Abstract
The generation of terminal 2′, 3′-cyclic phosphates on RNA oligomers is an important process in the study of tRNA splicing and repair, ribozyme catalysis, and RNA circularization. Here, we describe a simple method for producing 2′, 3′-cyclic phosphate functionalized RNA by the deoxyribozyme-catalyzed cleavage of a short 3′-RNA overhang in frozen solution. This method avoids the nonspecific modification and degradation of RNA and attached functional groups (e.g., fluorophores) inherent in other methods, and the use of frozen conditions enables cleavage at very low divalent metal ion concentrations, limiting RNA hydrolysis.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Tanaka N, Chakravarty AK, Maughan B, Shuman S (2011) Novel mechanism of RNA repair by RtcB via sequential 2′,3′- cyclic phosphodiesterase and 3′-phosphate/5′-hydroxyl ligation reactions. J Biol Chem 286(50):43134–43143. https://doi.org/10.1074/jbc.M111.302133
Tanaka N, Shuman S (2011) RtcB is the RNA ligase component of an Escherichia coli RNA repair operon. J Biol Chem 286(10):7727–7731. https://doi.org/10.1074/jbc.C111.219022
Tanaka N, Meineke B, Shuman S (2011) RtcB, a novel RNA ligase, can catalyze tRNA splicing and HAC1 mRNA splicing in vivo. J Biol Chem 286(35):30253–30257. https://doi.org/10.1074/jbc.C111.274597
Lutay AV, Chernolovskaya EL, Zenkova MA, Vlasov VV (2005) Nonenzymatic template-dependent ligation of 2′,3′-cyclic phosphate-containing oligonucleotides catalyzed by metal ions. Dokl Biochem Biophys 401:163–166. https://doi.org/10.1007/s10628-005-0060-4
Lutay AV, Chernolovskaya EL, Zenkova MA, Vlassov VV (2006) The nonenzymatic template-directed ligation of oligonucleotides. Biogeosciences 3:243–249
Dolinnaya NG, Sokolova NI, Ashirbekova DT, Shabarova ZA (1991) The use of BrCN for assembling modified DNA duplexes and DNA-RNA hybrids; comparison with water-soluble carbodiimide. Nucleic Acids Res 19(11):3067–3072. https://doi.org/10.1093/nar/19.11.3067
McLeod AC, Lilley DMJ (2004) Efficient, pH-dependent RNA ligation by the VS ribozyme in trans. Biochemistry 43(4):1118–1125. https://doi.org/10.1021/bi035790e
Hoadley KA, Purtha WE, Wolf AC et al (2005) Zn2+-dependent deoxyribozymes that form natural and unnatural RNA linkages. Biochemistry 44(25):9217–9231. https://doi.org/10.1021/bi050146g
Mutschler H, Wochner A, Holliger P (2015) Freeze-thaw cycles as drivers of complex ribozyme assembly. Nat Chem 7:502–508. https://doi.org/10.1038/nchem.2251
Mutschler H, Holliger P (2014) Non-canonical 3′-5′ extension of RNA with prebiotically plausible ribonucleoside 2′,3′-cyclic phosphates. J Am Chem Soc 136:5193–5196. https://doi.org/10.1021/ja4127714
Hertel KJ, Uhlenbeck OC, Herschlag D (1994) A kinetic and thermodynamic framework for the hammerhead ribozyme reaction. Biochemistry 33(11):3374–3385. https://doi.org/10.1021/bi00177a031
Obianyor C, Newnam G, Clifton BE et al (2020) Towards efficient nonenzymatic DNA ligation: comparing key parameters for maximizing ligation rates and yields with carbodiimide activation. Chembiochem 21(23):3359–3370. https://doi.org/10.1002/cbic.202000335
Shabarova ZA, Dolinnaya NG, Drutsa VL et al (1981) DNA-like duplexes with repetitions. III. Efficient template-guided chemical polymerization of d(TGGCCAAGCTp). Nucleic Acids Res 9(21):5747–5761. https://doi.org/10.1093/nar/9.21.5747
Cruz RPG, Withers JB, Li Y (2004) Dinucleotide junction cleavage versatility of 8-17 deoxyribozyme. Chem Biol 11:57–67. https://doi.org/10.1016/S1074-5521(03)00284-9
Breslow R, Huang DL (1991) Effects of metal ions, including Mg2+ and lanthanides, on the cleavage of ribonucleotides and RNA model compounds. Proc Natl Acad Sci U S A 88:4080–4083. https://doi.org/10.1073/pnas.88.10.4080
Yu T, Zhou W, Liu J (2018) An RNA-cleaving catalytic DNA accelerated by freezing. Chembiochem 19:1012–1017. https://doi.org/10.1002/cbic.201800049
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Le Vay, K.K., Mutschler, H. (2022). Generation of RNA with 2′, 3′-Cyclic Phosphates by Deoxyribozyme Cleavage in Frozen Solutions. In: Steger, G., Rosenbach, H., Span, I. (eds) DNAzymes. Methods in Molecular Biology, vol 2439. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2047-2_19
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2047-2_19
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2046-5
Online ISBN: 978-1-0716-2047-2
eBook Packages: Springer Protocols