Eur. J. Biochem. 210,205-210 (1992) 0 FEBS 1992

Study of polynucleotide conformation by resolution-enhanced ultraviolet spectroscopy Poly(rC) and poly(dC)

Pere GARRIGA, David GARCIA-QUINTANA and Joan MANYOSA Unitat de Biofisica, Departament de Bioquimica i de Biologia Molecular, Facultat de Medicina, Universitat Autonoma de Barcelona, Bellaterra, Spain

(Received August 20,1992) - EJB 92 1200

Self-deconvolution and the fourth derivative of ultraviolet absorption spectra have been used to study stacked single-stranded and double-helix structures of different cytosine-containing poly- nucleotides for the first time. These compounds were studied under different solution conditions (pH and organic solvents) and at low temperatures. The red shift of the lower band (B2" band plus possibly some n + n* transition) of the absorption spectra in the cytosine-containing polynucleotides and the appearance of new peaks in the deconvoluted and derivative spectra in the 280-310 nm region are attributed mainly to cytosine-cytosine stacking interactions. In particular, the fourth-derivative peaks at wavelengths higher than 290 nm can be associated to coupling of electronic transitions of cytosine bases. The nature of the electronic transitions producing the absorption bands which are resolved in the aforementioned fourth-derivative peaks is discussed.

It is concluded that the resolution-enhancement techniques used in this work, i. e. self-deconvolu- tion and fourth derivative, complement each other and are useful methods to study structural changes of single-stranded and double-stranded polynucleotides allowing, at the same time, more information to be obtained about specific stacking interactions than classical absorption spectrophotometry.

Absorption spectrophotometry has been a classical tech- nique with important applications in the study of nucleic acid conformational transitions and interactions [l]. This tech- nique, however, yields valuable but limited information about these structural changes at the molecular level. Its main in- terest has been focused on the analysis of overall changes in stacking interactions as measured from the percentage of hyperchromicity or hypochromicity. The absorption spectra of nucleic acids in the 220 - 320-nm region are mainly due to the absorption of the nucleic-acid-base moieties. These spectra usually consist of broad bands resulting from the overlap of different individual electronic transitions. The high com- plexity of the absorption spectra from an electronic point of view is due to the low symmetry of the bases and also to the presence of non-bonded electrons [2]. This fact has prevented a detailed study of the different electronic contributions to the whole absorption spectrum of nucleic acids. In spite of these limitations, early studies on nucleic acid bases, on nucleosides and their derivatives [3 - 51 gave some interesting resolution in spectral fine structure, particularly those using magnetic circular dichroism [6].

An attempt has been made to analyze the individual elec- tronic contributions to the absorption spectra of homopoly-

Correspondence to J. Manyosa, Unitat de Biofisica, Departament de Bioquimica i de Biologia Molecular, Universitat Autbnoma de Barcelona, E-08193 Bellaterra, Catalonia, Spain

Abbreviations. Me,P, trimethylphosphate; HWHH, half-width at half-height.

nucleotides under different solution conditions. The strategy applied to overcome the aforementioned difficulties has been the study of model compounds (bases, nucleotides, etc.) and the use of resolution-enhancement techniques, i. e. self- deconvolution and fourth-derivative spectrophotometry.

Fourth-derivative spectrophotometry has been success- fully applied to the study of conformational properties of proteins [7]. Recently, we have applied this technique to inves- tigate conformational transitions of both poly[d(A-T)] . poly[d(A-T)] and poly[d(n2A-T)] . poly[d(n2A-T)] (n, amino) [8] and also to study the effect of 9-aminoacridine on the BjZ transition of poly[d(A-T)] . poly[d(A-T)] [9]. The fourth-derivative technique allows the partial resolution of electronic (n + n* and rc --f z*) and vibrational bands which otherwise would be hidden in the broader absorption spectra. Thinner bands are favored compared to broader bands since the fourth-derivative peak intensity is proportional to the inverse of the fourth power of the bandwith at half-height [lo]. In addition, low temperatures have been used to enhance the visual features of spectra obtained at room temperature, gaining new insight into the behavior of the polynucleotides under such conditions. Taking all these facts into account, a detailed analysis of the electronic features of the absorption spectra of poly(rC) and poly(dC) has been carried out. This has allowed us to associate some of the fourth-derivative peaks in the 280 - 310-nm region to cytosine-cytosine stacking inter- actions. The fourth-derivative technique is thus shown to be a useful technique to detect and follow transitions between

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nucleic acid conformations. Furthermore, deconvoluted spectra confirm that fourth-derivative peaks observed actually correspond to component bands of the absorption spectra and hence they are not artifactual. The assignment of the derivative peaks in the long-wavelength region (280 - 310 nm) has been tentatively attempted using different solution conditions (pH and organic solvents). The nature of the electronic transitions that give rise to these characteristic fourth-derivative peaks is discussed.

