Template switching enables chemical probing of native RNA structures
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ABSTRACT:
PROVIDER: PRJNA1144991 | ENA |
REPOSITORIES: ENA
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Project description:RNAs are often studied in non-native sequence contexts to facilitate structural studies. However, seemingly innocuous changes to an RNA sequence may perturb the native structure and generate inaccurate or ambiguous structural models. To facilitate the investigation of native RNA secondary structure by selective 2′ hydroxyl acylation analyzed by primer extension (SHAPE), we engineered an approach that couples minimal enzymatic steps to RNA chemical probing and mutational profiling (MaP) reverse transcription (RT) methods - a process we call template switching and mutational profiling (Switch-MaP). In Switch-MaP, RT templates and additional library sequences are added post-probing through ligation and template switching, capturing reactivities for every nucleotide. For a candidate SAM-I riboswitch, we compared RNA structure models generated by the Switch-MaP approach to those of traditional primer-based MaP, including RNAs with or without appended structure cassettes. Primer-based MaP masked reactivity data in the 5′ and 3′ ends of the RNA, producing ambiguous ensembles inconsistent with the conserved SAM-I riboswitch secondary structure. Structure cassettes enabled unambiguous modeling of an aptamer construct but introduced non-native interactions in the full-length riboswitch. In contrast, Switch-MaP provided reactivity data for each nucleotide in each RNA and enabled unambiguous modeling of secondary structure, consistent with the conserved SAM-I fold. Switch-MaP is an alternative approach to primer-based and cassette-based chemical probing methods that precludes primer masking and the formation of alternative secondary structures due to non-native sequence elements.
2024-12-26 | GSE274121 | GEO
Project description:We deployed CapTS-seq for sequencing synthetic miRNAs, human total brain RNA, and liver FFPE RNA, and demonstrated that chemical capping in conjunction with template switching consistently reduces sequencing bias and improves library quality in comparison with commercially available RNA-seq kits. Finally, we showed the simultaneous detection of miRNAs and mRNAs in FFPE derived samples, underscoring the potential of this workflow for dissecting regulatory networks between miRNAs and their target gene transcripts.
2021-09-13 | GSE171049 | GEO
Project description:RNAseq experiments of Enterovirus A71 wild type demonstrate that the pyrazine-carboxamide ribonucleotide stimulates catalyzed intra- and intermolecular template switching. These results suggest that pyrazine-2 carboxamide ribonucleotides do not induce lethal mutagenesis or chain termination, but function by promoting template switching and formation of defective viral genomes. We conclude that RdRp-catalyzed intra- and intermolecular template switching can be induced by pyrazine-carboxamide ribonucleotides, defining an additional mechanistic class of antiviral ribonucleotides with potential for broad-spectrum activity.
2021-09-14 | GSE183959 | GEO
Project description:Chemical capping improves template switching and enhances sequencing of small RNAs
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Project description:The reverse transcriptases (RTs) encoded by mobile group II intron and other non-LTR-retro-elements differ from retroviral RTs in being able to template switch from the 5' end of one template to the 3' end of another without pre-existing complementarity between the donor and acceptor nucleic acids. Here, we used the ability of a thermostable group II intron RT (TGIRT) to template switch directly from synthetic RNA template/DNA primer duplexes having either a blunt end or a 3'-DNA overhang end to establish a complete kinetic framework for the reaction and identify conditions that more efficiently capture acceptor RNAs or DNAs. The rate and amplitude of template switching are optimal from starter duplexes with a single nucleotide 3'-DNA overhang complementary to the 3' nucleotide of the acceptor RNA, suggesting a role for non-templated nucleotide addition of a complementary nucleotide to the 3’ end of cDNAs synthesized from natural templates. Longer 3'-DNA overhangs progressively decrease the rate of template switching, even when complementary to the 3' end of the acceptor template. Although dependent upon only a single base pair between the donor and acceptor, template switching discriminates against mismatches, which coupled with the high processivity of the enzyme, enables the synthesis of full-length DNA copies of acceptor nucleic acids beginning directly at their 3' end. We discuss possible biological functions of the template-switching activity of group II intron and other non-LTR-retroelements RTs, as well as the optimization of this activity for adapter addition in RNA-and DNA-seq.
2019-11-11 | GSE138200 | GEO
Project description:Zhao et al. Amplification Table 1 This experiment was designed to determine the effects of template switching (TS) primer and the type of columns used in ds cDNA cleanup on the fidelity of the T7 based RNA linear amplification. BC91 total RNA was amplified with or without TS primer and with two different ds cDNA cleanup protocols. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Computed
2005-11-03 | E-GEOD-3560 | biostudies-arrayexpress
Project description:Zhao et al. Amplification Table 1 This experiment was designed to determine the effects of template switching (TS) primer and the type of columns used in ds cDNA cleanup on the fidelity of the T7 based RNA linear amplification. BC91 total RNA was amplified with or without TS primer and with two different ds cDNA cleanup protocols. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set
2005-11-04 | GSE3560 | GEO
Project description:Induced intra- and intermolecular template switching as a therapeutic mechanism against RNA viruses
| PRJNA762534 | ENA
Project description:Chemical cross-linking of reactive groups in native proteins and protein complexes in combination with the identification of cross-linked sites by mass spectrometry has been in use for more than a decade. Recent advances in instrumentation, cross-linking protocols, and analysis software have led to a renewed interest in this technique, which promises to provide important information about native protein structure and the topology of protein complexes. In this article, we discuss the critical steps of chemical cross-linking and its implications for (structural) biology: reagent design and cross-linking protocols, separation and mass spectrometric analysis of cross-linked samples, dedicated software for data analysis, and the use of cross-linking data for computational modeling. Finally, the impact of protein cross-linking on various biological disciplines is highlighted.
| S-EPMC2938055 | biostudies-literature
Project description:We combined electrophoresis and nanopore sequencing to analyze MarathonRT velocity over the heterogeneous sequences and structures of HOTAIR RNA template and reveal that the local sequences and structures of the template have negligible effect to MarathonRT, and MarathonRT can copy the long RNA in a single turnover, which leads to unusually synchronized primer extension at a constant speed of 25 nt/sec. We further demonstrate that ultra-stable RNA structure insertions do not obstruct MarathonRT, suggesting that MarathonRT can immediately disrupt any RNA structures within the template.
2022-06-12 | GSE195979 | GEO