Synthesis of modified nucleotide polymers by the poly(U) polymerase Cid1: Application to direct RNA sequencing on nanopores.

To this end we explored the ability of available 3′ nucleotidyl transferases to incorporate nucleotides other than the standard four that make up most transcripts in the cell

Vo

2022

Scholarcy highlights

  • RNA plays dual roles during gene expression, both carrying information and interpreting it along the way
  • Distinct homopolymers or mixed polymers offer the potential for barcoding and pooled sequencing of multiple samples on nanopores. To this end we explored the ability of available 3′ nucleotidyl transferases to incorporate nucleotides other than the standard four that make up most transcripts in the cell
  • We first confirmed that the commercial preparations had the expected nucleotide adding specificities by incubating them with various rNTPs and a 24-nucleotide oligomer of adenosine under the reported conditions
  • We set out to test the ability of 3′ nucleotidyl transferases to incorporate modified nucleotides in order to explore potential applications for nanopore direct RNA sequencing
  • We showed that I-tailing by Cid1 does not distort measurement of poly(A)+ mRNAs, while simultaneously enabling robust detection of essential non-polyadenylated RNAs such as snRNAs, telomerase, and RNAse P RNA
  • Our results indicate that addition of modified nucleotides is a feasible alternative to the standard method and that distinct current signatures allow their discrimination from native nucleotides
  • Modified UTP analogs were purchased from TriLink, Inc. Polynucleotide tailing with S. cerevisiae Poly(A) Polymerase To add a homopolymer to the 3′ ends of RNAs using Poly(A) Polymerase, Yeast a reaction containing is incubated at 37°C for 30 minutes, two volumes of Gel Loading Buffer II are immediately added to stop the reaction

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