Extended Data Fig. 2: Recombinant TENT4B (rTENT4B) can add 2′OMeATP at the 3’end of RNA oligonucleotide to generate an exonuclease-resistant block. | Nature Biomedical Engineering

Extended Data Fig. 2: Recombinant TENT4B (rTENT4B) can add 2′OMeATP at the 3’end of RNA oligonucleotide to generate an exonuclease-resistant block.

From: Extension of replicative lifespan by synthetic engineered telomerase RNA in patient induced pluripotent stem cells

Extended Data Fig. 2

A, Extension of a 20-mer RNA oligonucleotide (Oligo 1) using rTENT4B and ATP analogs demonstrating differential incorporation. (ADP: Adenosine-5’-diphosphate; ATP: Adenosine-5’-triphosphate; Azido-ATP: 3’-O-azidomethyl-ATP; AlphaS-ATP: Adenosine-5’-(α-thio)-triphosphate; GammaS-ATP: Adenosine-5’-(γ-thio)-triphosphate; ApCpp: Adenosine-5’-[(α,β)-methyleno]triphosphate; AppCp: Adenosine-5’-[(β,γ)-methyleno]triphosphate; AppNhp: Adenosine-5’-[(β,γ)-imido]triphosphate; 2’OMe-ATP: 2’-O-Methyladenosine-5’-triphosphate; Cordycepin-TP: Cordycepin-5’-triphosphate; dd-ATP: dideoxyadenosine-5’-triphosphate; LNA-ATP: Locked nucleic acid-adenosine-5’-triphosphate). n = 3 replicates, one representative shown. B, Upper: Structure of 2’-O-methyladenosine-5’-triphosphate. Bottom: Structure of Locked nucleic acid-adenosine-5’-triphosphate. C, 3’exonuclease activity detection via fluorescence-based assay using exonuclease T (ExoT) and recombinant PARN (PARN) for Oligo 3 alone or Oligo 3 treated with 2’OMeATP or LNA-ATP using rTENT4B. Fluorescently-labeled Oligo 3 is quenched by the addition of complementary anti-sense probe at the end of exonuclease reaction. Degradation of the Oligo 3 prevents hybridization and quenching by the anti-sense oligonucleotide, resulting in higher fluorescence. Degradation-resistant Oligo 3 will have a lower fluorescence. n = 3 replicates, mean ± S.D., p values calculated by 2way-ANOVA. n.s. not significant D, Self-limiting extension of a 20-mer RNA oligonucleotide (Oligo 1) using rTENT4B and 2’OMeATP. n = 3, one representative shown. E,F, RNA oligonucleotide degradation assay using rPARN (E) or exonuclease T (F) for Oligo 2 variants (Oligo 2, 2-mA, 2-mA2, 2-mA3) which have no (0), one, two or three 2’OMeA terminal residues at the 3’ ends, +/- extension with rTENT4B and 2’OMeATP. n = 3, one representative shown. G, RNA oligonucleotide degradation assay using exonuclease T (ExoT) and recombinant PARN (PARN) for Oligo 2 alone or Oligo 2 treated with 2’OMeATP using rTENT4B versus E. Coli poly(A)-polymerase (EPAP). n = 3 replicates, one representative shown.

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