Extended Data Figure 6. Selection scheme for RNA ligase XNAzymes.

a, XNA library preparation using DNA-dependent XNA polymerases, primed by a 5′ triphosphorylated (5′ppp) RNA primer (LigS2^R), which serves as one of the substrates for RNA ligation in cis. Libraries are synthesized with 3′ biotinylated DNA template, allowing capture and removal by streptavidin beads. b, Single-stranded libraries (unbiotinylated) are annealed and incubated in reaction buffer (see Method) together with a biotinylated chimeric DNA-RNA substrate (tag1_LigS1^R), which successful XNAzymes ligate to RNA substrate LigS2^R in cis. c, Size separation of reacted XNA pools using Urea-PAGE. Ligated XNA pools are gel-extracted and captured by streptavidin beads. d, Reverse transcription of XNA pools using XNA-dependent DNA polymerase RT521L, which is also able to transcribe RNA across the ligation junction (i.e. [RNA-RNA-XNA] -> cDNA). e, Amplification of transcribed cDNA by successive PCR reactions; out-nest reaction depends on priming site (tag1) from ligated substrate tag1_LigS1^R. f, PCR reaction generating templates for XNA synthesis (now 5′ biotinylated) for further rounds of selection.