MATERIALS AND METHODS

Chemicals and experimental procedures

Cytosine, cytidine, 2'-deoxycytidine, poly(rC) and poly- (dC) were purchased from P-L. Biochemicals and used with- out further purification. All other chemicals used [trimethyl- phosphate (Me,P), MgC12, glycerol] were of reagent grade.

Ultraviolet absorption spectra were recorded on a 320 Perkin-Elmer spectrophotometer interfaced to a micro- computer to allow posterior treatment of the spectra. The absorption of the polymer solutions was measured in 1-cm pathlength cells placed in a thermostated holder.

The low-temperature experiments were performed using an Oxford DN 1704 cryostat connected to an Oxford DTC 2 temperature controller. The samples used in this case contained 70% glycerol to avoid crystallization and were ex- tensively degassed. The temperature was slowly lowered (about 2"C/min) in order to avoid the formation of cracks. Concentration of the samples before the addition of glycerol was calculated to obtain a final absorbance similar to that of the room-termperature samples.

Fourth-derivative spectroscopy

The use of the techniques applied in this work is rapidly expanding, mainly in the determination of structural proper- ties of proteins [ll]. The fourth-derivative technique used in this work is based on an iterative least-squares program de- veloped by Savitzky and Golay [12]. The first and the follow- ing orders of derivation of an absorption spectrum are obtained using a mathematical method. The main operation of this procedure is to obtain (as many times as indicated by the derivation order) the difference between the absorption of a sample at a certain wavelength A ( A ) and that of the same sample at a wavelength shifted a finite interval ( A i , a differencing interval), A ( i + A i ) . The difference value obtained is assigned to the mid-point between A ( i ) and A ( i + Ai l ) , that is at ( A + 442) . For Gaussian bands, the intensity of the nth derivative is inversely proportional to the nth power of the band-width [lo]. This is the reason why derivative spectrophotometry favors narrow bands to the det- riment of broader bands.

On a theoretical basis, an increase in the derivation order should result in an increase in the resolution of the spectral absorption bands. Although this is essentially true, there is also a concomitant signal/noise ratio decrease. The best compromise between these two parameters is obtained in the case of the fourth derivative, which presents the advantage that the maxima of the fourth-derivative peaks coincide with those of the absorption spectra [7].

In the present study, the fourth-derivative spectra were obtained with a BASIC program developed in our laboratory, based on the Savitzky and Golay's algorithm [12]. The absorp- tion spectra were the result of four or eight scans, coadded in

order to increase the signal/noise ratio, depending on the concentration of the sample. These spectra were previously smoothed, with interval 1 and group of points 6, and after- wards derivated with interval 3 and group of points 11 [12].

Fourier self-deconvolution technique

Fourier self-deconvolution is a mathematical procedure that uses Fourier transforms as means of spectral-data pro- cessing to attain band narrowing [13]. The main problem encountered in spectra of condensed-phase samples is band overlap. The width of individual absorption bands is often greater than the separation between neighboring bands. In this situation, increasing the instrumental resolution does not achieve band separation and in order to 'resolve' such band contours into their individual components, one must use other mathematical methods like Fourier self-deconvolution [ 141.

The basic principles of the Fourier self-deconvolution method have been previously published [13 - 151. Two param- eters play an important role in this method. These are y (half- width at half-height; HWHH) and K , the resolution enhance- ment factor. The latter parameter can be defined as K = y/y' where y is the original HWHH and y' is the HWHH obtained after the band-narrowing procedure. Thus, an increase in K would result in an increased resolution. However, the self- deconvolution method also enhances the noise in the spec- trum. This limits the maximal value that can be attained by K [14]. Also, the need for spectra with high signal/noise ratios has to be emphasized in order to extract the maximum infor- mation from experimental data.

The successful application of the method relies basically on the careful choice of the two aforementioned parameters. This is important in order to avoid overdeconvolution or to avoid a decrease in the signal/noise ratio which may eventually lead to the appearance of artifactual bands not present in the original absorption spectrum.

