of deoxyriboguanylic acid. According to a crystallographic analysis by Sato,21 these two crystals are nearly isomorphous: both belong to C2 space group, the guanosine conformation is C-3'-endo-anti in both crystals and the manner of packing of the molecules in the crystals is similar. As expected from these similarities, the Raman spectra of these two crystals are certainly similar as far as the frequencies and intensities of prominent lines are concerned. In detail, however, these two spectra are surprisingly different from each other. It is especially surprising that some of the Raman bands (1316, 978 and 723 cm-I), which are single peaks for 5'-GMP-Ni-8H20, are split into two bands in the deoxy analog, which is so similar in every other respect. A probable explanation for this splitting is the occurrence of Fermi resonance; the 1310cm-' band, for example, is located close to the overtone frequency of the 664cm-I vibration. The molecular packing and geometry are so similar in these two crystals as to be essentially identical. It seems that no plausible explanation for the occurrence of these splittings which occur in dGMP but not in GMP is possible which is based on crystal-field arguments since the crystal fields of the two crystals must be essentially identical, as are the unit cells of the crystals. Disregarding these splittings, we find very similar spectral features in the 1400-1300 cm-' region of the spectra for both the GMP and dGMP crystals. In each case we have 1391 cm-' (strong),

224 Y . NISHIMURA E T A L .

00 1600 I400 1200 loo0 800 600 I I I 1 I I 1 - I + , I

5'GMP/dGMP soln

266 nm exc

Wavenumber. cm-l

Figure 3. Above: classical Raman spectra of guanosine-5'- monophosphoric acid (5'-GMP) and 2'-deoxyguanosine-5- monophosphoric acid (5'-dGMP) in their Ni salt crystals and in their concentrated (0.5 M) aqueous solutions at pH 7. Below: ultraviolet resonance Raman spectra of 5'-GMP and 5'-dGMP in their dilute ( 1 rnM) aqueous solutions at pH 7.

1344 cm-' (weak) and 1316 cm-' (medium). This intensity pattern is just what was proposed to assign to a C-3'-endo-anti conformation of the guanosine residue.839 The strong peak at 667 cm-' (or 664 cm-') was also proposed to be characteristic of the C-3'-endo- anti conformation.'

On going from crystal to solution, drastic spectral changes take place in both the 1400-1300 and 700 cm-' regions for both GMP and dGMP (see the middle por- tion of Fig. 3). In the 1400-1300cm-' region two medium-intensity broad peaks are observed at 1365 and 1325 cm-', and in the 700-600 cm-' region a broad band with its center at about 680cm-'. These observations are interpreted as indicating that neither GMP nor dGMP has the C-3'-endo-anti conformation in solution as they do in the crystal. Instead, they are fluctuating among the 0-4'-endo-anti, the C-1'-exo-anti, and the C-2'-endo-anti conformations. These conclusions are based on the conformation-Raman spectra correlations proposed previously based on classical Raman measure- m e n t ~ . ~ - ~ On reducing the excitation wavelength from 514.5 to 266 nm, the strong 981 cm-' band disappears (see the bottom spectra in Fig. 3). This fact indicates that this line is caused by a nearly pure PO:-- symmetric stretching vibration and is not coupled to the excited electronic states of the guanine. Likewise, the weak 811 cm-* line must also be assigned to a nearly pure sugar-phosphate vibration. Of the two diagnostic regions

in the classical Raman spectra of guanosine residues, the 1400-1300 cm-' region remains diagnostic even with the 266 nm excitation. The two Raman lines at 1365 and 1325 cm-I, characteristic of the higher pseudo-rotation angle P (which covers the 0-4'-endo to C-2'-endo range) with an anti glycosidic bond, are clearly observed here. Their relative intensities with 266 nm excitation are different from those with 514.5 nm excitation. The other diagnostic region, 700-600 cm-', seems still to be avail- able with 266 nm excitation, but the relative intensity of the 680cm-' band is much lower here. Therefore, the signal-to-noise ratio may sometimes be lower with 266 nm excitation and low (millimolar) concentrations than that obtained using 514.5 nm excitation at 20- 50 mM concentration. In solution with 266 nm excitation, no significant differences in the UVRR spectra are found between GMP and dGMP.

