NEGATIVE CHARGE AT 3' TERMINUS OF OLIGONUCLEOTIDES AND RESISTANCE TO VENOM EXONUCLEASE

Oligonucleotides bearing negatively charged groups at the ω(right) end have been tested between pH 4 and 9 for resistance to hydrolysis by venom exonuclease. Resistance conferred by the 3′-phosphoryl group is highly dependent on pH and chain length. The compounds dNpNp and dNpNpNp are hydrolyzed optimally near pH 6, with hydrolysis of dNpNp proceeding at a 30-fold slower rate. Compared with dNpN, which shows the usual pH optimum of 9, the hydrolysis of dNpNp is inhibited by a factor varying between 40 at pH 5 and 1700 at pH 9. The hydrolysis of ApA>p and ApApA>p occurs with a pH optimum of approximately 9. Compared with ApA, the hydrolysis of ApA>p is inhibited by a relatively constant factor varying between 7 at pH 6 and 2 at pH 8. The hydrolysis of CpC is inhibited 18 times more than that of dNpN by borate buffer at pH 9. These observations indicate that resistance to exonuclease attack is conferred largely by negative charge, particularly a double charge, at the ω end of an oligonucleotide. A practical result of these observations is that the complete hydrolysis of dNpNp can be carried out with less enzyme and with less danger of dephosphorylation at pH 5-6 than at the usual pH 9. With the presently available preparation of exonuclease (Richards, G. M., Tulas, D. J., Wechter, W. J., and Laskowski, M., Sr. (1967), Biochemistry 6, 2908), the complete hydrolysis of dNpNpNp can be carried out at any pH between 5 and 8 with no significant dephosphorylation. The previous conclusion (Laskowski, M. (1967), Advan. Enzymol. 29, 165) that venom exonuclease hydrolyzes substrates bearing an ω-3′-monophosphoryl group by releasing an a nucleoside first is revoked. Trinucleotides dNαpNβpNγp are hydrolyzed by first releasing dpNγp in agreement with the order established for all types of chains. © 1969, American Chemical Society. All rights reserved.