Synthesis of a nitrobenzeneboronic acid substituted polyacrylamide and its use in purifying isoaccepting transfer ribonucleic acids.
Highly purified isoaccepting species of transfer ribonucleic acid (tRNA) were prepared by use of a polyacrylamide substituted with nitrobenzeneboronic acid functional groups. This method exploits the well-known ability of boronic acids to complex with RNA cis-diols. tRNA isoacceptors were obtained by enzymatically acylating a mixture of tRNA species with a single amino acid and passing the mixture over a solid-state nitrobenzeneboronic acid at pH 6.5 or 7.0. Pure aminoacyl-tRNA eluted at the column liquid volume, and unacylated tRNA species were bound. The bound species were recovered by lowering the pH of the eluant to 4.5. This procedure is uncomplicated, rapid, and applicable to nearly all tRNA isoacceptors. It does not chemically modify the tRNA(s) of interest or adversely affect their ability to be aminoacylated. Since boronic acids must be ionized to complex with cis-1,2-diols, boronic acid derivatives were prepared which ionize at a pH compatible with the stability of the aminoacyl bond. Two isomeric benzeneboronic acids with pKas of 6.8 and 7.4 were synthesized by introducing electron-withdrawing nitro groups into the aromatic ring. The addition of succinyl side chains permitted the nitrobenzeneboronic acids to be coupled to aminoethylpolyacrylamide. The properties of the nitrobenzeneboronic acid substituted acrylamide were illustrated by enriching phenylalanyl-tRNA at pH 7.0 to greater than 95% purity (1.63 nmol of phenylalanine accepted per A260 unit of tRNA) and seryl-tRNA isoacceptors at pH 6.5 to essentially theoretical purity (1.58 nmol of serine accepted per A260 unit of tRNA.
D000180:Acrylic Resins; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D001897:Boronic Acids; D004926:Escherichia coli / Q000032:analysis*; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy; D008722:Methods; D009578:Nitrobenzenes; D012343:RNA, Transfer / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000032:analysis; D000099166:Polyacrylamides
D000180:Acrylic Resins; D000604:Amino Acyl-tRNA Synthetases; D001897:Boronic Acids; D007202:Indicators and Reagents; D009578:Nitrobenzenes; D012343:RNA, Transfer; D000099166:Polyacrylamides; C017562:3-nitrobenzeneboronic acid