Fig. 3: Optimization of SCAF4_LS to improve its affinity and inhibitory capacity.

a Workflow illustrating the sequence optimization procedures through the addition of supporting nucleotides to SCAF4_LS. b Schematic representation of the competition assay used for the selection of extended sequences. c Evaluation of the inhibitory efficacy of the expanded 64 sequences, utilizing a competitive FP assay. d Direct interactions of the expanded 64 sequences with SCAF4 assessed by SPR spectroscopy. e Selection of DNA sequences exhibiting both binding affinity and inhibitory efficacy. f Assessments of the inhibitory efficacy of the second round of expanded 64 sequences on the SCAF4 and RNAP2 interaction. g Direct interactions of the second round of expanded 64 sequences to SCAF4. h Selection of DNA sequences demonstrating both binding affinity and inhibitory efficacy. i Standard kinetics SPR assay demonstrating the binding of SCAF4_F3_11-nt (SRiApt-1) to immobilized SCAF4. Average affinity value is 58.40 ± 5.80 nM (biological replicates n = 3, mean ± SD). Source data are provided as a Source Data file. j Dose-response curves depicting the inhibition of RNAP2 interaction with SCAF4 by SCAF4_F3_11-nt (SRiApt-1). The IC₅₀ is determined from three biological replicates, each containing three technical replicates (biological replicates n = 3, mean ± SD). Source data are provided as a Source Data file. k Standard kinetics SPR assay demonstrating the binding of SRiApt-3 to immobilized SCAF4. Average affinity value is 22.67 ± 1.00 nM (biological replicates n = 3, mean ± SD). Source data are provided as a Source Data file. l Dose-response curves depicting the inhibition of RNAP2 interaction with SCAF4 by SRiApt-3. The IC₅₀ is determined from three biological replicates, each containing three technical replicates (biological replicates n = 3, mean ± SD). Source data are provided as a Source Data file. m Summary of aptamers optimized from the lead sequence. Both affinities and IC₅₀s of selected aptamers are labeled on the left, and the sequence information was indicated on the right. Residues iterated from the previous sequence are labeled in gray. n Iterations of affinities and IC₅₀s for aptamers, with affinity shown in red and IC₅₀ displayed in blue (biological replicates n = 3, mean ± SD). Source data are provided as a Source Data file.