Promoter-independent synthesis of chemically modified RNA by human DNA polymerase θ variants

Polθ steric-gate variant efficiently incorporates ribonucleotide analogs with base modifications. (A,C,E,G) Denaturing gels showing PolθRP2 time-dependent incorporation of the indicated canonical and base-modified ribonucleotides on the indicated RNA/DNA primer–templates. (B,D,F) Scatter plots comparing the relative rates of PolθRP2 incorporation of the indicated canonical and base-modified ribonucleotides. n = 3, ±SD. (H) Schematic representation of a method for modifying the 3′-terminal end of RNA by Polθ steric-gate variants. (I) Denaturing gels showing time courses of DNA-dependent RNA synthesis by PolθRP2 in the presence of the indicated NTPs.