title: Polysome shift assay for direct measurement of miRNA inhibition by anti-miRNA drugs.
abstract: Anti-miRNA (anti-miR) oligonucleotide drugs are being developed to inhibit overactive miRNAs linked to disease. To help facilitate the transition from concept to clinic, new research tools are required. Here we report a novel method--miRNA Polysome Shift Assay (miPSA)--for direct measurement of miRNA engagement by anti-miR, which is more robust than conventional pharmacodynamics using downstream target gene derepression. The method takes advantage of size differences between active and inhibited miRNA complexes. Active miRNAs bind target mRNAs in high molecular weight polysome complexes, while inhibited miRNAs are sterically blocked by anti-miRs from forming this interaction. These two states can be assessed by fractionating tissue or cell lysates using differential ultracentrifugation through sucrose gradients. Accordingly, anti-miR treatment causes a specific shift of cognate miRNA from heavy to light density fractions. The magnitude of this shift is dose-responsive and maintains a linear relationship with downstream target gene derepression while providing a substantially higher dynamic window for aiding drug discovery. In contrast, we found that the commonly used 'RT-interference' approach, which assumes that inhibited miRNA is undetectable by RT-qPCR, can yield unreliable results that poorly reflect the binding stoichiometry of anti-miR to miRNA. We also demonstrate that the miPSA has additional utility in assessing anti-miR cross-reactivity with miRNAs sharing similar seed sequences.
authors.affiliation: Regulus Therapeutics Inc., 3545 John Hopkins Ct, San Diego, CA 92121, USA johnandrosavich@gmail.com.
authors.forename: John R
authors.initials: JR
authors.lastname: Androsavich
authors.affiliation: Department of Chemical Engineering, University of Michigan, Ann Arbor, MI, USA.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Sobczynski
authors.affiliation: Regulus Therapeutics Inc., 3545 John Hopkins Ct, San Diego, CA 92121, USA.
authors.forename: Xueqing
authors.initials: X
authors.lastname: Liu
authors.affiliation: Regulus Therapeutics Inc., 3545 John Hopkins Ct, San Diego, CA 92121, USA.
authors.forename: Shweta
authors.initials: S
authors.lastname: Pandya
authors.affiliation: Regulus Therapeutics Inc., 3545 John Hopkins Ct, San Diego, CA 92121, USA.
authors.forename: Vivek
authors.initials: V
authors.lastname: Kaimal
authors.affiliation: Regulus Therapeutics Inc., 3545 John Hopkins Ct, San Diego, CA 92121, USA.
authors.forename: Tate
authors.initials: T
authors.lastname: Owen
authors.affiliation: Regulus Therapeutics Inc., 3545 John Hopkins Ct, San Diego, CA 92121, USA.
authors.forename: Kai
authors.initials: K
authors.lastname: Liu
authors.affiliation: Regulus Therapeutics Inc., 3545 John Hopkins Ct, San Diego, CA 92121, USA.
authors.forename: Deidre A
authors.initials: DA
authors.lastname: MacKenna
authors.affiliation: Regulus Therapeutics Inc., 3545 John Hopkins Ct, San Diego, CA 92121, USA.
authors.forename: B Nelson
authors.initials: BN
authors.lastname: Chau
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism; D001681:Biological Assay; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005634:Fructose-Bisphosphate Aldolase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D008099:Liver / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011132:Polyribosomes / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D015703:Antigens, CD; D035683:MicroRNAs; C544354:Mirn122 microRNA, mouse; D009841:Oligonucleotides; D005634:Fructose-Bisphosphate Aldolase