Eiko Ohtsuka

The Synthesis of Polynucleotides

Publisher Summary This chapter focuses on the chemical synthesis of oligo and poly-nucleotides. Some of the ways in which synthetic polynucleotides have been used in clarifying molecular-biological problems, and comments on areas in which such compounds are currently under investigation are summarized in the chapter. The parallelism between the development of polynucleotide synthesis and that of polypeptide synthesis is obvious, and many of the early approaches were derived from consideration of analogous types of reaction in the peptide field. It remains to be seen whether advances in the techniques of polynucleotide synthesis will ever make this type of compound more accessible than synthetic poly peptides in the way that protein sequences are now often more readily accessible indirectly by determination of DNA or RNA sequence than by amino acid sequencing. After protection and joining of nucleotide units, removal of protecting groups must be effected without cleavage or migration of the phosphoric diester linkages and without attack at the intrinsically reactive positions of the nucleoside residues.

Chemical synthesis of the 5-́half molecule of E.coli tRNA2GLY

Abstract A tritriacontanucleotide which has the sequence of the 5 - half molecule of E.coli glycine tRNA2, was synthesized by the phosphotriester method involving p-anisidate protection for the 3 - phosphate ends. Di- and trinucleotide units were prepared from 5 - dimethoxytrityl-2 - O-tetrahydrofuranyl-3-O-(o-chlorophenyl)phosphoryl derivatives of uridine, N-benzoylcytidine, N-benzolyadenosine and N-iso-butyrylguanosine by condensation with 3 , 5 - unprotected nucleosides followed by phosphorylation to give 3 - phosphodiester blocks. The 3 - terminal dimers and trimers were synthesized by using 3 - (o- chlorophenyl)phosphoro-p-anisidates instead of 3,5-unprotected nucleosides. The 3-phosphodiesters of oligonucleotides with a chain length of larger than 5 were obtained by removal of the 3-phosphoro-p-anisidate with isoamyl nitrite. The 5 - dimethoxytrityl group was removed by treatment with zinc bromide under anhydrous conditions. Fragments were designed to use common dimer blocks and to reduce the step for 5 - deblocking of larger fragments. Finally a 3 - phosphodiester block with a chain length of 20 was condensed with a 5 - OH component (tridecanucleotide). The fully protected 33 mer was deblocked and purified by chromatography. The structural integrity of the product was confirmed by mobility shift analysis and complete digestion with RNase T2.

Interaction of methionine-specific tRNAs from Escherichia coli with immobilized elongation factor Tu

The interaction of three different Met‐tRNAsMet from E. coli with bacterial elongation factor (EF) Tu · GTP was investigated by affinity chromatography. Met‐tRNAfMet which lacks the base pair at the end of the acceptor stem binds only weakly to EF‐Tu·GTP, while Met‐tRNAmMet has a high affinity for the elongation factor. A modified Met‐tRNAfMet which has a C1‐G72 base pair binds much more strongly to immobilized EF‐Tu·GTP than the native aminoacyl(aa)‐tRNA with non‐base‐paired C1A72 at this position, demonstrating that the base pair including the first nucleotide in the tRNA is one of the essential structural requirements for the aa‐tRNA·EF‐Tu·GTP ternary complex formation.

Photo CIDNP study on the complex formation of λ- cro protein with OR3

A photo CIDNP spectrum of λ cro repressor protein showed that one of the three tyrosines and His 35 are quite accessible to the photosensitive dye. For the remaining two tyrosine residues one is slightly accessible, but the other is inaccessible. In comparison with the result of differential nitration at tyrosine side chains followed by the peptide analysis, it can be concluded that Tyr 26 is mostly exposed and Tyr 51 is slightly exposed on the surface of the cro dimer. On the addition of OR3 17mer, His 35 and Tyr 26 are no longer accessible to the dye, which indicates that they are involved in interaction. However, a similar phenomenon was observed by adding CAP binding site 22mer. The interaction mechanism will be discussed.