Guanosine-3',5'-cyclic phosphoric acid

The largest change in a UVRR spectrum of a mononucleotide due to a change in the furanose ring structure might be expected to occur when the attached furanose ring is held in a specific conformation, as is the case with the cyclic mononucleotides. Figure 4(a) shows the classical Raman spectrum of 3',5'-cyclic GMP (cGMP) in a crystal where the atomic coordinates have been determined by x-ray diffraction measurements.22 In this crystal, the cGMP is known to have a C-4'-exo-syn conformation, It is of interest to see from the Raman spectri if this conformation is conserved in solution. Figure 4(b) shows a comparison of the classical Raman spectra of cyclic 3',5'-GMP in solution (dotted line) with 5'-GMP (full line). The Raman spectra taken with visible excitation at 514.5 nm shows that the three peaks at 620, 635 and 645 cm-', which are considered to be caused by the C-4'-exo-syn conformer, remain unchanged on going from the crystal to solution. In the 660-700 cm-' region, however, in addition to the 677 and 690cm-' peaks, assignable to the C-4'-exo-syn-guanosine, other Raman lines around 682 and 698 cm-' overlap in solution. The latter two are ascribed to the C-4'-exo-anti-guanosine. The pair of frequency shifts, from 1319 to 1325cm-' and from 1360 to 1365 cm-', found on going from crystal to solution are compatible with a partial conformational change from C-4'-exo-syn to C-4'-exo-anti. A fairly strong Raman line is observed at 775 cm-' in solution, which is not far from the 761 cm-' line of the crystal. This line is considered to correspond to a main chain vibration characteristic of a conformation in which the torsional angles, beta and gamma, are given by the values p = 60", y = -60", which has been previously named as a type d main chain.'

Figure 4(c) and (d) show the same comparison but using UV excitation at 266 and 218nm. The 775 and 1083 cm-' main chain vibrations are not observed here. Both of the 677 and 635 cm-' bands, on the other hand, are observed with nearly the same relative intensities with 218 nm excitation as in the classical Raman spectra. This fact indicates that both the 677 and the 635 cm-' bands involve appreciable contributions from the guanine ring-breathing vibrations which derive their resonance intensities from the first and second strongly allowed electronic states of the guanine residue. The 3',


1600 1400 1203 1030 Bw 600

514 5 nm exc

514 5nrn exc

MP/cGMP E 266 nm erc

21anm 8 ° C

I GMP _r..o

, I , , < I 1600 1400 I200 1000 Bw 600

Wavenumber, cm-’

Figure 4. (a) Classical Raman spectrum of guanosine-3’,5-cyclic phosphoric acid (cGMP) in a crystal of known atomic coordinates.’’ (b) Classical Raman spectrum of cGMP in 0.1 M aqueous solution at ph7, and the classical Raman spectrum of guanosine-5’- monophosphoric acid (GMP) under similar conditions for comparison. (c) Ultraviolet resonance Raman spectrum (UVRRS) of a 5 mM aqueous solution (pH 7) of cGMP and that of GMP taken with 218 nm excitation. (e) UVRRS of a 1 mM (in nucleotide) solution at pH 7 of poly[d(GC).poly[d(GC)J at 0.5 M NaCl (6 form) and at 4 M NaCl ( Z form), taken with 266 nm excitation.

5’-cyclic-GMP concentration here is as low as 0.5 mM; nevertheless, strong peaks are observed at 1325 and 1365 cm-’, at exactly the same frequencies as those observed with 514.5 nm excitation for a concentrated (30%) solution. Therefore, the ratio of the number of molecules in the C-4’-exo-syn conformation to the number in the C-4-exo-anti conformation (which is about 3: 1) may be considered to be independent of the concentration. In addition to these differences observed in the 600-700 and 1300-1400cm-’ regions, a few new

spectral differences have been found between GMP and cGMP. The 817 cm-’ band, which is sharp in cGMP, is broad and centered about 820 cm-’ band, which is sharp in cGMP, is broad and centered about 820cm-’ in 5’-GMP. Similar changes are observed in the bands at 870, 1034, 1077 and 1326 cm-’. These differences seem to show that GMP in solution is a mixture of a number of conformers (mostly anti) which give rise to broad bands, while cGMP in solution has mainly (about two thirds) the C-4’-exo-syn conformation. Also these differences should reflect the difference between anti and syn conformations in the Raman spectra.

