title: Dissecting the target specificity of RNase H recruiting oligonucleotides using massively parallel reporter analysis of short RNA motifs.
abstract: Processing and post-transcriptional regulation of RNA often depend on binding of regulatory molecules to short motifs in RNA. The effects of such interactions are difficult to study, because most regulatory molecules recognize partially degenerate RNA motifs, embedded in a sequence context specific for each RNA. Here, we describe Library Sequencing (LibSeq), an accurate massively parallel reporter method for completely characterizing the regulatory potential of thousands of short RNA sequences in a specific context. By sequencing cDNA derived from a plasmid library expressing identical reporter genes except for a degenerate 7mer subsequence in the 3'UTR, the regulatory effects of each 7mer can be determined. We show that LibSeq identifies regulatory motifs used by RNA-binding proteins and microRNAs. We furthermore apply the method to cells transfected with RNase H recruiting oligonucleotides to obtain quantitative information for >15000 potential target sequences in parallel. These comprehensive datasets provide insights into the specificity requirements of RNase H and allow a specificity measure to be calculated for each tested oligonucleotide. Moreover, we show that inclusion of chemical modifications in the central part of an RNase H recruiting oligonucleotide can increase its sequence-specificity.
authors.affiliation: Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark.
authors.forename: Jakob Lewin
authors.initials: JL
authors.lastname: Rukov
authors.affiliation: Roche Pharmaceutical Discovery and Early Development, Roche Innovation Center Copenhagen A/S, Fremtidsvej 3, DK-2970 Hørsholm, Denmark.
authors.forename: Peter H
authors.initials: PH
authors.lastname: Hagedorn
authors.affiliation: Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark.
authors.forename: Isabel Bro
authors.initials: IB
authors.lastname: Høy
authors.affiliation: Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan, Hubei Province 430070, P.R. China.
authors.forename: Yanping
authors.initials: Y
authors.lastname: Feng
authors.affiliation: Roche Pharmaceutical Discovery and Early Development, Roche Innovation Center Copenhagen A/S, Fremtidsvej 3, DK-2970 Hørsholm, Denmark.
authors.forename: Morten
authors.initials: M
authors.lastname: Lindow
authors.affiliation: Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark jvinther@bio.ku.dk.
authors.forename: Jeppe
authors.identifier: http://orcid.org/0000-0002-3847-3853
authors.initials: J
authors.lastname: Vinther
mesh_terms: D020413:3' Untranslated Regions; D005786:Gene Expression Regulation; D015723:Gene Library; D017930:Genes, Reporter; D006367:HeLa Cells; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D035683:MicroRNAs; D059372:Nucleotide Motifs; D009841:Oligonucleotides / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000378:metabolism; D038621:Regulatory Sequences, Ribonucleic Acid; D016914:Ribonuclease H / Q000378:metabolism*; D017423:Sequence Analysis, RNA / Q000379:methods*; D013816:Thermodynamics
chemical_list: D020413:3' Untranslated Regions; D035683:MicroRNAs; D009841:Oligonucleotides; D016601:RNA-Binding Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; D016914:Ribonuclease H