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. 2019 Feb 13;17(7):1778-1786.
doi: 10.1039/c8ob02467g.

Fluorescent peptide displacement as a general assay for screening small molecule libraries against RNA

Neeraj N Patwardhan  1 , Zhengguo Cai, Colby N Newson, Amanda E Hargrove
Affiliations

Fluorescent peptide displacement as a general assay for screening small molecule libraries against RNA

Neeraj N Patwardhan et al. Org Biomol Chem. .

Abstract

A prominent hurdle in developing small molecule probes against RNA is the relative scarcity of general screening methods. In this study, we demonstrate the application of a fluorescent peptide displacement assay to screen small molecule probes against four different RNA targets. The designed experimental protocol combined with statistical analysis provides a fast and convenient method to simultaneously evaluate small molecule libraries against different RNA targets and classify them based on affinity and selectivity patterns.

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Conflict of interest statement

Conflicts of Interest: There are no conflicts of interest to declare.

Figures

Figure 1:
Figure 1:
Sequence of FRET pair labeled Tat peptide used in displacement assays against HIV-1-TAR RNA. Structures of fluorophores 5-FAM, and TAMRA used at the N and C-termini shown. Amino acid abbreviations: A = Ala, R = Arg, K = Lys, Q = Gly.
Figure 2:
Figure 2:
A) Sequences and secondary structures of 4 RNAs used in assays. B) Binding curves for the 4 RNA targets against Tat peptide. Error bars shown are the standard errors of mean from three replicates. Conditions: 50 nM Tat, 0–500 nM RNA, Incubation 30 min, Ex = 485 nm, Em = 590 nm; Buffer: 50 mM Tris, 50 mM KCl, 0.01% Triton-X-100, pH = 7.4.
Figure 3:
Figure 3:
Results of screening of 30 small molecules against HIV-1-TAR (Panel 1: Red), HIV-2-TAR (Panel 2: Green), A-Site (Panel 3: Blue), RRE-IIB (Panel 4: Orange) at 10 μM small molecule concentration. Conditions: 50 nM Tat-peptide; 40 nM HIV-1-TAR, 40 nM HIV-2-TAR, 120 nM A-Site, 75 nM RRE-IIB; 0, 10, 50 μM Small molecule; λ(Ex): 485 nm, λ(Em): 590 nm; Buffer: 50 mM Tris, 50 mM KCl, 5% DMSO, 0.01% Triton-X-100, pH = 7.4. %FID calculated using Equation 2 shown in the SI. See tables S4 and S5 and Figure S2 for other results of the Tat peptide displacement based screening experiments.
Figure 4:
Figure 4:
A) Heat map and agglomerative hierarchical clustering (AHC) data with the displacement screening (%FID) data obtained at 10 μM small molecule concentration. B) Loading plots from the principal component analysis (PCA) data along PC1 and PC2 C) Scatter plot of the data along the PC1 and PC2 axes.
See this image and copyright information in PMC

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