Sequence analysis of low-molecular-weight RNAs by the use of non-radioactive labeling

A new procedure for non-radioactive labeling of the 3'-terminal -OH was developed to facilitate the sequencing works on low-molecular-weight RNAs. At first, biotinylated pCp (cytidine 3',5'-bisphosphate) was synthesized and ligated to the 3'-terminal of yeast tRNA(Phe) with the aid of RNA ligase. Although the labeling efficiency was high enough, this labeling resulted in a lack of the sequence ladders around the 3'-terminal 10 nucleotides. A longer oligonucleotide, p(dC)(dT)16-Biotin was designed to overcome this defect and proved to be a better non-radioactive 3'-labeling probe.