Fig 2: In vitro binding assays with purified recombinant proteins. (A) CBB stained polyacrylamide gel depicting purified recombinant ANXA5 and CA8 (left panel) and immunoblotting of recombinant CA8 proteins with either an anti-CA8 antibody or anti-His antibody (right panel). (B and C) Binding assays of recombinant ANXA5 (0, 250, 500 and 1000 nM) and CA8 (0, 500, 1000 and 2000 nM) with (B) Toc-Malat1 and (C) Toc-DMPK. (D) CBB stained polyacrylamide gel depicting purified recombinant APEX1 and FEN1. (E and F) In vitro binding assays of Toc-HDO substrates incubated with recombinant APEX1 and FEN1 in (E) N buffer (APEX1: 50, 100 and 150 nM; FEN1: 50, 100 and 150 nM), (F) M buffer with APEX1, FEN1 and RNase H (APEX1: 50, 100 and 150 nM; FEN1: 50, 100 and 150 nM; RNase H: 5, 10 and 20 mU). (G) Complete binding of Toc-HDO substrates by high dose of APEX1 and FEN1 in M buffer (APEX1: 100, 200 and 400 nM; FEN1: 100, 200 and 400 nM; RNase H: 1, 2.5, 5 mU). (H) Cation-dependent binding pattern of APEX1 (APEX1: 2.7, 27 and 270 nM). (I) Substrate length-dependent binding assays with ANXA5, CA8, APEX1 and FEN1 (1000, 1000, 150, 150 nM, respectively). (J) In vitro binding assays with or without Toc 13-mer substrates with ANXA5, CA8, APEX1 and FEN1 (1000, 1000, 150, 150 nM, respectively).

Fig 3: Toc-HDO-dependent gene silencing of the identified proteins. (A) Hepa1–6 cells were used to generate subcell lines stably expressing shRNA against no target (ctrl), luciferase, ANXA5, CA8, RNase H1, APEX1 and FEN1. Immunoblots performed with the extracts from shRNA knocked down Hepa1–6 cells. (B) Toc-Malat1 dose-dependent gene expression regulation analyzed by RT-qPCR (n = 3). (C and D) Quantitative analysis of the substrates uptake of (C) Toc-Malat1 effect (n = 4), (D) ASO effect (n = 4). (E–H) Quantitative analysis of the (E) Toc-Malat1 effect with 25 nM (n = 3, light gray), 50 nM (n = 5, gray), and 100 nM (n = 4, dark gray), (F) 100 nM of Toc-DMPK effect (n = 4), (G) 100 nM of Toc-ApoB effect (n = 5) or (H) 500 nM of ASO effect against Malat1 (n = 4).

Fig 4: Characterization of identified proteins. (A) Fluorescence polarization assays of Toc-HDO substrates incubated with recombinant ANXA5, CA8, APEX1, and FEN1 proteins (10-9 to 10-5.5 M). (B and C) In vitro binding assays with ANXA5, CA8, APEX1, and FEN1 (1000, 1000, 150 and 150 nM, respectively) and Toc-HDO substrates with (B) PS ASO backbone and (C) PO ASO backbones. (D) Pull-down experiments using Toc-biotin–HDO and biotin–ASO of Malat1.