Aminoacyl-oligonucleotides

The important feature of this methodology is that fidelity of the amide bond formation is achieved by specific and template-directed coupling of the desired acyl transfer. Therefore, proper juxtaposition of peptidyl- and aminoacyl-oligonucleotides on a polynucleotide template control the direction of polypeptide synthesis. This is essentially the way that fidelity of translation in protein biosynthesis is taking place in natural systems. 

Particularly noteworthy is the Mg -dependent, sequence-specific aminoacyl esterase activity demonstrated with a Tetrahymena ribozyme which was engineered at the IGS (GGAGGG to GGGUUG) to recognize the aminoacylated oligonucleotide CAACCA derived from the 3 end of N-formylmethionyl-... 

Fig. 4 Peptide bond formation by reaction of an aminoacyl (anhydride) oligonucleotide as described in [171]...

A similar system was also capable to promote acyl transfer allowing the synthesis of a 2/,3/-aminoacyl (ester) oligonucleotide from an aminoacyl (anhydride) 5 -phosphale oligonucleotide [172],... 

In the fragment reaction, the ability of puromycin to mimic the aminoacyl-tRNA in the peptidyl transferase reaction was exploited to measure catalytic activity. Puromycin was subsequently used to design a transition-state analog for the peptidyl transferase reaction, known as the Yarns inhibitor, in which it is linked to the oligonucleotide CCdA by a phosphoramide group [73]. In a complex with the 50S ribosomal subunit, the Yarns inhibitor was used to define the catalytic site in a high-resolution crystal structure. No protein was found within 18 A of this site [74]. This result demonstrated conclusively that the catalytic activity indeed resides in the ribosomal RNA. 

There are fewer catalytic ribozymes compared to deoxyribozymes. Examples include a trara-splicing ribozyme, an alcohol dehydrogenase, a ligase capable of functioning at low temperature, " a ribozyme that will ligate the 5 -terminus of RNA to a polypeptide, a transcriptional activator and a tRNA aminoacylation catalyst. An RNA aptamer bearing 5 -CoA has been selected to catalyse thioester formation in the presence of biotin-AMP. In vitro selection has also been used to identify allosteric hairpin ribozymes, activated in the presence of short oligonucleotides, and a ribozyme that catalyses amide bond formation from a 2 -amino nucleotide. " ... 

The hydrolytic stability of branched RNA where the branch-point is a phospho-triester unit revealed that when the triester was embedded within the oligonucleotide it was stable to base-mediated hydrolysis by more than an order of magnitude compared to the simple trinucleotide unit. 5 -Aminoalkyl phosphate nucleotides have been synthesised as analogues of aminoacyl adenylates, and salicyl phospho-diesters of adenylates as potential inhibitors of Mycobacterium tuberculosis. ... 

Another criterion for functionality of short oligonucleotide templates is the stimulation of their binding to ribosomes by cognate aminoacyl-tRNAs."... 

Labeling Reaction. Labeling media were buffered at pH 7.2 to 7.4 and usually contained 6-20 vaM Mg, 80-150 mil/ NH4CI, and radioactively labeled aflSnity reagent at 10- to 100-fold excess over ribosomes added in the 70 S form or as isolated 30 S subimits (sometimes preactivated ). To enhance the specificity and stability of the reversible complex of oligonucleotide and ribosome, some reaction mixtures were supplemented with the cognate aminoacyl-tRNAs. > Labeling mixtures were usually incubated for 1-2 hr at 0°-37°. 

Another criterion for specificity is based on the stimulation of covalent binding by cognate aminoacyl-tRNAs. Reduced covalent reaction to ribosomes precomplexed with poly(U) and Phe-tRNA ° and competition by nonmodified oligonucleotides have also been taken as indications for the specificity of labeling. 

As indicated above, it is possible that codons bind to ribosomes at two positions corresponding to donor and acceptor sites. The nature of the site occupied by attached oligonucleotides has only been tentatively determined by testing the reactivity in the puromycin reaction of the aminoacyl-tRNA bound to the modified ribosomes. In another case, the ability of ApUpGpU -modified 70 S ribosomes to bind Met-tRNA in the presence of EF-Tu has been considered indicative of acceptor site location of the bound codon. ... 

Table 19.2a Variation of aminoacyl-tRNA using T. thermophilus EF-Tu GTP. Asp-tRNA originates from yeast and is fully modified. A minihelix consists of two RNA oligonucleotides derived from aminoacyl arm of tRNA. The number of base pair counted from aminoacyl end is indicated in parentheses. His-TYMV RNA is an aminoacylated tRNA-like structure originating from turnip yellow mosaic virus. AEDANS indicates that the fluorescence reporter group was used as an indicator for the measurements of the interaction with EF-Tu GTP whereas 2 or 3 dA, 3 NH, and oxi/red denote different non-isomerisable aminoacyl-tRNAs [28].

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