PURIFICATION OF T4 RNA LIGASE BY 2',5'-ADP SEPHAROSE CHROMATOGRAPHY

RNA hgase was discovered in T4 infected Escherichia coli cells [ 11. This enzyme catalyzes the intramolecular loinmg of 5’.P and 3’.OH termini of polynucleotides and the intermolecular joining of various combmations of oligoand polynucleotides at their 5’.P and 3’.OH termmi [2-81. RNA ligase also catalyzes the addition of dinucleoside pyrophosphates [9] and mononucleotides [lO,l l] to oligonucleotide acceptors. Nucleoside 3’,5’-diphosphates are the shortest donor substrates and their 3’.phosphates are essential for the reaction because nucleoside S’-phosphates and nucleoside 2’,5’-drphosphates are unable to serve as donors [9-l 11. We found that 2’,5’-ADP inhibited the RNA hgase reaction. The procedures for punfication of RNA ligase have been improved by several groups [2,3,5,12,13]. However, it was sometrmes difficult to remove a trace of nuclease activity from RNA ligase preparations. Recently, we found that RNA hgase bound to 2’S’ADP Sepharosein the presence of Mg2* but not in its absence, and we introduced the 2’S’-ADP Sepharose chromatography at the last step of our purification procedure. The enzyme so obtained had no detectable RNase activity and was suitable for the synthesis of oligoribonucleotides with defined sequences.

New condensing reagents for stereospecific synthesis of dinucleoside monophosphate aryl esters

Abstract Condensation of 5′-O-dimethoxytritylnucleoside 3′-O-(o-chlorophenyl) phosphates and 3′-O-benzoylnucleosides with a new condensing reagent, 2,4,6-triisopropylbenzenesulfonyl 5-(pyridin-2-yl) tetrazolide gave o-chlorophenyl ester of protected dinucleoside monophosphates which had a stereospecific configuration. The corresponding mesitylenesulfonyl derivative gave similar results.

Solid phase synthesis of ribo-oligonucleotides on a polyacrylmorpholide support

Abstract A riboheptanucleotides G-C-A-A-C-C-A which has a sequence of the 3′-end of E. coli formylmethionine tRNA has been synthesized on a polyacrylmorpholide resin by the triester approach using dinucleotide blocks.

Semiautomated synthesis of oligonucleotides on a silica gel support

Abstract Dodecadeoxynucleotides have been synthesized by the phosphotriester method on a column of γ-aminopropyl silica gel or benzylaminopolystyrene by using a programmed synthesizer.

Synthesis of a protein biosynthesis inhibitor, 5′-tri-phosphoryladenylyl-(2′-5′)-adenosine

Abstract A protein biosynthesis inhibitor, 5′-triphosphoryladenylyl-(2′-5′)-adenylyl-(2′-5′)-adenosine was synthesized by polymerization of N 6 -benzoyl-3′-O-(o-nitrobenzyl)adenosine 5′-phosphate follwed by reaction with pyrophosphate using 1,1′-carbonyldiimidazole.

Inhibition of a novel subspecies of DNA polymerase α by 2′-deoxy-2′-azidoadenosine 5′-triphosphate

Abstract DNA polymerase α1, a subspecies of DNA polymerase α of Ehrlich ascites tumor cells, was associated with a novel RNA polymerase activity and utilized poly(dT) and single-stranded circular fd DNA as a template without added primer in the presence of ribonucleoside triphosphates and a specific stimulating factor. DNA synthesis in the above system was inhibited by the ATP analogue, 2′-deoxy-2′-azidoadenosine 5′-triphosphate more than the DNA synthesis with poly(dT)·oligo(rA) by DNA polymerase α1 and RNA synthesis by mouse RNA polymerases I and II. Kinetic analysis showed that the analogue inhibited DNA polymerase α1 activity on poly(dT) competitively with respect to ATP, suggesting that the analogue inhibited RNA synthesis by the associated RNA polymerase activity.