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Template switching enables chemical probing of native RNA structures

ABSTRACT: Template switching enables chemical probing of native RNA structures

PROVIDER: PRJNA1144991 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR30153237_1.fastq.gz	Fastqsanger.gz
	SRR30153237_2.fastq.gz	Fastqsanger.gz
	SRR30153238_1.fastq.gz	Fastqsanger.gz
	SRR30153238_2.fastq.gz	Fastqsanger.gz
	SRR30153239_1.fastq.gz	Fastqsanger.gz

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Template switching enables chemical probing of native RNA structures

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.

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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.

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