Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 118, Issue 42, Pages -Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.2107900118
Keywords
non-LTR retroelement reverse transcriptase; RNA sequencing; miRNA; tRNA; noncoding RNA
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Funding
- NIH [T32 GM007232, R35 GM130315, DP1 HL156819]
- Bakar Fellows Program
- Howard Hughes Medical Institute
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Research found that the non-long terminal repeat (non-LTR) retroelements' enzyme supports robust and continuous template jumping, and also discovered the unexpected terminal deoxynucleotidyl transferase activity of the reverse transcriptases (RTs).
Selfish, non-long terminal repeat (non-LTR) retroelements and mobile group II introns encode reverse transcriptases (RTs) that can initiate DNA synthesis without substantial base pairing of primer and template. Biochemical characterization of these enzymes has been limited by recombinant expression challenges, hampering understanding of their properties and the possible exploitation of their properties for research and biotechnology. We investigated the activities of representative RTs using a modified nonLTR RT from Bombyx mori and a group II intron RT from Eubacterium rectale. Only the non-LTR RT supported robust and serial template jumping, producing one complementary DNA (cDNA) from several templates each copied end to end. We also discovered an unexpected terminal deoxynucleotidyl transferase activity of the RTs that adds nucleotide(s) of choice to 3 ' ends of singleand/or double-stranded RNA or DNA. Combining these two types of activity with additional insights about nontemplated nucleotide additions to duplexed cDNA product, we developed a streamlined protocol for fusion of next-generation sequencing adaptors to both cDNA ends in a single RT reaction. When benchmarked using a reference pool of microRNAs (miRNAs), library production by Ordered Two-Template Relay (OTTR) using recombinant non-LTR retroelement RT outperformed all commercially available kits and rivaled the low bias of technically demanding home-brew protocols. We applied OTTR to inventory RNAs purified from extracellular vesicles, identifying miRNAs as well as myriad other noncoding RNAs (ncRNAs) and ncRNA fragments. Our results establish the utility of OTTR for automation-friendly, low-bias, end-to-end RNA sequence inventories of complex ncRNA samples.
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