Screening of RNAP2 Ser2ph-specific scFv-sfGFP and development of RNAP2 Ser2ph-mintbody. (A) scFvs tagged with sfGFP (scFv-sfGFP) were transiently expressed in HeLa cells, and the fluorescence patterns were imaged using a confocal microscope. Single confocal sections are shown. Among the 18 different clones tested, 42B3 was most accumulated in the nucleus, where the target RNAP2 Ser2ph is present. (B) Amino acid sequence of RNAP2 Ser2ph (42B3) and H4K20me1 (15F11) scFvs. Framework regions (FRs), complementarity-determining regions (CDRs), and the linker region are indicated. Mutated sites in 42B3 are indicated in colors. The final construct, named “RNAP2 Ser2ph-mintbody,” contains R78K, A80T, and M95V substitutions. (C) Example images of mutant scFv-mCherry that were stably expressed in HeLa cells. The image acquisition setting and contrast adjustment (70–2,000) are the same. (D) Comparison of model structures of 42B3 and 42B3(R78K/A80T/M95V). The models were generated using 15F11 (Protein Data Bank accession no. 5B3N). The M95V mutation appears to strengthen the hydrophobic core more than the original 42B3. Scale bars, 5 µm.