Enhanced detection of tRNA isoacceptors by combinatorial oligonucleotide hybridization

Abstract

A method that greatly enhances the detection of tRNA by oligodeoxyribonucleotide probe hybridization has been developed. Because highly structured tRNA regions often preclude heteroduplex formation, we have tested the ability of cold oligodeoxyribonucleotides called unfolders to disrupt the tRNA secondary/tertiary structures and promote hybridization of a second labeled oligonucleotide complementary to the anticodon loop. Here we show that an excess of unfolders in the pre/hybridization reaction can enhance a barely detectable hybridization signal by more than 200-fold without affecting probe specificity. This sensitive assay makes it possible to easily study and monitor changes in tRNA isoacceptor expression.

Human tRNASecassociates with HeLa membranes, cell lipid liposomes, and synthetic lipid bilayers

“…1) in the presence of lipid bilayers to see whether it might have an unusual association with HeLa membranes. Therefore, we prepared membrane fractions from HeLa cells and measured tRNA Sec by double-probe hybridization (Buvoli et al 2000), a technique which gives greatly sensitized detection of structured RNAs, including tRNA. As we will show below, this centrifugation technique is adequate to detect membrane affinity, but is likely too slow to recover RNAmembrane complexes quantitatively.…”

“…The black arrow marks the isopentenylated A37. The nucleotides in blue are the region complementary to the 37-nt oligoDNA unfolder and the nucleotides in red are the region complementary to the 20-nt adjacent unlabeled oligonucleotide ''unfolder'' (Buvoli et al 2000), is both linear and specific to tRNA Sec .…”

“…2) were precipitated. Northern blot analysis was performed according to the adjacent oligonucleotide technique of Buvoli et al (2000). A 37-nt oligoDNA unfolder (59-GTGGAATTGAACCACTCTGTCG CTAGACAGCTACAGG-39) was applied during prehybridization (see Fig.…”

“…1). Both oligoDNA unfolder and oligoDNA probe were similarly constructed as in Buvoli et al (2000) where the double-oligo hybridization signal was %100-fold greater than probe alone.…”

The many applications of acid urea polyacrylamide gel electrophoresis to studies of tRNAs and aminoacyl-tRNA synthetases

“…5' or 3' terminal region, variable loop) are advised. Alternatively, hybridization signals can be enhanced using either 'combinatorial oligonucleotide hybridization' [22], which uses a combination of [ 32 P]-labeled DNA oligonucleotide and a molar excess of a non-labeled DNA oligonucleotide acting as an 'unfolder', or [ 32 P]-labeled RNA oligonucleotides, providing for a more stable RNA:RNA hybrid. In both cases, specificity of hybridization has to be carefully monitored on a case-bycase basis.…”