Attachment of protein affinity-labeling reagents of variable length and amino acid specificity to E. coli tRNA^fMet

Abstract

Transamination with bifunctional amines in the presence of bisulfite has been used to attach side chains of variable length to the N4-position of single stranded cytidine residues in E. coli tRNAfMet. Such side chains, terminating in reactive primary amino groups, have been coupled to a variety of N-hydroxysuccinimide esters. The resulting modified tRNAs carry protein affinity labeling groups capable of covalent reaction with a variety of amino acids.

DNA-labelled cytidine assay for the quantification of CAG repeats

“…The CAG repeat model sequences were labelled, specifically binding the BLC5, as immunochemical label, to cytidine by means of transamination-acylation reactions (Avignolo et al, 1990;Draper, 1984;Higginson et al, 2005;Schulman et al, 1981). The labelling efficiency obtained was a 3-8%, similar to the results described by Avignolo (7%), using biotin for labelling cytidine in herring sperm DNA (Avignolo et al, 1990(Avignolo et al, , 1991.…”

“…3), using the p-bromobenzoyl radical as label, in order to introduce it into CAG repeat model sequences (AA1-2, AA1-2-BB1-2 for ATXN2 and NAL, PAL for ATXN3), at denaturalization temperatures (Draper, 1984;Higginson et al, 2005;Schulman et al, 1981).…”

In situ detection of human .BETA.1-interferon mRNA with a chemically biotinylated probe.

“…DNA relatively free of protein is needed to avoid crosslinking between nucleic acids and proteins during the transamination reaction (11). The plasmid DNA was sonicated to give 300-500 by fragments and then treated as follows (12): DNA was denatured with boiling water for 5 min (for the 37°C reaction, DNA was rapidly cooled in ice, and then placed in a 37°C water bath). Then 1 M ethylenediamine-2 M bisulfite, pH 7.5, was added, and boiling was continued (or incubated at 37°C) for various lengths of time.…”

Method for Confirming Cytoplasmic Delivery of RNA Aptamers

“…In the event that placing a biotin on the 5′ end of an aptamer disrupts its function, we advise positioning the biotin on the 3′ end. Additional linking chemistries could also be explored [26–29]. …”