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. Author manuscript; available in PMC: 2023 Jul 1.
Published in final edited form as: Nat Chem. 2022 Oct 13:10.1038/s41557-022-01050-8. doi: 10.1038/s41557-022-01050-8

Fig. 1. The two-residue steric gate.

Fig. 1

a) Chemical structure of 2’-O-methyl (2’OMe)-RNA. The 2-methoxy substituent is highlighted in cyan. b) Sequence alignment showing polymerases Tgo wildtype and engineered polymerases and respective key mutations in TGK (blue), TGLLK (green) and 2M (red). c) Space-filling model of the ternary structure of KOD DNA polymerase (PDB ID 5OMF) with respective mutations in TGK (blue), TGLLK (green) and 2M (red). d) Structural model of the active site of KOD DNA polymerase (PDB ID 5OMF) with DNA template strand (orange), active site 2’OMe-ATP (carbons shown in grey, nitrogens in blue, oxygens in red and phosphorus atoms in orange) and 2’OMe-RNA nascent strand (cyan) with 2’-methoxy groups of terminal 3’ and + 1 nucleotide shown as space-filling envelope. Key steric gate mutations (T541G, K592A) are displayed in pink (sticks) with wild-type side-chain residues shown as space-filling envelope highlighting the reduction in steric bulk. e) Denaturing PAGE of 2’OMe-RNA synthesis (DNA primer FD, template TempNpure, full length +72 nt) of steric gate single and double mutations. This experiment was performed once. Note the synergistic effect of T541G and K592A double mutation. Vertical black lines separate non-adjacent lanes from the same gel, see Source Data for full gel. f) Denaturing PAGE of DNA (H), RNA (OH) and 2’OMe-RNA (OMe) synthesis by TGK, TGLLK or 2M on defined-sequence template (DNA/2’OMe-RNA primer FD, template TempNpure, full length +72 nt). Similar, consistent results have been obtained twice as well as three times with 2M only. Vertical black lines separate non-adjacent lanes from the same gel, see Source Data for full gel. g) Denaturing PAGE of RNA (OH) and 2’OMe-RNA (OMe) synthesis by TGK, TGLLK or 2M on random N40 template (RNA/2’OMe-RNA primer A-Test2, template Tag3.3-N40-Test2, full length +79 nt), densitometry of N40 synthesis yield: TGLLK 2’OMe-RNA 0%, 2M 2’OMe-RNA 90% (SI Fig. 17). Similar, consistent results have been obtained twice. Vertical black lines separate non-adjacent lanes from the same gel, see Source Data for full gel. h) Denaturing PAGE of RNA (OH) and 2’OMe-RNA (OMe) long-range synthesis of a GFP transcript by TGLLK or 2M (2’OMe-RNA primer Synth-out1mm, template sfGFP, full length +752 nt). Similar, consistent results have been obtained three times (with 2M only).