In the present work, several combinations of the two pa- rameters have been tried. The parameters found that produced the best resolution and the less distortion of the original spectra were y = 10 and K = 2.5. The deconvolution procedure was carried out, in practice, using the programs of Moffatt et al. [16].

Thus, a more detailed analysis of absorption spectra has been carried out, taking advantage of the enhancement power of these techniques. It must be noted, however, that these techniques do not increase the intrinsic nominal resolution achieved with the spectrophotometer, but make evident over- lapping component bands, hidden under the broad envelope of the absorption band centered at approximately 260 nm, thus improving the visual features of the spectra.

RESULTS

Cytosine monomer

Absorption, deconvoluted and fourth-derivative spectra of cytosine at neutral pH and low ionic strength, are presented in Fig. 1. The wide band of cytosine absorption spectrum with a maximum at 267 nm (BZu band) is resolved in the derivative spectrum in five peaks of different intensities at 281,274,267, 263 and 257 nm.

The position of the Bzu band can be modified by changing the solution conditions [17- 191. Acidification of a cytosine solution to pH 1 .O results in an 8-nm red shift of the absorp- tion maximum and also in the modification of the position

207

240 260 280 300 320 Wavelength (nrn)

Fig. 1. Absorption and resolution-enhanced spectra of neutral cytosine. Cytosine in 50 mM Tris/HCl, pH 7.4. Temperature, 20°C. Upper trace, fourth-derivative spectra (-); lower trace, absorption (-) and deconvoluted (---) spectra.

x TI \ Q 77

W 0 C

In 4

0

4 U \ 4 D

240 260 280 300 320

Wave leng th (nrn)

Fig. 2. Absorption and resolution-enhanced spectra of cytosine at acid pH and in Me3P. (A) Cytosine at pH 1.0. (B) Cytosine in Me3P solution. All spectra at 20 “C. Upper trace, fourth-derivative spectra (-); lower trace, absorption (-) and deconvoluted (---) spectra.

and the ratio between the intensities of the three major peaks in the derivative spectra, which are red shifted from 281, 274 and 267 nm to 291, 284 and 277 nm, respectively (Fig. 2A). Protonation of cytosine occurs at the endocyclic N3 of the molecule and the observed red shift emerges as a result of the large effect on the energy and the shape of orbitals of a molecule upon formation of ions [20]. In fact, the main elec- tronic transitions accounting for Bzu absorption (n + n* and .n + .n* electronic transitions) must be profoundly affected by protonation [20]. Similar changes can be observed in Me3P solution (Fig. 2B). In the latter case, the derivative spectrum shows three major peaks at 293.5, 284 and 276 nm, but with an intensity about seven-times lower than the corresponding peaks in the neutral cytosine spectrum. This intensity decrease can be interpreted in terms of a broadening of the components of the long-wavelength band in Me3P. Similar results are

240 260 280 300 320

Wave l e n g t h (nrn)

Fig. 3. Absorption and resolution-enhanced spectra of 2’-deoxycytidine at neutral and acid pH. (A) 2’-Deoxycytidine at pH 7.4. (B) 2‘- Deoxycytidine at pH 2.5. All spectra at 20°C. Upper trace, fourth- derivative spectra (-); lower trace, absorption (-) and deconvoluted (---) spectra.

obtained if the solvent used is dioxane. It is known that the principal factor in determining the position and the intensities of the first-absorption bands in cytosine is hydrogen bonding [17]. Thus, in the case of cytosine, thc changes in the position and intensity of the fourth-derivative peaks in the vicinity of the 290-nm long-wavelength region can be mainly associated to the lack of hydrogen bonding between cytosine and the organic solvent in comparison with cytosine in aqueous solu- tion.

The analysis of the long-wavelength region in the deriva- tive spectra is interesting in view of its sensitivity to local environment and to changes in the electronic features of the cytosine chromophore. This fact is being used in the structural study of single-stranded and double-stranded polynucleotides.

Cytidine and 2’-deoxycitidine

It has been shown, in the case of the cytosine monomer, that the resolution-enhanced techniques provide a finer reso- lution than absorption spectrophotometry. In particular, the fourth-derivative peaks are sensitive to changes in the environ- mental conditions of the cytosine chromophore. However, the aim of this present study is to analyze spectra of polynucleo- tides. In this respect, it has to be taken into account that nucleosides and nucleotides are more appropriate as models for polynucleotides.

