RNA displacement assays enable the study of RNA interactions, typically involving the displacement of a pre-bound RNA strand by another nucleic acid-based molecule such as DNA, RNA, or a small molecule. RNA displacement assays allow investigation of RNA binding proteins, RNA secondary structure stability, and the efficiency of competitive nucleic acid interactions.
In RNA displacement assays, an RNA strand is labeled with a fluorescent or radiolabeled marker. The labeled RNA is hybridized to a complementary strand or bound to a protein. A competitor molecule, such as another RNA, DNA, protein, or small molecule, is introduced to test if it can displace the labeled RNA. The displacement is monitored using the following methods: fluorescence changes, gel electrophoresis, or other detection methods. RNA fluorescent indicator displacement (RFID) assays enable the detection and quantification of interactions between RNA and other molecules without requiring fluorescent labeling of both the RNA and the binding molecule.
RNA displacement assays allow the study of various biological interactions and mechanisms, including RNA-protein interactions, investigation of RNA secondary structures and their stability, evaluating the efficacy of RNA-targeting drugs, and assessing oligonucleotide-based therapeutics such as antisense oligonucleotides or siRNAs.
The following is a list of applications for RNA displacement assays.
Antisense & siRNA Therapeutics: RNA displacement assays can test how well an antisense oligonucleotide (ASO), or siRNA can displace or bind to its target RNA by assessing the effect of ASOs designed to block translation of a disease-related mRNA.
CRISPR & Gene Regulation Studies: RNA displacement assays evaluate how guide RNAs (gRNAs) interact with DNA or RNA targets by displacing pre-bound strands, for example, by testing the efficiency of Cas-based RNA editing systems.
Drug Screening for RNA-Targeting Molecules: RNA displacement assays allow screening if small molecules bind to RNA and displace a previously bound probe, for example, to identify inhibitors of viral RNA structures such as HIV or SARS-CoV-2.
RNA Secondary Structure Analysis: RNA displacement assays help confirm the presence of RNA hairpins, G-quadruplexes, or other secondary structures by introducing a complementary strand to displace a labeled RNA strand, for example, by testing the stability of a siRNA or aptamer structure.
RNA-Protein Interactions: RNA displacement assays allow scientists to determine if a protein binds to an RNA sequence and whether another molecule, such as an inhibitor, can displace it. An example is the study of RNA helicases that unwind RNA structures by displacing bound RNA strands or the interaction of viral proteins with RNA oligonucleotides.
Fluorescence polarization-based RNA displacement assay
Perveen et al. (2021) developed a fluorescence polarization-based RNA displacement assay suitable for high-throughput screening of inhibitors that bind to the nsp10-nsp16 complex to identify RNA competitive inhibitors of nsp16 methyltransferase. The assay utilizes fluorescein amidite (FAM)-labeled RNA (5′-N7-meGpppACCCCC-FAM 3′), biotinylated RNA (5′-N7-meGpppACCCCC-Biotin 3′) together with S-adenosyl-l-methionine (SAM), S-adenosyl-l-homocysteine (SAH), and sinefungin. This assay also allowed the researchers to triage potential inhibitors from alternative screens competing with RNA substrates and determine their inhibition mechanism.
In a coronavirus such as SARS-CoV-2, RNA capping is essential for viral mRNA's stability and evading the host's immune system. Uncapped RNA molecules are identified as "foreign" by the host's innate immune response and undergo degradation. The virus's non-structural proteins (NSPs) are critical parts of the replication and transcription complex (RTC) and are essential for immune system evasion. NSPs help the virus hijack the endoplasmic reticulum (ER) membrane and establish the RTC, inducing ER stress after membrane rearrangement, which can cause severe neuronal disturbance.
Proteins catalyzing RNA capping are attractive targets for antiviral drug development. Nsps involved in viral mRNA capping include nsp10 (cofactor for nsp14 and nsp16), nsp13 (RNA 5′-triphosphatase, helicase), nsp14 (guanine-N7 methyltransferase), and nsp16 (2′-O-methyltransferase).
The capping process begins with removing the 5′-γ-phosphate of newly synthesized RNA chains (pppN-RNA) by the nsp13 protein. In the following step, a guanylyltransferase (GTase) catalyzes the formation of GpppN-RNA by transferring a guanosine monophosphate (GMP) molecule to the 5′-diphosphate of the RNA chains (ppN-RNA). Next, the Nsp14 protein methylates the cap structure at the N7 position of the guanosine, formatting cap-0 (N7-meGpppN-RNA). Ultimately, the nsp10-nsp16 complex catalyzes the addition of a methyl group on the ribose 2′-O position of the first transcribed nucleotide of the cap-0 to form cap-1 (N7-meGpppNme-RNA).
References
Asare-Okai PN, Chow CS. A modified fluorescent intercalator displacement assay for RNA ligand discovery. Anal Biochem. 2011 Jan 15;408(2):269-76. [PMC]
Blazer L.L., Li F., Kennedy S., et al. A Suite of Biochemical Assays for Screening RNA Methyltransferase BCDIN3D. SLAS Discov. 2017;22:32–39. [PubMed]
Perveen S, Khalili Yazdi A, Devkota K, Li F, Ghiabi P, Hajian T, Loppnau P, Bolotokova A, Vedadi M. A High-Throughput RNA Displacement Assay for Screening SARS-CoV-2 nsp10-nsp16 Complex toward Developing Therapeutics for COVID-19. SLAS Discov. 2021 Jun;26(5):620-627. [PMC]
Picard-Jean, F., Brand, C., Tremblay-Létourneau, M., Allaire, A., Beaudoin, M., Boudreault, S., Duval, C., Rainville-Sirois, J., Francis, R., Pelletier, J., Geiss, B., & Bisaillon, M. (2018). 2'-O-methylation of the mRNA cap protects RNAs from decapping and degradation by DXO. PLoS One, 13(3), e0193804. [PMC]
Ray A, Frick DN. Fluorescent probe displacement assays reveal unique nucleic acid binding properties of human nudix enzymes. Anal Biochem. 2020 Apr 15;595:113622. [PMC]
Santerre M, Arjona SP, Allen CN, Shcherbik N, Sawaya BE. Why do SARS-CoV-2 NSPs rush to the ER? J Neurol. 2021 Jun;268(6):2013-2022. [PMC]
Tam D, Lorenzo-Leal AC, Hernández LR, Bach H. Targeting SARS-CoV-2 Non-Structural Proteins. Int J Mol Sci. 2023 Aug 20;24(16):13002. [PMC]
Wicks SL, Hargrove AE. Fluorescent indicator displacement assays to identify and characterize small molecule interactions with RNA. Methods. 2019 Sep 1;167:3-14. [PMC]
Zhang, J., Umemoto, S., and Nakatani, K.; Fluorescent Indicator Displacement Assay for Ligand−RNA Interactions. J. Am. Chem. Soc. 2010, 132, 11, 3660–3661. [ACS]
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