Raman-spectral studies of nucleic acids XVII. Conformational structures of polyinosinic acid

volume 4 Number 7 July 1977 Nucleic Acids Research Raman-spectral studies of nucleic acids X V I I . Conformational structures of polyinosinic acid C. H. Chou, G. J. Thomas, Jr., Struther Arnott*, and P. J. Campbell Smith* Department of Chemistry, Southeastern Massachusetts University, North Dartmouth, MA 02747 and * Department of Biological Sciences, Purdue University, West Lafayette, IN 47907, USA Received 6 May 1977 Laser-Raman spectra of poly(rl) show the formation of an ordered complex in aqueous solutions of high ionic strength. This structure exhibits the A-helix geometry, contains stacked bases and is apparently stabilized by specific hydrogen bonding involving hypoxanthine C6=0 groups. Thermal dissociation of the poly(rl) complex (Tm=45°C) yields single-stranded and disordered poly(rl) chains. A disordered structure also occurs for poly(rl) in aqueous solutions of low ionic strength. In oriented films, poly (rI) forms an ordered structure probably the same as that which occurs in solutions of high ionic strength. Raman intensities measured at 815 and 1100 cm'l in spectra of poly(rl) and poly(rll)-poly(rA)•poly(rU) indicate that the correlation previously established for single- and double-stranded ribopolymer structures is valid also for these multi-stranded structures. X-ray diffraction and model-building studies confirm the A-helix structure. INTRODUCTION Polyinosinic acid [poly(rl)] associates to form a multistranded complex, the structure of which has been investigated by various techniques. The first X-ray diffraction data*, obtained from fibers of poly(rI), led to the proposal that the ordered molecular structure was probably a triple-stranded and right-handed helix stabilized by hydrogen bonding between C6=0 and Nl-H groups of the hypoxanthine bases in adjacent strands (Fig. la). Later independent X-ray diffraction and molecular model-building studies by Arnott et al.2 and by Zimmerman et al.3 led to the conclusion that the complex contains four righthanded helical chains. important respect's. However the models differed in two In the Arnott et al.2 model the sugar rings are puckered C2'-endo (i.e. the conformation is of the B-type) and individual bases in the hydrogen-bonded quartet are not perpendicular to the common helix axis. In the Zimmerman et al. © Information Retrieval Limited 1 Falconberg Court London W1V5FG England 2407 ABSTRACT Nucleic Acids Research model the sugar rings are C3'-endo (i.e. A-type) and the quartet of bases share a common plane perpendicular to the helix axis. In both models G can replace I and additional N2-H bonds formed. N7 hydrogen Polyriboguanylic acid is therefore isostructural with poly(rl). 5 Aqueous solutions of poly(rl) have been studied by optical rotatory dispersion (ORD) 6 and circular dichroism (CD) techniques. 6,7 j n the earlier spectroscopic study^ the data were interpreted in terms of a structure with left-handed helical chains. Cech and Tinoco 7 however were able to show that the obtained with right-handed helices provided that the individual bases are, like the Arnott et al.^ model, not perpendicular to the helix axis. Nevertheless the ORD and CD results do indicate that the multi-stranded complex of poly(rl) is stable in solutions of high ionic strength (0.1 M NaCl). A single-stranded and "poorly stacked" structure was proposed for poly(rl) in aqueous solutions containing less than 0.1 M NaCl. 6 Infrared** and Raman spectroscopy9>10 have been employed previously to identify the keto tautomeric structure of hypoxanthine and to demonstrate the stacking of hypoxanthine bases in poly(rl) and related model compounds. Of the techniques employed to study aqueous poly(rl), Raman spectroscopy would appear to offer the greatest promise for distinguishing A and B-helix geometries as well as to detect base stacking and hydrogen bonding interactions.H Raman data of both aqueous and solid samples may also be compared to determine whether conformational properties of poly(rl) are the same or different in the two states. addressed in this communication. These and similar questions are We also report the Raman spectrum of a triple-stranded and right-handed polyribonucleotide complex, viz. poly(rU)•poly(rA)-poly(rU), to show that multistrandedness of itself does not invalidate the conformational correlations established previously for double-stranded ribopolymer complexes. i2 .13 MATERIALS AND METHODS Details of the preparations of films of poly(rl) and fibers 2408 characteristic negative circular dichroism of poly(rl) is Nucleic Acids Research of poly(rU)'poly(rA)«poly(rU) are discussed elsewhere. 2 . 14 In all samples, the polynucleotide molecules were oriented more or less unidirectionally so that the helical axes were roughly parallel to one another along a known direction of the film or along the fiber axis. Samples were deuterated by storage for several days in a sealed hygrostatic cell (751 relative humidity) which contained a saturated solution of NaC10 3 in D2O.I 5 After spectral determinations of films or fibers were completed the samples were dissolved to about 40 ug/yl Na2SO^ was added to solutions standard. Salt concentration was adjusted in the range 0 to 1.5 M by direct addition of NaCl to the solutions. Spectra were recorded in the temperature range 0 to 80°C by use of a thermostatically controlled cell, described previously. 1 ^ Solution pH (or pD) was always within the range 6.8 ±. 0.4. The Raman instrumentation used in this work consists of a Coherent Model CR2 argon-ion laser and Spex Ramalog spectrometer. Further details are given elsewhere.10»12,16 Spectra of solids were excited with approximately 200 milliwatts of 514.5 nm radiation and of solutions with 400 mW of 488.0 nm radiation. Figure 1: Hydrogen-bonding schemes proposed in (a) the triplestranded model of poly(rl), ref. 1, and (b) the four-stranded models of poly(rl), refs. 2 and 3. Possible differences in furanose ring puckering are not shown. (N and 0 atoms are indicated by filled and open circles; see also refs. 1-5). 2409 in either H£O or D2O, as required. for use of its 980 cm~l line as a Raman intensity and frequency Nucleic Acids Research RESULTS AND DISCUSSION 1. Poly(rl). Raman spectra of non-deuterated and deuterated films of poly(rl) are shown in Fig. 2. Raman lines in the region 750-850 cm'l, which are due to vibrations of the phosphodiester backbone, are informative of the polynucleotide chain conformation.H Thus the lines of comparable intensity near 815 and 795 cm~l in each spectrum of Fig. 2 show that the film of poly(rl) consists of a mixture of roughly equal amounts of ordered (A-helix) and Figure 2: Raman spectra of (a) non-deuterated and (b) deuterated films of polyCrl). In both (a) and (b), the incident beam was at 45° to tke plane of the film and the scattered radiation was collected at 90° to the incident beam direction. Raman frequencies of the prominent (...truncated)