In the lowest part of Fig. 4, a UVRR spectrum of synthetic DNA with an alternating sequence of guanylic and cytidylic acids with 266 nm excitation is shown. This has previously been examined by Fodor and Spiro,” but we have carried out a re-examination in connection With the syn- and anti-guanosine problem now in question. It is known that poly[d(GC)]. poly[d(GC)] has the B form in 0.5 M NaCl solution (full line in Fig. 4(e)), whereas it has the Z form in 4 M NaCl solution (dotted line in Fig. 4(e)). The guanosine should have a 100% O-4’-endo-anti conformation in the former solution, whereas it should have 100% C-3’-endo-syn conformation in the latter. The Raman spectral feature^^-^ in the 1400-1300 and 700-600 cm-’ regions characteristic of these guanosine conformations are found to be preser- ved on going from 514.5 nm to 266nm excitation. It should be emphasized that in the 266 nm UVRR spectrum two sharp lines at 1318 and 624cm-’ are seen, which may be taken as good evidence of the occurrence of guanosine in the syn conformation.

Adenosine derivatives

Just as the positions and intensities of some guanine Raman bands reflect the guanosine conformation, it is expected that the positions and the intensities of some adenine lines will reflect the adenosine conformation. With this expectation in mind, we examined the UVRR spectra of adenosine-5‘-phosphoric acid (AMP), 2’- deoxyadenosine-5’-phosphoric acid (dAMP) and adenosine-3’,5’-cyclic phosphoric acid (CAMP). The results are shown in Figs 5 and 6. As can be seen in Fig. 5, the UVRR spectra of AMP and dAMP are very similar both for 266 and 218 nm excitation. Therefore, these two adenosine derivatives are considered to have essentially the same conformation in dilute neutral aqueous solution. A comparison of the AMP and cAMP spectra, on the other hand, reveals a number of appreciable differences (see Fig. 6). It is probable that these spectral differences are mostly caused by the difference in the conformation of these molecules which occur around the glycosidic bond. Because a cGMP crystal has a syn-guanosine conformation and this conformation seems to remain in solution, it is likely that cAMP has syn-adenosine in its solution. Therefore, it is suggested that the frequency differences 1310 vs 1303 cm-’ and 730 vs 725 cm-’ (see Fig. 6) are correlated with a conformational difference of adenosine, anti vesus syn.

It is interesting that such a spectrum-structure correla- tion is also suggested by the classical Raman spectra of the DNA oligomer, [d(CGCATGCG)], , reported by Benevides et u L * ~ The x-ray diffraction measurements

226 Y. NISHIMURA E T A L .

I 1 1 8 1 I 1 I I I I I :mn om I I-

266 nrn A M P / dAMP

N

f -

a exc.

218nm exc.

Wavenumber, cm-'

ure 5. Above: UVRR sDectrurn of an aaueous IDH 7. 5 rnMI so?ution of adenosine-5-monophosphoric acib (AMP)' and that o i 2'-deoxyadenosine-5'-~onophosphoric acid (dAMP), taken with 266 nm excitation. Below: UVRR spectrum of the same AMP and dAMP solutions taken with 218 nm excitation.

indicate that this octamer duplex involves C-3'-endo-syn- adenosine in the crystal, and its Raman spectrum indicates a strong scattering at about 1300 cm-'. Because the Raman spectra of [d(CGCGCG)], crystals (which contains syn-guanosine) do not show this 1300 cm-' band, this band must be assigned to the syn-adenosine. Unfortunately, this was not observed separated from the strong 1318 cm-' line of syn-guanosine, which also occurs in this [d(CGCATGCG)], crystal.

It is also interesting that by using the ultraviolet excita- tions, a number of AMPICAMP spectral differences are newly found (see Fig. 6). One of them, found in the 700-600 cm-' region, is remarkable. Here, the 633 cm-' line of anti-adenosine seems to be replaced by two lines at 665 and 610cm-' on going to syn-adenosine. Some

P li 266 nm

ex c

!I 2i8nrn

e x c

Wavenumber. cm- l

Figure 6. Above: UVRR spectrum of a pH7 aqueous solution of adenosine-3',5'-cyclic phosphoric acid (CAMP), and that of a pH 7 aqueous solution of adenosine-5'-monophosphoric acid (AMP) for comparison. Below: UVRR spectrum of the same CAMP and AMP solutions taken with 218 nm excitation.

of these spectral differences in the UVRR spectra may serve to detect the existence of a syn-adenosine conformation in a synthetic DNA, for example in the poly[d(GT)] poly[d(CA)] sequence, if syn-adenosine were to occur.

Acknowledgements

T h i s work was partly supported by the US-Japan Cooperative Science Program (1984-86) under the US National Science Foundation grant No. INT 8312052 and Japan Society for the Promotion o f Science, and also a grant No. 57060004 from the Ministry of Education, Culture and Science of Japan. USA NSF grant No. DMB-841799 and USA NIH grant No. GM 15547 provided partial support for instrumentation.

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ultraviolet resonance raman bands of guanosine and adenosine residues useful for the determination...

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