Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Dec 24:71:13.17.1-13.17.38.
doi: 10.1002/cpnc.41.

3'-O-Caged 2'-Deoxynucleoside Triphosphates for Light-Mediated, Enzyme-Catalyzed, Template-Independent DNA Synthesis

Anu Stella Mathews  1   2 , Haikang Yang  1   2 , Carlo Montemagno  1   2
Affiliations

3'-O-Caged 2'-Deoxynucleoside Triphosphates for Light-Mediated, Enzyme-Catalyzed, Template-Independent DNA Synthesis

Anu Stella Mathews et al. Curr Protoc Nucleic Acid Chem. .

Abstract

Synthesis, purification, and characterization of 3'-O-caged 2'-deoxyribonucleoside triphosphates (dNTPs), namely 3'-O-(2-nitrobenzyl)-2'-deoxy ribonucleoside triphosphates (NB-dNTPs) and 3'-O-(4,5-dimethoxy-2-nitrobenzyl)-2'-deoxy ribonucleoside triphosphates (DMNB-dNTPs), are discussed in detail. A total of eight 3'-O-caged dNTPs are synthesized with specific protocols depending on the nitrogenous base on the first carbon, i.e., adenine, guanine, thymine, and cytosine, as well as the photo-cleavable group, i.e, 2-nitrobenzyl and 4,5- dimethoxy-2-nitrobenzyl, to be attached in the 3'-O position. The purification of the synthesized compounds is done using ion-exchange and flash chromatography; this is followed by structural confirmation by nuclear magnetic resonance (NMR) and mass spectroscopy (MS). The efficiency of the designed compounds is tested by conducting and evaluating UV-cleaving experiments at 365 nm with proton NMR and LC-MS curves. Finally, the application of the 3'-O-cagged dNTPs in template-independent, enzyme-catalyzed, photo-mediated oligonucleotide synthesis is demonstrated. © 2017 by John Wiley & Sons, Inc.

Keywords: TdT enzyme; photo-cleavable nucleotides; template independent DNA synthesis; terminal deoxynucleotidyl transferase.

PubMed Disclaimer

References

Literature Cited

    1. Bochet, C. G. (2002). Photolabile protecting groups and linkers. Journal of the Chemical Society. Perkin Transactions, 1, 125-142. doi: 10.1039/B009522M.
    1. Castleberry, C. M., Chou, C.-W., & Limbach, P. A. (2008). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of oligonucleotides. Current Protocols in Nucleic Acid Chemistry, 33, 10.1.1-10.1.21. doi: 10.1002/0471142700.nc1001s33.
    1. Ellington, A., & Pollard, J. D. (2001). Introduction to the synthesis and purification of oligonucleotides. Current Protocols in Nucleic Acid Chemistry, 00, A.3C.1-A.3C.22. doi: 10.1002/0471142700.nca03cs00.
    1. Guo, J., Xu, N., Li, Z., Zhang, S., Wu, J., Kim, D. H., … Ju, J. (2008). Four-color DNA sequencing with 3′-O-modified nucleotide reversible terminators and chemically cleavable fluorescent dideoxynucleotides. Proceedings of the National Academy of Sciences of the United States of America, 105, 9145-9150. doi: 10.1073/pnas.0804023105.
    1. Hermanson, G. T. (2013). Bioconjugate technologies (3rd Ed.). Amsterdam: Elsevier.

Publication types

LinkOut - more resources