title: Nucleoside triphosphates--from synthesis to biochemical characterization.
abstract: The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues. In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern; marcel.hollenstein@dcb.unibe.ch.
authors.forename: Marcel
authors.initials: M
authors.lastname: Hollenstein
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern.
authors.forename: Christine Catherine
authors.initials: CC
authors.lastname: Smith
authors.affiliation: Department of Chemistry and Biochemistry, University of Bern.
authors.forename: Michael
authors.initials: M
authors.lastname: Räz
mesh_terms: D009705:Nucleosides / Q000138:chemical synthesis / Q000737:chemistry*; D009711:Nucleotides / Q000138:chemical synthesis / Q000737:chemistry*; D011122:Polyphosphates / Q000138:chemical synthesis / Q000737:chemistry*
chemical_list: D009705:Nucleosides; D009711:Nucleotides; D011122:Polyphosphates
references.citation: Nucleic Acids Res. 2009 Apr;37(5):1638-49
references.pmid: 19153138
references.citation: Chembiochem. 2012 Jan 2;13(1):19-25
references.pmid: 22162282
references.citation: Angew Chem Int Ed Engl. 2008;47(23):4346-50
references.pmid: 18435522
references.citation: J Am Chem Soc. 1999;121(42):9781-9
references.pmid: 11543572
references.citation: Chembiochem. 2009 Aug 17;10(12):1988-92
references.pmid: 19591189
references.citation: Acc Chem Res. 2007 Feb;40(2):141-50
references.pmid: 17309194
references.citation: Angew Chem Int Ed Engl. 1998 Nov 2;37(20):2872-2875
references.pmid: 29711104
references.citation: J Am Chem Soc. 2010 Mar 31;132(12):4141-51
references.pmid: 20201573
references.citation: Org Biomol Chem. 2011 Mar 7;9(5):1366-71
references.pmid: 21203615
references.citation: Nucleic Acids Res. 2004 Mar 05;32(4):1591-601
references.pmid: 15004246
references.citation: Bioorg Med Chem Lett. 2012 Jul 1;22(13):4428-30
references.pmid: 22633686
references.citation: Angew Chem Int Ed Engl. 2001 Nov 5;40(21):3990-3993
references.pmid: 29712240
references.citation: Chem Commun (Camb). 2008 Nov 30;(44):5675-85
references.pmid: 19009049
references.citation: J Biol Chem. 2001 Jun 29;276(26):23616-23
references.pmid: 11319228
references.citation: Molecules. 2012 Nov 15;17(11):13569-91
references.pmid: 23154273
references.citation: Bioorg Med Chem. 2004 Mar 1;12(5):1111-20
references.pmid: 14980623
references.citation: Chembiochem. 2006 Apr;7(4):669-72
references.pmid: 16502476
references.citation: Chem Rev. 2000 Jun 14;100(6):2047-60
references.pmid: 11749283
references.citation: Chemistry. 2012 Mar 26;18(13):4080-7
references.pmid: 22337599
references.citation: Chemistry. 2012 Oct 15;18(42):13320-30
references.pmid: 22996052
references.citation: Eur J Biochem. 2003 Apr;270(8):1628-44
references.pmid: 12694176
references.citation: J Am Chem Soc. 2007 Feb 7;129(5):1456-64
references.pmid: 17263432
references.citation: Chem Soc Rev. 2011 Dec;40(12):5802-14
references.pmid: 21625726
references.citation: Chem Biol. 1997 Sep;4(9):675-83
references.pmid: 9331408
references.citation: Org Lett. 2004 Feb 19;6(4):489-92
references.pmid: 14961605
references.citation: J Am Chem Soc. 2000 Mar 22;122(11):2433-9
references.pmid: 11543272
references.citation: Org Biomol Chem. 2008 Jul 7;6(13):2233-41
references.pmid: 18563253
references.citation: Org Biomol Chem. 2011 Oct 21;9(20):6949-54
references.pmid: 21670811
references.citation: Proc Natl Acad Sci U S A. 2012 Dec 4;109(49):19971-6
references.pmid: 23139410
references.citation: Nucleic Acids Res. 2007;35(4):1222-9
references.pmid: 17267413
references.citation: Nat Chem. 2012 Jan 10;4(3):183-7
references.pmid: 22354431
references.citation: Org Biomol Chem. 2010 Mar 7;8(5):1194-201
references.pmid: 20165813
references.citation: J Am Chem Soc. 2001 Feb 28;123(8):1556-63
references.pmid: 11456753
references.citation: Curr Opin Chem Biol. 2007 Dec;11(6):588-94
references.pmid: 17967435
references.citation: Chem Soc Rev. 2011 Dec;40(12):5669-79
references.pmid: 21431120
references.citation: Nucleic Acids Res. 1994 Jul 25;22(14):2817-22
references.pmid: 7519769
references.citation: Angew Chem Int Ed Engl. 2004 Jun 21;43(25):3337-40
references.pmid: 15213968
references.citation: Curr Opin Chem Biol. 2006 Dec;10(6):607-14
references.pmid: 17049298
references.citation: J Am Chem Soc. 2005 Nov 2;127(43):15071-82
references.pmid: 16248646
references.citation: Chem Asian J. 2011 Oct 4;6(10):2747-52
references.pmid: 21887745
references.citation: Bioorg Med Chem Lett. 2009 Jun 1;19(11):2941-3
references.pmid: 19414263
references.citation: Biochim Biophys Acta. 2010 May;1804(5):1151-66
references.pmid: 19596089
references.citation: Org Biomol Chem. 2011 Apr 7;9(7):2266-73
references.pmid: 21311779
references.citation: Molecules. 2010 Aug 09;15(8):5423-44
references.pmid: 20714306
references.citation: ACS Comb Sci. 2013 Apr 8;15(4):174-82
references.pmid: 23485334
references.citation: Org Biomol Chem. 2013 Aug 21;11(31):5162-72
references.pmid: 23817514
references.citation: Angew Chem Int Ed Engl. 2012 Jan 2;51(1):254-7
references.pmid: 22083884
references.citation: Org Biomol Chem. 2011 Nov 7;9(21):7482-90
references.pmid: 21931884
references.citation: Bioorg Med Chem. 2006 Apr 15;14(8):2518-26
references.pmid: 16359870