Dependence of purine 8C-H exchange on nucleic acid conformation and base-pairing geometry: a dynamic probe of DNA and RNA secondary structures.

Abstract

AbstractLaser Raman spectroscopy is employed as a probe of hydrogen isotope exchange in nucleic acids exhibiting different secondary structures. The rates for deuterium exchange of 8C‐H groups in adenine (A), hypoxanthine (I), and guanine (G) residues of ribo‐ and deoxyribopolynucleotides are compared with corresponding rates of mononucleotides. In general, nucleic acid secondary structure significantly retards the rate of purine 8C‐H exchange. Specifically, the exchange kinetics are strongly dependent on both the kind and amount of secondary structure. The retardation factor (R), defined as the quotient of rate constants for monomer and polymer exchanges, is greatest for the A‐helix (9.5 ± 1), intermediate for the B‐helix (2.8 ± 0.6), and smallest for the Z‐helix (1.5), thus permitting the three authenticated DNA structures to be distinguished from one another using the Raman dynamic probe. Polyribonucleotide complexes of the A‐helix family that contain the same backbone conformation and ribosyl pucker (C3′ ‐ endo/anti) but that differ in the geometry of base pairing or number of helix strands are also clearly distinguished by their different deuterium exchange rates. The extraordinarily large retardation of 8C‐H exchange (R > 200) that occurs in multihelical structures is attributed to hydrogen bonding by the purine 7N acceptor. These results indicate that 8C‐H exchange may be exploited to detect Hoogsteen base pairing and possibly interactions of nucleic acid‐binding proteins that involve the 7N site as hydrogen‐bond acceptor. The feasibility of the method for evaluation of solvent penetration to encapsidated genomes of DNA and RNA viruses is considered. The present results also reveal a number of new vibrational band assignments for identification of DNA and RNA secondary structures from equilibrium Raman spectra.