Absorption, fourth-derivative and deconvoluted spectra of 2’-deoxycytydine, at neutral and acid pH, are shown in Fig. 3. Spectra of cytydine are virtually indistinguishable (not shown). The absorption spectrum of 2‘-deoxycytidine at neu- tral pH (Fig. 3 A), shows the characteristic absorption maximum at 271 nm, corresponding to the BZu transition, and a band at about 230 nm associated previously to an n + n* transition [5]. The corresponding fourth-derivative spectrum shows main peaks at 275 nm and 285 nm. At pH 2.5, absorp- tion and resolved spectra undergo important changes

208

I -

A

\

220 240 260 280 300 320

Wavelength (nrn)

Fig. 4. Absorption and fourth-derivative spectra of poly(rC) and poly(dC). (A) Absorption spectra of poly(rC) (-) in 50 mM Tris/ HCl, pH 7.4, and of poly(dC) (---) in the same buffer but at pH 9.0 (to prevent the formation of the self-complex). Temperature, 20 "C. (B) Fourth-derivative spectra of poly(rC) (-) and poly(dC) (---) from the absorption spectra in (A). Temperature, 20°C.

(Fig. 3B). The absorption maximum of the spectrum of the acid form displays the same behavior as cytidine [5], that is a red shift to about 280 nm and hyperchromism with regard to the neutral form, while the band at about 230 nm disappears. The fourth-derivative spectrum shows main peaks at 282 nm and 295 nm, arising from the shift of the BzU band in unprotonated 2'-deoxycytidine. The spectral changes ob- served upon protonation of 2'-deoxycytidine (which are the same as those of cytidine) are similar to those observed in the case of cytosine. However, protonation of the deoxynucleotide leads to bands red shifted 3 - 4 nm with respect to those of cytosine.

Poly(rC) and poly(dC) in unprotonated form The absorption spectrum of poly(rC) and poly(dC) have

the same overall features with maxima at about 269 ~ 270 nm (Fig. 4A) but important differences appear in the correspond- ing derivative spectra; poly(dC) shows a main peak at 286 nm whereas poly(rC) shows two main peaks, at 288 nm and 269 nm respectively (Fig. 4B). The analysis of these spectral differences has special interest due to its known different struc- ture in solution [21, 221. Despite the close similarity of their structural constitutions [they only differ in the absence of the 02' atom in the deoxyribose moiety of poly(dC)] no clear evidence has been found that can account for their different stacking patterns. At neutral pH and low ionic strength, poly(rC) has been proposed to be a monohelix structure stabilized by stacking interactions between bases, whereas poly(dC) seems devoid of an ordered structure [22]. It is interesting to note here that the long-wavelength band of both polynucleotides is red shifted about 6 - 8 nm with respect to cytosine, and about 3 -4 nm with respect to cytidine and 2'- deoxycytidine, under similar solution conditions, and so are the corresponding fourth-derivative peaks appearing in this region.

220 240 260 280 300 320 Wavelength (nm)

Fig. 5. Fourth-derivative spectra of poly(dC) at neutral and acidic pH. The polynucleotide in 50 mM Tris/HCl, at pH 9.0 (-, single- stranded form) and at pH 5 (---, self-complex). Temperature, 20°C.

Poly(rC) and poly(dC) in protonated form

The techniques used in this work are useful in monitoring conformational changes of polynucleotides, like those caused by changes in pH. In our case, we have been able to follow the formation of the protonated self-complexes of poly(rC) and poly(dC) obtaining pK values that are in good agreement with those previously published [23-251. Fig. 5 shows the derivative spectra of poly(dC) at pH 9, where it is assumed to be single stranded, and at acid pH where it has adopted the form of the self-complex poly(dC+ . dC) [22, 251. Interest- ingly, the spectrum at acid pH shows two new peaks at 295 nm and at 306 nm respectively. Similar results are obtained in the case of poly(rC). When the polymer is heated to 90"C, the 306-nm peak virtually disappears, while the intensity of the peak at 295 nm is importantly reduced (not shown), in an analogous way as that previously observed upon melting of other synthetic polynucleotides [8]. It has to be noted that the peak at 295 nm does not disappear completely due to the fact that, in addition to stacking interactions, it is reflecting the protonated state of the 2'-deoxycytidine moiety, as can be seen in Fig. 3 B. In view of its behavior at high temperature, we propose that the peaks in this long-wavelength region to be related to the stacking of adjacent C . C + base pairs as stated by Gray et al. using CD spectroscopy [26, 271. These authors reported an important increase in the positive CD signal at wavelengths as long as 290 - 3 10 nm (where the ab- sorption is dominated by cytosine) upon acidification of cytosine-containing polymers [26, 271.

