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Incorporation of Pseudouridine into RNA for Biochemical and Biophysical Studies

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RNA Structure and Function

Part of the book series: RNA Technologies ((RNATECHN,volume 14))

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Abstract

Modification of RNA molecules has a significant effect on their structure and function. Many different examples of RNA modifications have been observed and each contributes in various ways to ensure proper biological activity. One of the most common modifications is pseudouridine which occurs in key dynamic locations in many RNAs. Despite its prevalence in natural RNA sequences, organic synthesis of pseudouridine has been challenging because of the stereochemistry requirement and the sensitivity of reaction steps to moisture. Herein, we describe the semi-enzymatic synthetic route for the synthesis of pseudouridine using adenosine-5’-monophosphate and uracil as the starting materials and a reverse reaction catalyzed by a pseudouridine monophosphate glycosidase. Moreover, we describe the conversion from nucleoside (pseudouridine) to nucleotide triphosphate (pseudouridine-5’-triphosphate) to incorporate into RNA via in vitro transcription for biochemical and biophysical studies.

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Abbreviations

ACE:

Bis(2-acetoxyethoxy)methyl

AMP:

Adenosine-5′-Monophosphate

DMT:

Dimethoxytrityl

Et3NHHCO3:

Triethylammonium Bicarbonate

(HNBu3)2H2P2O7:

bis-(Tri-n-butylammonium)pyrophosphate

NTP:

Nucleoside Triphosphate

POCl3:

Phosphorous Oxychloride

PSCl3:

Thiophosphoryl Chloride

R5P:

Ribose-5′-Phosphate

Ψ:

Pseudouridine

ΨMP:

Pseudouridine-5′-Monophosphate

ΨMP Glycosidase:

Pseudouridine Monophosphate Glycosidase

Ψ Synthase:

Pseudouridine Synthase

ΨTP:

Pseudouridine-5′-Triphosphate

snoRNA:

Small Nucleolar RNA

snoRNP:

Small Nucleoprotein Particle

TBDMS:

tert-Butyldimethylsilyl

TFAA:

Trifluoroacetic Anhydride

TLR7:

Toll-like Receptor 7

TOM:

2′-O-triisopropylsilyloxymethyl

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Acknowledgements

This work was supported by faculty startup funds and graduate student research grants from Southern Illinois University Edwardsville.

Author information

Authors and Affiliations

  1. Department of Chemistry, Southern Illinois University Edwardsville, Edwardsville, IL, 62026, USA

    Tristan Sanford, Andrew Riley, Kylie Raasch & Minako Sumita

  2. Department of Biological Science, Southern Illinois University Edwardsville, Edwardsville, IL, 62026, USA

    Melanie Clawson, Ridwan Oyebamiji & Minako Sumita

  3. Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, 73104, USA

    Tristan Sanford

  4. Department of Cell and Developmental Biology, University of Illinois Urbana-Champaign, Urbana, IL, 61801, USA

    Andrew Riley

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  1. Tristan Sanford
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  5. Ridwan Oyebamiji
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Correspondence to Minako Sumita .

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  1. NanoBioMedical Centre of Adam Mickiewicz University, Poznań, Poland

    Jan Barciszewski

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Sanford, T., Riley, A., Clawson, M., Raasch, K., Oyebamiji, R., Sumita, M. (2023). Incorporation of Pseudouridine into RNA for Biochemical and Biophysical Studies. In: Barciszewski, J. (eds) RNA Structure and Function. RNA Technologies, vol 14. Springer, Cham. https://doi.org/10.1007/978-3-031-36390-0_25

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