The Evaluation of SHAPE-MaP RNA Structure Probing Protocols Reveals a Novel Role of Mn2+ in the Detection of 2′-OH Adducts

Grzywacz, Kamilla; Chełkowska-Pauszek, Agnieszka; Plucinska-Jankowska, Marianna; Żywicki, Marek

doi:10.3390/ijms24097890

Open AccessArticle

The Evaluation of SHAPE-MaP RNA Structure Probing Protocols Reveals a Novel Role of Mn²⁺ in the Detection of 2′-OH Adducts

by

Kamilla Grzywacz

^1,*

,

Agnieszka Chełkowska-Pauszek

²

,

Marianna Plucinska-Jankowska

²

and

Marek Żywicki

^2,*

¹

Institute of Bioorganic Chemistry Polish Academy of Sciences, 61-704 Poznań, Poland

²

Department of Computational Biology, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, 61-614 Poznań, Poland

^*

Authors to whom correspondence should be addressed.

Int. J. Mol. Sci. 2023, 24(9), 7890; https://doi.org/10.3390/ijms24097890

Submission received: 5 March 2023 / Revised: 22 April 2023 / Accepted: 24 April 2023 / Published: 26 April 2023

(This article belongs to the Special Issue Molecular Biology of RNA: Recent Progress)

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Abstract

Chemical probing, for decades, has been one of the most popular tools for studying the secondary structure of RNA molecules. Recently, protocols for simultaneous analysis of multiple RNAs have been developed, enabling in vivo transcriptome-wide interrogation of the RNA structure dynamics. One of the most popular methods is the selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP). In this study, we describe the evaluation of this protocol by addressing the influence of the reverse transcription enzymes, buffer conditions, and chemical probes on the properties of the cDNA library and the quality of mutational profiling-derived structural signals. Our results reveal a SuperScript IV (SSIV) reverse transcriptase as a more efficient enzyme for mutational profiling of SHAPE adducts and shed new light on the role of Mn²⁺ cations in the modulation of SSIV readthrough efficiency.

Keywords: RNA structure probing; SHAPE-MaP; 1M7; BzCN; mutational profiling

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MDPI and ACS Style

Grzywacz, K.; Chełkowska-Pauszek, A.; Plucinska-Jankowska, M.; Żywicki, M. The Evaluation of SHAPE-MaP RNA Structure Probing Protocols Reveals a Novel Role of Mn²⁺ in the Detection of 2′-OH Adducts. Int. J. Mol. Sci. 2023, 24, 7890. https://doi.org/10.3390/ijms24097890

AMA Style

Grzywacz K, Chełkowska-Pauszek A, Plucinska-Jankowska M, Żywicki M. The Evaluation of SHAPE-MaP RNA Structure Probing Protocols Reveals a Novel Role of Mn²⁺ in the Detection of 2′-OH Adducts. International Journal of Molecular Sciences. 2023; 24(9):7890. https://doi.org/10.3390/ijms24097890

Chicago/Turabian Style

Grzywacz, Kamilla, Agnieszka Chełkowska-Pauszek, Marianna Plucinska-Jankowska, and Marek Żywicki. 2023. "The Evaluation of SHAPE-MaP RNA Structure Probing Protocols Reveals a Novel Role of Mn²⁺ in the Detection of 2′-OH Adducts" International Journal of Molecular Sciences 24, no. 9: 7890. https://doi.org/10.3390/ijms24097890

APA Style

Grzywacz, K., Chełkowska-Pauszek, A., Plucinska-Jankowska, M., & Żywicki, M. (2023). The Evaluation of SHAPE-MaP RNA Structure Probing Protocols Reveals a Novel Role of Mn²⁺ in the Detection of 2′-OH Adducts. International Journal of Molecular Sciences, 24(9), 7890. https://doi.org/10.3390/ijms24097890

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The Evaluation of SHAPE-MaP RNA Structure Probing Protocols Reveals a Novel Role of Mn²⁺ in the Detection of 2′-OH Adducts

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