Poly(rC) and poly(dC) at 153 K

Fig. 6 shows spectra of poly(rC) and poly(dC) at 153 K. Important differences are observed between their correspond- ing derivative spectra. The more striking feature is the im- portant increase of the peaks in the derivative spectrum of poly(rC) respective to those of poly(dC). In fact, a certain increase in the derivative peaks is to be expected when lowering the temperature, arising from the narrowing of the absorption bands, which reflects a reduction of the possible vibrational states of a given electronic transition. However, taking into account that the electronic states of both homopolymers, i. e. poly(rC) and poly(dC), must be very similar, the important difference observed between the derivative spectra of both polynucleotides at 153 K can be attributed, not only to the above mentioned narrowing of the absorption component bands of the whole spectrum, but also to the different stacking

209

I

220 240 260 280 300 320 Wave length (nm)

Fig. 6. Fourth-derivative spectra of poly(rC) and poly(dC) at low tem- perature. Poly(rC) (---) and poly(dC) (-) in the same conditions as for Fig. 3 in 70% glycerol solution at the temperature of 153 K.

lo-

can be seen. It is noteworthy that there is a linear relationship between the intensity of the 269-nm derivative peak and tem- perature. This would reflect the effect of the temperature alone upon the vibrational features of the absorption spectra. The particular behavior of the peak at 290 nm would be related, in turn, not only to the effect of temperature but to a change in the stacking interactions present in the polynucleotide helix. Thus, our derivative spectra can be interpreted as an increase in the cytosine-cytosine interactions of poly(rC) under low- temperature conditions, provided that it has a somewhat ordered helical structure (this arrangement would allow exten- sive stacking of the cytosine base residues) and that stacking interactions are assumed to strengthen when the temperature is lowered. The absence of such a definite increase in the fourth-derivative peaks of the poly(dC) derivative spectrum would reflect a structureless conformation devoid of the afore- mentioned interactions acting in the case of the homoribo- polymer counterpart.

DISCUSS I 0 N

140 180 220 260 300 Temperoture ( K )

Fig. 7. Plot of the heights of fourth-derivative peaks in poly(rC) spectrum versus temperature. The differential effect of temperature on the inten- sity of the fourth-derivative peaks located at 290 nm and 269 nm in the poly(rC) spectrum (see Fig. 6) is shown.

and/or hydrogen-bonding patterns in solution. In the case of poly(dC), a remarkable feature of its derivative spectrum at 153 K is the appearance of weak peaks at 295 nm and 306 nm, analogous to those obtained in the self-complex spectrum at acidic pH (see Fig. 5). The appearance of these peaks, attri- buted previously to stacking interactions of adjacent C . C + in the self-complex, suggests that poly(dC) at low temperature, although in an overall structureless conformation, undergoes a slight increase in the base-base interactions but to a lesser extent than poly(rC).

Regarding the properties of the solution at low tempera- tures, it has to be noted that the dielectric constant is lowered in 70% glycerol/water solutions at room temperature. How- ever, the lowering of the temperature produces the recovery of the dielectric constant value [28]. Taking this into account, the pK values and the state of the ionizable groups are not expected to be significantly affected by the low temperature. Furthermore, the proton activity of the glycerol solutions at 153 K in Tris buffer can be significantly increased, possibly over 10, as extrapolated from Douzou [28]. Thus, in this situation, poly(dC) should not be protonated and therefore it cannot form a weak hemiprotonated duplex, taking into account that in aqueous solution poly(dC) has a pK of 7.2 (ionic strength independent) [24].

Fig. 7 shows the plot of the heights, normalized with re- gard to molar concentration of the sample, of the fourth- derivative peaks at 290 nm (corresponding to that at 288 nm in the absence of glycerol) and 269 nm, in the spectra of poly(rC) (see Fig. 6) versus temperature, where a particular behavior of the peak at 290 nm, with regard to that at 269 nm,

It has been shown that the mathematical procedures used in this work (deconvolution and differentiation) allow a more detailed analysis of the changes in the absorption spectra of polynucleotides as a result of modification of the solution conditions. The aim of this study has been to gain new insight in the structural features of polynucleotides undergoing con- formational transitions as a result of changes in the solution conditions. The techniques used have been applied to the study of the structural differences between poly(rC) and poly(dC). This has been accomplished with a progressive ap- proach starting from the study of cytosine, cytidine and 2’- deoxycytidine under different solution conditions.

