Abstract
Abstract The ability of ribonuclease T1 to catalyze transesterification of guanosine 2',3'-cyclic phosphate groups to the free 5'-hydroxyl groups of nucleosides or (oligo)nucleotides has been exploited for the synthesis of short oligonucleotides of previously determined sequence. This reaction, the reverse of the first step in RNase T1-catalyzed hydrolysis of RNA, has an equilibrium constant of 2.2 m-1 (pH 7.0, 0°). In the presence of high concentrations (0.3 to 1.0 m) of the 5'-hydroxyl-bearing component, transesterification (synthesis) proceeds more rapidly than hydrolysis of the cyclic phosphate. Thirty-two different oligonucleotides of chain length 2 to 8 have been prepared in yields which compare very favorably with other methods of synthesis. Oligonucleotides terminating in guanosine 2',3'-cyclic phosphate residues were produced by reaction of 2'(3')-phosphates with ethyl chloroformate. Dithioerythritol in aqueous ammonia was used to inactivate RNase T1 when synthetic reactions were complete. RNase A has been shown to be intrinsically less satisfactory as a catalyst for oligonucleotide synthesis than RNase T1, as a result of its more rapid hydrolysis of cyclic substrates.
Highlights
Realizing that synthesis would be favored by increasing the concentration of reactants as much as possible we developed two special techniques for doing this
T1 in oligonucleotide synthesis were largely unsuccessful until we observed that the enzyme remained active during paper chromatography
Synthetic product formed in concentrated reaction mixtures at 0” was broken down during subsequent chromatography in neutral solvents at room temperature
Summary
Source of materials-The barium salt of G-cyclic-p was purchased from Sigma. Dinucleoside monophosphates were purchased from Sigma, the Gallard-SchlesingerChemical Manufacturing Company, and ZellstofffabrikWaldhof (Mannheim, Germany). Source of materials-The barium salt of G-cyclic-p was purchased from Sigma. Dinucleoside monophosphates were purchased from Sigma, the Gallard-Schlesinger. Oligoadenylates were prepared by partial alkaline hydrolysis of poly A followed by dephosphorylation with Escherichia coli alkaline phosphatase and separation on a DEAE-. UpUpUp was donated by Dr Frank Martin. Yeast RNA, 2’(3’)-UMP, ADP, all radioactive ribonucleoside diphosphates, and 2(3’)-GMP-8-14C came from Schwarz Bio-.
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