Detecting tRNA modifications with fluorescent-labeled DNA probes

Transfer RNAs (tRNAs) undergo extensive post-transcriptional modifications on their nucleobases and sugar moieties. Here, we describe methods to detect tRNA modifications using DNA oligonucleotide probes labeled with fluorescent dyes as an alternative to radioactive labeling. Unlike radioactive probes, fluorescent probes maintain their sensitivity after long-term storage and do not require relabeling between experiments. The first method combines Northern blotting with differential probe hybridization based upon the presence or absence of a modification in the tRNA target. In addition to detecting modifications, this method provides insight into the effects of tRNA modification on tRNA processing and abundance. The second approach uses a viral reverse transcriptase to detect primer extension blocks caused by certain tRNA modifications during cDNA synthesis of the target tRNA. The primer extension approach provides a quantitative readout of modification status in tRNAs at nucleotide resolution. Overall, the fluorescence-based approaches described in this Chapter provide a convenient and safer alternative to radioactive probes while maintaining sensitivity and resolution.