Quantamaster qm 4 spectrofluorometer

For generation of RNA in vitro, the HIV 5′-UTR (nucleotides 1–497) was cloned into pSP72 vector (Promega) using BglII and EcoRI sites under the control of T7 promoter. All constructed plasmids were verified by DNA sequencing. Primers were obtained from IDT and are reported in Feng et al [73 (link)]. Fluorescently labeled RNA was produced by transcribing pSP72 DNA cut with EcoRI in vitro using T7 RNA polymerase with a nucleotide mixture containing fluorescein-12-UTP (Roche Applied Science). Steady-state rotational anisotropy reactions (60 μL) were conducted in buffer containing 50 mM Tris, pH 7.5, 40 mM KCl, 10 mM MgCl₂, and 1 mM DTT and contained 10 nM fluorescein-labeled 5’UTR RNA and increasing amounts of A3 (A3H_{hap II}, 0.1–61 nM; A3C-A3H_{hap II}, 0.0045–6.040 nM; and A3G, 0.36202 nM). A QuantaMaster QM-4 spectrofluorometer (Photon Technology International) with a dual emission channel was used to collect data and calculate anisotropy. Measurements were performed at 21°C. Samples were excited with vertically polarized light at 495 nm (6-nm band pass), and vertical and horizontal emissions were measured at 520 nm (6-nm band pass). The K_d was obtained by fitting to a hyperbolic decay curve equation using SigmaPlot version 11.2 software.

Steady state fluorescence depolarization (rotational anisotropy) was used to measure enzyme-DNA binding affinities using the same fluorescence labeled forked double stranded (ds) DNA substrates used for the helicase assays (S1 Table). Measurements were performed in a quartz cuvette of 1 cm path length. Reactions of 70 μL contained F-labeled forked dsDNA (10 nM) in helicase assays buffer (without ATP); ChlR1-WT (0–1900 nM) or ChlR1-Q23A (0–2300 nM) was added via titration. A QuantaMaster QM-4 spectrofluorometer (Photon Technology International) with a dual emission channel was used to collect data and calculate anisotropy. Measurements were made at 21°C. Samples were excited with vertically polarized light at 495 nm (6 nm band pass) and vertical and horizontal emissions were measured at 520 nm (6 nm band pass). Apparent dissociation constants (Kd) were obtained by fitting to a sigmoidal curve using Sigma Plot 11.2 software.

Steady state fluorescence depolarization (rotational anisotropy) was used to measure enzyme-ssDNA binding affinities using the same F-labeled ssDNA substrates (with cytosines 63 nt apart) that were used for deamination reactions (Table S1). Reactions were 60 µL and contained F-labeled ssDNA (10 nM) in RT buffer and A3G (0–650 nM), A3F (0–80 nM), A3F CTD (0–600 nM), A3G NPM (0–350 nM), or A3F NGM (0–650 nM) were titrated into the reaction. A QuantaMaster QM-4 spectrofluorometer (Photon Technology International) with a dual emission channel was used to collect data and calculate anisotropy. Measurements were made at 21°C. Samples were excited with vertically polarized light at 495 nm (6 nm band pass) and vertical and horizontal emissions were measured at 520 nm (6 nm band pass). Apparent dissociation constants (K_d) were obtained by fitting to a sigmoidal curve using Sigma Plot 11.2 software.