Extended Data Fig. 2: RT mutation screen.
From: Discovery and evolution of RNA and XNA reverse transcriptase function and fidelity
a, Space filling surface model of the ternary structure of KOD polymerase (PDB ID 5OMF) with primer (nascent) strand (red) and template strand (green) shown. The position of mutations selected for screening are shown in blue. b, ELONA (enzyme-linked oligonucleotide assay)-based RT activity assay scheme: (from left to right) RT reactions are performed with a biotinylated primer bbTest7 (Supplementary Table 7), bound to wells in a streptavidin plate and hybridized to 2′-O-Me RNA template (cyan) TFRst 2′-O-Me (Supplementary Table 7). RT synthesized cDNA (red), which remains bound to the plate after template removal. The presence of the (correct) cDNA can be detected by a specific oligonucleotide probe FitcFd (Supplementary Table 7) labelled with FITC (green), which in turn is detected by an anti-FITC antibody (blue) conjugated to horse-radish peroxidase (yellow star). c, RT mutation activity screen: only mutations S383K, N735K and I114T show an improved signal and when combined (RT-TKK) show more than double the signal of wt (RT521K) (NTC, no template negative control, n = 3).
