The use of terminal blocking groups for the specific joining of oligonucleotides in RNA ligase reactions containing equimolar concentrations of acceptor and donor molecules
Open Access
- 1 November 1976
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 3 (11), 3157-3166
- https://doi.org/10.1093/nar/3.11.3157
Abstract
Under the conditions that RNA ligase converts the tetranucleotide, pA-A2-A, to larger polynucleotides, no such polymerization can be detected with the derivative, pA-A2-A(MeOEt), that possesses a terminal 2′-0-(α-methoxyethyl) group. The protection against self condensation offered by the methoxyethyl group in this system allows the specific joining of donor and acceptor oligonucleotides in reaction mixtures containing equimolar concentrations of the two species. Thus, the enzyme, together with ATP, converts equimolar quantities of A-A2-A and pA-A2-A(Me0Et) to A-A6-A(MeOEt) in 55% yield, while a similar reaction with A-A2-A and pU-U2-U(MeOEt) results in a 40% yield of A-A3-U3-U(MeOEt). The intermediate in these ligations is a disubstltuted pyrophosphate composed of the donor molecule and the adenylate moiety deriving from ATP. In the case of the intermediate arising from the blocked adenosine tetranucleotide, the assigned structure, A5′pp5′A-A2-A(MeOEt), has been confirmed by chemical synthesis. The pyrophosphate derivative is able to participate in joining reactions in the absence of ATP. These observations constitute an efficient approach to the synthesis of larger polynucleotides from a specific series of oligonucleotide blocks since (i), the methoxyethyl group can be easily introduced into each oligonucleotide using the single addition reaction catalyzed by polynucleotide phosphorylase in the presence of a 2′-0-(α-methoxyethyl) nucleoside 5′-diphosphate, and (ii), the blocking group may be readily removed under mild conditions after each successive ligation reaction. Two other octanucleotides, I-I2-A-U3-U and U-U2-C-I3-A, have also been synthesized by this method, and these molecules correspond (with I substituting for G) to sequences appearing near the 3′ terminus of the 6S RNA transcribed from phage lambda DNA. The terminal 3′-phosphate group serves equally well as a blocking group for specific ligation reactions in that the ligase converts equimolar amounts of A-A2-A and pA-A2-Ap to A-A6-Ap in 50% yield.Keywords
This publication has 17 references indexed in Scilit:
- Catalysis of DNA joining by bacteriophage T4 RNA ligaseBiochemical and Biophysical Research Communications, 1976
- Separation of oligonucleotides, nucleotides, and nucleosides on columns of polystyrene anion-exchangers with solvent systems containing ethanolAnalytical Biochemistry, 1976
- Single addition substrates for the synthesis of specific oligoribonucleotides with polynucleotide phosphorylase. Synthesis of 2'-O-(α-methoxyethyl)nucleoside 5'-diphosphatesBiochemistry, 1975
- Studies on ribonucleic acid ligase. Characterization of an adenosine triphosphate-inorganic pyrophosphate exchange reaction and demonstration of an enzyme-adenylate complex with T4 bacteriophage-induced enzyme.1974
- T4 RNA ligase: Substrate chain length requirementsFEBS Letters, 1974
- Stepwise degradation of polyribonucleotidesBiochemistry, 1974
- RNA Ligase Activity in Phage-Infected Bacteria and Animal CellsEuropean Journal of Biochemistry, 1974
- ‘Single addition’ and ‘transnucleotidation’ reactions catalyzed by polynucleotide phosphorylase. Effect of enzymatic removal of inorganic phosphate during reactionNucleic Acids Research, 1974
- 2'-0-(α-Methoxyethyl)nucleoside 5'-diphosphates as single-addition substrates in the synthesis of specific oligoribonucleotides with polynucleotide phosphorylaseBiochemistry, 1973
- Purification and Properties of Bacteriophage T4-Induced RNA LigaseProceedings of the National Academy of Sciences of the United States of America, 1972