RNA Structure Analysis by Chemical Probing with DMS and CMCT

2019 ◽  
pp. 209-223
Author(s):  
José M. Andrade ◽  
Ricardo F. dos Santos ◽  
Cecília M. Arraiano
Keyword(s):  
Structure Analysis ◽  
Rna Structure ◽  
Chemical Probing

scholarly journals Large-Scale in Vitro Transcription, RNA Purification and Chemical Probing Analysis

2018 ◽  
Vol 48 (5) ◽  
pp. 1915-1927 ◽  
Author(s):  
Fariha Kanwal ◽  
Ting Chen ◽  
Yunlon Zhang ◽  
Altaf Simair ◽  
Cai Rujie ◽  
...  
Keyword(s):  
Structure Analysis ◽  
Rna Structure ◽  
In Vitro Transcription ◽  
Size Exclusion ◽  
Nucleoside Triphosphate ◽  
Separation Procedure ◽  
Rna Purification ◽  
Chemical Probing ◽  
Rna Interaction

Background/Aims: RNA elements such as catalytic RNA, riboswitch, microRNA, and long non coding RNA (lncRNA) play central roles in many cellular processes. Studying diverse RNA functions require large quantities of RNA for precise structure analysis. Current RNA structure and function studies can benefit from improved RNA quantity and quality, simpler separation procedure and enhanced accuracy of structural analysis. Methods: Here we present an optimized protocol for analyzing the structure of any RNA, including in vitro transcription, size-exclusion chromatography (SEC) based denaturing purification and improved secondary structure analysis by chemical probing. Results: We observed that higher Mg2+, nucleoside triphosphate (NTP) concentrations and longer reaction duration can improve the RNA yield from in vitro transcription, specifically for longer and more complicated constructs. Our improved SEC-based denaturing RNA purification effectively halved the experiment duration and labor without introducing any contaminant. Finally, this study increased the accuracy and signal-to-noise ratio (SNR) of selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) chemical probing for analyzing RNA structure. Conclusion: Part or all of our modified method can improve almost any RNA-related study from protein-RNA interaction analysis to crystallography.

scholarly journals Viral RNA structure analysis using DMS-MaPseq

Methods ◽  
2020 ◽  
Vol 183 ◽  
pp. 68-75 ◽  
Author(s):  
Phillip Tomezsko ◽  
Harish Swaminathan ◽  
Silvi Rouskin
Keyword(s):  
Structure Analysis ◽  
Rna Structure ◽  
Viral Rna

scholarly journals RNA structure analysis of human spliceosomes reveals a compact 3D arrangement of snRNAs at the catalytic core

2013 ◽  
Vol 32 (21) ◽  
pp. 2804-2818 ◽  
Author(s):  
Maria Anokhina ◽  
Sergey Bessonov ◽  
Zhichao Miao ◽  
Eric Westhof ◽  
Klaus Hartmuth ◽  
...  
Keyword(s):  
Structure Analysis ◽  
Rna Structure ◽  
Catalytic Core

scholarly journals RNA base pairing complexity in living cells visualized by correlated chemical probing

2019 ◽  
Author(s):  
Anthony M. Mustoe ◽  
Nicole Lama ◽  
Patrick S. Irving ◽  
Samuel W. Olson ◽  
Kevin M. Weeks
Keyword(s):  
Single Molecule ◽  
Rna Structure ◽  
Dimethyl Sulfate ◽  
Detection Methods ◽  
Base Pairing ◽  
Base Pairs ◽  
Chemical Probing ◽  
Pairing Interactions ◽  
In Cells

ABSTRACTRNA structure and dynamics are critical to biological function. However, strategies for determining RNA structure in vivo are limited, with established chemical probing and newer duplex detection methods each having notable deficiencies. Here we convert the common reagent dimethyl sulfate (DMS) into a useful probe of all four RNA nucleotides. Building on this advance, we introduce PAIR-MaP, which uses single-molecule correlated chemical probing to directly detect base pairing interactions in cells. PAIR-MaP has superior resolution and accuracy compared to alternative experiments, can resolve alternative pairing interactions of structurally dynamic RNAs, and enables highly accurate structure modeling, including of RNAs containing multiple pseudoknots and extensively bound by proteins. Application of PAIR-MaP to human RNase MRP and two bacterial mRNA 5'-UTRs reveals new functionally important and complex structures undetectable by conventional analyses. PAIR-MaP is a powerful, experimentally concise, and broadly applicable strategy for directly visualizing RNA base pairs and dynamics in cells.

scholarly journals RNA base-pairing complexity in living cells visualized by correlated chemical probing

2019 ◽  
Vol 116 (49) ◽  
pp. 24574-24582 ◽  
Author(s):  
Anthony M. Mustoe ◽  
Nicole N. Lama ◽  
Patrick S. Irving ◽  
Samuel W. Olson ◽  
Kevin M. Weeks
Keyword(s):  
Single Molecule ◽  
Rna Structure ◽  
Messenger Rna ◽  
Dimethyl Sulfate ◽  
Detection Methods ◽  
Base Pairing ◽  
Chemical Probing ◽  
Pairing Interactions ◽  
In Cells

RNA structure and dynamics are critical to biological function. However, strategies for determining RNA structure in vivo are limited, with established chemical probing and newer duplex detection methods each having deficiencies. Here we convert the common reagent dimethyl sulfate into a useful probe of all 4 RNA nucleotides. Building on this advance, we introduce PAIR-MaP, which uses single-molecule correlated chemical probing to directly detect base-pairing interactions in cells. PAIR-MaP has superior resolution compared to alternative experiments, can resolve multiple sets of pairing interactions for structurally dynamic RNAs, and enables highly accurate structure modeling, including of RNAs containing multiple pseudoknots and extensively bound by proteins. Application of PAIR-MaP to human RNase MRP and 2 bacterial messenger RNA 5′ untranslated regions reveals functionally important and complex structures undetected by prior analyses. PAIR-MaP is a powerful, experimentally concise, and broadly applicable strategy for directly visualizing RNA base pairs and dynamics in cells.

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