Use of ribonucleosides as protecting groups in synthesis of polynucleotides with phosphorylated terminals

Abstract

Two new protected 5'-ribonucleotides, 2'3'-O-bis(4,4'-dimethoxytrityl)uridine 5'-(4-chlorophenyl phosphate) and 2',3'-O-(methoxymethylene)uridine 5'-(4-chlorophenyl phosphate), form the basis of a chemical procedure for phosphorylating the 5'-ends of DNA fragments synthesized by the phosphotriester approach. Condensation of either of these mononucleotide units with the free 5'-hydroxyl of an otherwise fully protected oligomer results in high-yield formation of a 5'-5' triester linkage. Subsequently, the terminal ribonucleotide of the deprotected product rU5'-5'd(N-Nn-N) can be cleaved by periodate oxidation of its 2',3'-cis-hydroxyl system followed by beta-elimination, leaving its phosphate attached to the 5'-hydroxyl group of the oligodeoxyribonucleotide. This procedure together with a tactic employing a 2',3'-O-acylribonucleotide residue at the 3'-terminus of the chain allows the synthesis of oligomers possessing monophosphate groups at either end or both ends. Furthermore, oligonucleotide intermediates possessing a 5'-5'-linked uridine terminal are shown to have a special application as acceptors in RNA ligase reactions, where the presence of the ribonucleoside cap on the 5'-phosphate limits ligation specifically to the 3'-ends of the oligomers. Removal of the uridine residues to expose free 5'-phosphates would then enable the products to participate as donors in further elongation reactions.

Molecular Recognition of cAMP by an RNA Aptamer

“…This RNA was incapable of binding to the cAMP-agarose matrix, just as predicted by the original boundary analysis (data not shown). Interestingly, conversion of the 34-nucleotide RNA into a 33-nucleotide RNA with a 3′-terminal phosphate moiety (Figure 4B, structure ii), via periodate-mediated oxidation and β-elimination of the 3′ nucleoside (35), yielded an RNA product that also failed to bind the cAMP-agarose matrix. We speculated that perhaps the aptamer requires the 2′,3′cyclic phosphate group that is generated by alkaline-mediated cleavage, and that this requirement is not necessary in the context of the full-length RNA clone.…”

“…The integration of aptamers into mRNAs has recently proven to be an effective way to introduce ligand-dependent control over gene expression in living cells (43). In addition, RNA aptamers have been used as allosteric binding sites for engineered ribozymes that are specifically controlled by small effector molecules (21,22,(34)(35)(36)(37)(38)(39)(40)(41)(42)(43)(44)(45)(46). Class II aptamers do retain cAMP-binding function under conditions that approach those found in living cells (Figure 6).…”

Anomalous hairpin formation in an oligodeoxyribonucleotide

“…Oligonucleotides were prepared in 10-20 mg quantities by the solution phase phosphotriester strategy with block condensation procedures previously reported (8,9). The fully protected oligomers were purified by silica gel column chromatography and the blocking groups were removed by successive treatments with tetramethylguanidinium pyridine-2-aldoximate, ammonia, and acetic acid as described (9). The oligonucleotides were separated from salts and the various blocking groups by chromatography on a column (1 X 75 cm) of Sephadex G-10 gel filtration beads with EtOH-H20 (1:4, v/v) as eluting solvent.…”

Self-Incorporation of coenzymes by ribozymes

“…Removal of a terminal nucleoside that has both 2' and 3' hydroxyls exposed was carried out by oxidation of the vicinal hydroxyls followed by _-elimination of the modified nucleoside (Nadeau et al 1984). A 10-111 aliquot from the NAD+-dependent reaction, equivalent to 600 fmol of input precursor RNA, was mixed with 2.4 1110.2 M NalO,_ and incubated on ice for I h. Residual oxidant was quenched by adding 3.6 1110.2 M L-methionine and incubating on ice for 20 rain.…”