tRid, an enabling method to isolate previously inaccessible small RNA fractions

Futai, Kazuki; Terasaka, Naohiro; Katoh, Takayuki; Suga, Hiroaki

doi:10.1016/j.ymeth.2016.04.033

Cited by 5 publications

(3 citation statements)

References 24 publications

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“…S1A, RiboMinus, Life technologies). To remove total tRNAs, we used tRid methodology (Futai et al 2016), recently developed in our laboratory using a modified flexizyme. In this method, we used a 3 ′ -oxidized dialdehyde-flexizyme (dial-Fx) to aminoacylate the total tRNA fraction in the extract with N-biotinylated phenylalanine (Biotin-Phe) essentially irrespective of sequence, and the biotin-tagged tRNAs were then effectively removed from other small RNAs by the use of streptavidinimmobilized magnetic beads (Supplemental Figs.…”

Section: Construction Of the Rrna/trna-depleted Human Small Rna Librarymentioning

confidence: 99%

“…The Fx3 + N1 (Futai et al 2016) and RNAs for BLI analysis were prepared by in vitro transcription with T7 RNA polymerase and purified by denaturing PAGE (8 M urea, 1 × TBE, 8% acrylamide/ bis-acrylamide [19:1]). The primers for preparing transcription templates and RNAs are shown in Supplemental Tables S1, S2.…”

Section: In Vitro Transcriptionmentioning

confidence: 99%

“…Here we have adopted a commercial method using oligonucleo-tide probes that are complementary to rRNAs for rRNA removal, and the tRid method recently developed in our laboratory that enables us to remove total tRNA. The latter technique involves the use of a modified flexizyme, referred to as dial-Fx, which is utilized to deplete the RNA sample of total tRNA through charging with N-biotinylated phenylalanine (Biotin-Phe) (Futai et al 2016). In this work, we have demonstrated smaRt-SELEX against folic acid (Fig.…”

Section: Introductionmentioning

confidence: 99%

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A human microRNA precursor binding to folic acid discovered by small RNA transcriptomic SELEX

Terasaka

Futai

Katoh

et al. 2016

RNA

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RNA aptamers are structured motifs that bind to specific molecules. A growing number of RNAs bearing aptamer elements, whose functions are modulated by direct binding of metabolites, have been found in living cells. Recent studies have suggested that more small RNAs binding to metabolites likely exist and may be involved in diverse cellular processes. However, conventional methods are not necessarily suitable for the discovery of such RNA aptamer elements in small RNAs with lengths ranging from 50 to 200 nucleotides, due to the far more abundant tRNAs in this size range. Here, we describe a new in vitro selection method to uncover naturally occurring small RNAs capable of binding to a ligand of interest, referred to as small RNA transcriptomic SELEX (smaRt-SELEX). By means of this method, we identified a motif in human precursor microRNA 125a (hsa-pre-miR-125a) that interacts with folic acid. Mutation studies revealed that the terminal loop region of hsa-pre-miR-125a is important for this binding interaction. This method has potential for the discovery of new RNA aptamer elements or catalytic motifs in biological small RNA fractions.

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Section: Construction Of the Rrna/trna-depleted Human Small Rna Librarymentioning

confidence: 99%

Section: In Vitro Transcriptionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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