Nucleic Acids Research

Proton-NMR has been used to determine the activation energies and pre-exponential factors for the deuterium exchange of AH8 in poly(dA-dT)-poly(dA-dT). and for GH8 in poly(dG-dC)*poly(dG-dC). No simple relationship between the kinetic parameters and molecular conformation was found. By addition of 4.5 M NaCl a transition from the B to the Z conformation was Induced for poly(dG-dC)'poly(dG-dC). and an increased exchange rate was observed. The exchange rate for poly(dA-dT)'poly(dA-dT) also increased below 64°C. and a significant decrease in activation energy on addition of 4.5 M NaCl was observed. The exchange rates at T = 55.8°C were also measured for the AH8 and GH8 in random sequence calf thymus DNA. From the difference in exchange rates, a method of preferential labeling of either the AH8 or the GH8 in high molecular weight DNA is evaluated. INTRODUCTION We have recently started experiments aimed at an understanding of the dynamics and orientation of the bases in oriented solid calf thymus DNA using deuterium NMR (1). By heating DNA in a DgO solution at ~65°C the adenine H8 (AH8) and the guanine H8(GH8) exchange with deuterium. This has previously been observed for mononucleotides (2) and polynucleotides (3). Typically, the ratio of exchange rates between the GH8 and the AH8 has been found to range from 1.1 to 11.6 at T = 50°C depending on polynucleotide sequence and conformation (3). In this study, the effects of external factors that Influence this ratio of exhange rates are investigated. The optimum conditions could then be found for preferential deuteratlon of either the adenine or the guanine bases in high molecular weight, random sequence calf thymus DNA. Selective labeling enables the studies © IR L Press Limited, Oxford, England. 9 4 9 1 Nucleic Acids Research of the dynamics and orientation of these bases independently. It was decided first to study the effects of salt concentration and temperature on the exchange rates of the GH8 and AH8 In the polynucleotides poly(dG-dC)'poly(dG-dC) and poly(dA-dT)•poly(dA-dT). respectively. The poly(dG-dC)-poly(dG-dC) sample has been shown to undergo a salt Induced transition from the B-form to the Z-form (4). accompanied by an increase in the GH8 exchange rate (3). No such transition has been observed for poly(dA-dT)-poly(dA-dT), and one might therefore expect the exchange rate to change less on addition of large amounts of NaCl. The exchange rates from these two polynucleotides will then be compared with the rates in the heterogeneous calf thymus DNA sample (measured only at T = 55.8°C). Proton NMR was used to monitor the exchange since it can follow the Intensity changes of each of the exchanging protons in the two bases simultaneously. EXPERIMENTAL Materials The polynucleotides were purchased from P-L Biochemicals: poly(dA-dT)-poly(dA-dT) lot # 782-38 and poly(dG-dC)'poly(dG-dC) lot It 317910. Calf thymua DNA was purchased from Worthington (lot tt W33B996) and was determined to contain ~1.IX protein by a modified method of Lowry (5). Each sample was dissolved in a buffer (1.0 H NaCl. 0.04 M cacodylic acid. 0.01 M EDTA. pH = 7.0). and sonicated with a Branson sonicator (model W-350) to reduce the molecular weight. The DNA sizes after sonlcatlon were determined by electrophoresls on a vertical polyacrylamlde gel. Using PBR322 (Haelll digest) as standard, the average lengths of poly(dA-dT)-poly(dA-dT). poly(dG-dC)>poly(dG-dC) and calf thymus DNA were found to be 100+50 base pairs (~80X of material). After sonlcatlon. ethanol was used to precipitate the DNA. The samples were finally dissolved in a D_0 buffer (0.1 M NaCl or 4.5 M NaCl. 0.004 M cacodylic acid. 0.001 M EDTA. pD = 7.0). and kept at 4°C. All samples showed normal melting behavior when heated up to 95 C. The lowest melting temperature was found to be 66.5°C