Yau-Kwan Ho

POLYNUCLEOTIDES CONTAINING 5‐MERCAPTO‐SUBSTITUTED PYRIMIDINES: INHIBITION OF VIRAL DNA POLYMERASES AND THE BIOLOGICAL IMPLICATION*

Partially thiolated polycytidylic acids MPC I-III, containing 1.7%, 3.5% and 8.6% 5-mercaptocytidylate units, respectively) inhibited the DNA polymerase of Friend leukemia virus (FLV) in the endogenic reaction as well as in the presence of poly(A)-(dT)14 or poly[d(a-T)] templates; the inhibitory activities were directly related to the percent of thiolation. Various partially thiolated RNA and DNA isolates from Ehrlich ascites cells (containing one 5-mercaptopyrimidine nucleotide/50-100 nucleotide units) also inhibited the DNA polymerases of FLV in the endogenic reaction, and also in the presence of the synthetic templates. The thiolated DNA was the most active, but the thiolated tRNA also showed substantial inhibitory effects, while the thiolated ribosomal RNA was less effective. In a bacterial DNA polymerase (E. coli-K12, using denatured DNA as template), MPC I-III showed no activity. By contrast, MPC III and several partially thiolated nucleic acid isolates significantly inhibited a regenerating rat liver DNA polymerase (I) system; among those tested, the thiolated DNA from Ehrlich ascites cells showed the highest activity. Kinetic analysis of the inhibitory action of this thiolated DNA in the rat liver enzyme system, using as template the corresponding unmodified DNA, demonstrated that the thiolated DNA acts as a competitive inhibitor of the template, with a Ki/Km ratio of 0.5.

Oligonucleotides containing modified bases. I. Inhibition of DNA and RNA polymerases by partially thiolated oligocytidylic acids.

A series of oligomers of cytidylic acid were prepared and partially (6-9% of the bases) thiolated in the 5 positions. The modified oligomers showed increasing inhibition with increasing chain length of both the DNA polymerase-alpha from regenerating rat liver and the DNA-dependent RNA polymerase of E. coli, but the minimum chain length for observable inhibitory activity was 5 nucleotide units for the DNA polymerase-alpha and 16 units for the RNA polymerase.

Selectivity of antitemplates as inhibitors of deoxyribonucleic acid polymerases

DNA polymerase alpha from calf thymus was relatively insensitive to the action of partially thiolated polycytidylic acid (MPC) which had been shown previously to be a potent inhibitor of the corresponding enzyme from regenerating rat liver, competitive with the activated DNA template. In contrast, partially thiolated polyuridylic acid (MPU) strongly inhibited the calf thymus enzyme as well, but showed non-competitive kinetics with respect to the activated DNA template. The much more potent inhibitory activity of MPU compared to MPC was attributed to the less rigid conformation of the former. Methyl substitution on the 5-mercapto groups of MPU substantially decreased but did not abolish its inhibitory activity. MPU was also a potent inhibitor of the herpes virus (HSV-1) induced DNA polymerase which, too, showed little sensitivity toward MPC; in this case, the inhibition by MPU was uncompetitive with respect to the DNA template. In preliminary experiments, MPU showed significant (61%) inhibition of the replication of HSV-1, while MPC was inactive. The results demonstrate that the inhibitory activity of partially thiolated synthetic polynucleotides toward certain DNA polymerases is dependent on the base composition.