A quantitative framework for structural interpretation of DMS reactivity DOI Creative Commons

D. H. Sanduni Deenalattha,

Chris P. Jurich,

Bret Lange

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 22, 2024

Abstract Dimethyl sulfate (DMS) chemical mapping is widely used for probing RNA structure, with low reactivity interpreted as Watson-Crick (WC) base pairs and high unpaired nucleotides. Despite its widespread use, a quantitative understanding of how DMS relates to specific 3D structural features remains incomplete. To address this gap, we systematically analyzed patterns massive library 7,500 constructs containing two-way junctions known structures. Our results reveal that exists on continuous spectrum rather than discrete bins. Approximately 10% overlap in between WC non-WC nucleotides demonstrates simple thresholds cannot accurately determine base-pairing status. In flanking pairs, correlates stacking strength junction dynamics. For nucleotides, increased hydrogen bonding decreased solvent accessibility led WC-like protection. Most significantly, discover non-canonical strongly atomic distances pair geometry, enabling discrimination different conformations. These relationships establish novel metrics evaluating models provide new framework incorporating into structure prediction algorithms.

Language: Английский

Telomerase RNA structural heterogeneity in living human cells detected by DMS-MaPseq DOI Creative Commons
Nicholas M. Forino,

Jia Z. Woo,

Arthur J. Zaug

et al.

Nature Communications, Journal Year: 2025, Volume and Issue: 16(1)

Published: Jan. 22, 2025

Language: Английский

Citations

0
Dissecting telomerase RNA structural heterogeneity in living human cells with DMS-MaPseq DOI Creative Commons
Nicholas M. Forino,

Jia Z. Woo,

Arthur J. Zaug

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: Oct. 5, 2023

Abstract Telomerase is a specialized reverse transcriptase that uses an intrinsic RNA subunit as the template for telomeric DNA synthesis. Biogenesis of human telomerase requires its (hTR) to fold into multi-domain architecture includes template-containing pseudoknot (t/PK) and three-way junction (CR4/5). These two hTR domains bind (hTERT) protein are thus essential catalytic activity. Here, we probe structure in living cells using dimethyl sulfate mutational profiling with sequencing (DMS-MaPseq) ensemble deconvolution analysis. Unexpectedly, approximately 15% steady state population has CR4/5 conformation lacking features thought be required hTERT binding. The proportion folded primary functional does not require expression fraction assumes misfolded domain refolded by overexpression binding partner. This result suggests role folding cofactor other than during biogenesis. Mutagenesis demonstrates stabilization alternative detrimental assembly Moreover, found RNP complexes purified from via epitope tag on hTERT, supporting hypothesis only major conformer active. We propose this portion cellular pool either slowly or degraded. Thus, kinetic traps have been so well-studied vitro may also present barriers ribonucleoprotein vivo.

Language: Английский

Citations

4
DMS‐MapSeq Analysis of Antisense Oligonucleotide Binding to lncRNA PANDA DOI
Gabriel A. Romero Agosto,

Ethan Cox,

Silvi Rouskin

et al.

Current Protocols, Journal Year: 2024, Volume and Issue: 4(11)

Published: Nov. 1, 2024

Abstract While various methods exist for examining and visualizing the structures of RNA molecules, dimethyl sulfate‐mutational profiling sequencing (DMS‐MaPseq) stands out its simplicity versatility. This technique has been proven effective studying both in vitro complex biological settings. We present an updated protocol DMS‐MaPseq, as well methodology that enables it to be used detection antisense oligonucleotides (ASOs) binding RNA. By applying this protocol, we successfully characterized structural ensemble HIV1 Rev Response Element (RRE), along with two alternative structures. The findings align previously published research validating accuracy method. also demonstrate utility DMS‐MaPseq by resolving confirming ASO at complementary sites P21‐associated noncoding DNA damage‐activated (PANDA) long non‐coding via decreased DMS reactivity. © 2024 Wiley Periodicals LLC. Basic Protocol 1 : on HIV1‐RRE 2 PANDA probing

Language: Английский

Citations

1
Viral RNA Interactome: The Ultimate Researcher’s Guide to RNA–Protein Interactions DOI Creative Commons

W. B. Hanson,

Gabriel A. Romero Agosto,

Silvi Rouskin

et al.

Viruses, Journal Year: 2024, Volume and Issue: 16(11), P. 1702 - 1702

Published: Oct. 30, 2024

RNA molecules in the cell are bound by a multitude of RNA-binding proteins (RBPs) with variety regulatory consequences. Often, interactions these facilitated complex secondary and tertiary structures molecules. Viral RNAs especially known to be heavily structured interact many RBPs, roles including genome packaging, immune evasion, enhancing replication transcription, increasing translation efficiency. As such, RNA-protein interactome represents critical facet viral cycle. Characterization is necessary for development novel therapeutics targeted at disruption essential cycle events. In this review, we aim summarize various shaping interactome, interactions, as well up-to-date methods developed characterization directions novel, RNA-directed therapeutics.

Language: Английский

Citations

0
A quantitative framework for structural interpretation of DMS reactivity DOI Creative Commons

D. H. Sanduni Deenalattha,

Chris P. Jurich,

Bret Lange

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Nov. 22, 2024

Abstract Dimethyl sulfate (DMS) chemical mapping is widely used for probing RNA structure, with low reactivity interpreted as Watson-Crick (WC) base pairs and high unpaired nucleotides. Despite its widespread use, a quantitative understanding of how DMS relates to specific 3D structural features remains incomplete. To address this gap, we systematically analyzed patterns massive library 7,500 constructs containing two-way junctions known structures. Our results reveal that exists on continuous spectrum rather than discrete bins. Approximately 10% overlap in between WC non-WC nucleotides demonstrates simple thresholds cannot accurately determine base-pairing status. In flanking pairs, correlates stacking strength junction dynamics. For nucleotides, increased hydrogen bonding decreased solvent accessibility led WC-like protection. Most significantly, discover non-canonical strongly atomic distances pair geometry, enabling discrimination different conformations. These relationships establish novel metrics evaluating models provide new framework incorporating into structure prediction algorithms.

Language: Английский

Citations

0