title: tRid, an enabling method to isolate previously inaccessible small RNA fractions.
abstract: Detection of rare small RNA species whose sizes are overlapping with tRNAs often suffers from insufficient sensitivity due to the overwhelming abundance of tRNAs. We here report a method, named tRid (tRNA rid), for removing abundant tRNAs from small RNA fractions regardless of tRNA sequence species. By means of tRid, we are able to selectively enrich small RNAs which have been previously difficult to access due to mass existence of tRNAs in such fractions. A flexible tRNA-acylation ribozyme, known as flexizyme, is a key tool where the total tRNAs are aminoacylated with N-biotinylated phenylalanine regardless of tRNA sequences, and therefore the biotin-tagged tRNAs could be readily removed from the small RNA fractions by the use of streptavidin-immobilized magnetic beads. Next generation sequencing of the isolated small RNA fraction revealed that small RNAs with less than 200nt were effectively enriched, allowing us to identify previously unknown small RNAs in HeLa and E. coli.
authors.affiliation: Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Futai
authors.affiliation: Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Terasaka
authors.affiliation: Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Katoh
authors.affiliation: Department of Chemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address: hsuga@chem.s.u-tokyo.ac.jp.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Suga
mesh_terms: D000604:Amino Acyl-tRNA Synthetases; D059014:High-Throughput Nucleotide Sequencing; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics*; D058727:RNA, Small Untranslated / Q000235:genetics / Q000302:isolation & purification*; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics*; D046249:Transfer RNA Aminoacylation / Q000235:genetics
chemical_list: D016337:RNA, Catalytic; D058727:RNA, Small Untranslated; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases
keywords: Deep sequencing; Flexizyme; Ribozyme; Small RNAs; tRNA elimination