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Fig. 1 | BMC Biology

Fig. 1

From: Methyltransferase-directed orthogonal tagging and sequencing of miRNAs and bacterial small RNAs

Fig. 1

Selective DmHen1ΔC methyltransferase-directed attachment of a sequencing adapter to the 3′ end of single-stranded RNA. a A principal scheme of bioorthogonal attachment of 3′ alkyne-adapter/RT primer for cDNA synthesis. ssRNA is functionalized with a hexynyl-azide group by the action of the DmHen1ΔC methyltransferase in the presence of the cofactor analogue Ado-6-azide. The resulting RNA-azide is conjugated to a 3′ alkyne-adapter/RT primer in the presence of Cu(I) yielding a covalent RNA-DNA conjugate which serves as a template for the cDNA synthesis. b DmHen1ΔC-directed azide-tagging of a random pool of 21-mer ssRNAs. Reactions were performed with 2 μM of DmHen1ΔC, 0.2 μM 5′-32P-labelled N21 RNA and 0.1 mM Ado-6-azide. The fraction of modified RNA was estimated after sodium periodate-mediated oxidation/β-elimination of the 3′ terminal nucleotide of unmodified RNA as described in [19]. c Chemical conjugation of RNA-azide with a 3′ alkyne-adapter. Reactions were carried out in 55% DMSO using 0.2 μM 32P-RNA-azide, 10 μM 3′ alkyne-adapter/RT primer and 3.3 mM CuBr-TBTA. Outlined are the 5′ terminal sequences of the 3′ adapters with different alkyne moieties tested in this study. d Reverse transcription through different conjugation linkers. RNA-DNA conjugates were prepared in click reactions containing 20 μM of RNA-azide and 5 μM of 3′ alkyne-adapter/32P-RT primer. Each RNA-DNA conjugate (10 nM) was reverse transcribed in reaction containing 10 U/μl of RevertAid Reverse Transcriptase and 0.25 mM dNTP

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