The derivative spectra are shown to be more sensitive to the solution conditions, i. e. pH and organic solvents, than absorption spectra, thus allowing a more detailed visualiza- tion of the component bands which are overlapped in the absorption spectra. This study of the different homopoly- nucleotides has allowed the analysis of the formation of acidic self-complexes and to relate some of the fourth-derivative peaks observed to stacking interactions.

At present, two models coexist regarding the structure of neutral poly(rC) in solution. The first one proposed by Arnott et al. [29] postulated that the poly(rC) secondary structure is that of a right-handed RNA helix with extensive stacking of the cytosine-base residues. Later, however, Broido and Kearns [30] proposed a left-handed helix for the polymer, hydrogen bonded on the outside and with characteristic amino-carbonyl hydrogen bonds, using NMR spectroscopy. In this latter case, the bases are arranged with a very favorable head-to-tail align- ment of the dipoles. Recently, Annamalai and Keiderling [31] have not been able to distinguish between these two proposed poly(rC) structures in their vibrational circular dichroism study. Our derivative spectra are compatible with an increase in the stacking interactions in poly(rC) under low-temperature conditions, since stacking interactions are assumed to strengthen when the temperature is lowered, as stated above. It is interesting to note the particular behavior of the derivative spectra of neutral poly(rC) (Fig. 3B), when cooled down to 153 K (see Fig. 6), that is a large increase in the intensity of the peaks in the 270 - 300-nm region, particularly that at 290 nm in poly(rC); whereas poly(dC) (Fig. 3 B) undergoes less important changes in the intensities of its derivative peaks (see Fig. 6).

210

Let us now address the question of the assignment of the bands in the 270-310-nm region to iz+n* or to n+n* electronic transitions. Much work has been devoted to the analysis of the electronic spectra of the nucleic acid bases (see [32] and references therein). n + IT* electronic transitions have been postulated in a great variety of cases [20, 33, 341. These kind of electronic transitions would be very difficult to detect in the absorption spectra due to their intrinsically low intensity and would be lost among the more intense n + n* transitions [32]. Thus, Zadoulek et al. [35] observed no compelling evi- dence for an n+n* transition at wavelength longer than 225 nm in cytosine. However, Matos and Roos [36] questioned this interpretation and did not discard the contribution of the n + n* character in the region over 225 nm. Rich and Kasha [37] had proposed an n + n* transition in the 280-nm region of neutral poly(rC) and that a head-to-tail alignment of tran- sition moments should produce hyperchromism, this being the arrangement of the dipoles corresponding to the n + n* electronic transitions in this polynucleotide. We think that in some cases, taking advantage of the resolving power of the derivative technique (which evidentiates almost inappreciable shoulders in the absorption spectrum), these weak transitions could be observed or at least partially detected when coupled to the more intense n + n* bands. Thus, the 288-nm peak of neutral poly(rC) (not observed either in the case of cytosine or in that of cytidine and therefore associated to the polymeric structure) could be tentatively related to coupling of n + n* transitions associated to the C = 0 groups of cytosines, this being favored by a head-to-tail alignment of transition mo- ments.

In summary, the resolution-enhancement procedures used in this work allow the detailed study of polynucleotide struc- ture giving further information than classical absorption spectrophotometry. The low-temperature studies accentuate the already-resolved features in the derivative and decon- voluted spectra, allowing, at the same time, one to gain new insight in the behavior of polynucleotides at low temperature. It has been shown that the fourth-derivative peaks in the 270 - 31 0-nm region of cytosine-containing polynucleotides are mainly attributed to stacking interactions of cytosine resi- dues. In particular, some of the peaks in this long-wavelength region are attributed to coupling of carbonyl transition mo- ments of cytosines. This conclusion has been achieved from the study of cytosine, cytidine and the poly(rC) and poly(dC) homopolynucleotides as model compounds.

It has been demonstrated that fourth-derivative spectro- photometry, combined to self-deconvolution, opens new possibilities in the detailed analysis ofpolynucleotide structure as a result of changes in solution conditions. The application of these techniques to the study of different double-stranded polynucleotides is an interesting approach which can provide useful information about polynucleotides undergoing confor- mational transitions. In particular, the application of these techniques to different Z-DNA-forming polymers will be the subject of a posterior study to be published.

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