title: Post-synthesis DNA modifications using a trans-cyclooctene click handle.
abstract: Post-synthesis DNA modification is a very useful method for DNA functionalization. This is achieved by using a modified NTP, which has a handle for further modifications, replacing the corresponding natural NTP in polymerase-catalyzed DNA synthesis. Subsequently, the handle can be used for further functionalization after PCR, preferably through a very fast reaction. Herein we describe polymerase-mediated incorporation of trans-cyclooctene modified thymidine triphosphate (TCO-TTP). Subsequently, the trans-cyclooctene group was reacted with a tetrazine tethered to other functional groups through a very fast click reaction. The utility of this DNA functionalization method was demonstrated with the incorporation of a boronic acid group and a fluorophore. The same approach was also successfully used in modifying a known aptamer for fluorescent labelling applications.
authors.affiliation: Department of Chemistry, Center for Diagnostics and Therapeutics, Georgia State University, Atlanta, GA 30302-3965, USA. wang@gsu.edu.
authors.forename: Ke
authors.initials: K
authors.lastname: Wang
authors.forename: Danzhu
authors.initials: D
authors.lastname: Wang
authors.forename: Kaili
authors.initials: K
authors.lastname: Ji
authors.forename: Weixuan
authors.initials: W
authors.lastname: Chen
authors.forename: Yueqin
authors.initials: Y
authors.lastname: Zheng
authors.forename: Chaofeng
authors.initials: C
authors.lastname: Dai
authors.forename: Binghe
authors.initials: B
authors.lastname: Wang
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry; D001897:Boronic Acids / Q000737:chemistry; D057930:Click Chemistry; D034242:Cyclooctanes / Q000737:chemistry*; D004247:DNA / Q000737:chemistry*; D004256:DNA Polymerase I / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004261:DNA Replication; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D006367:HeLa Cells; D006573:Heterocyclic Compounds, 1-Ring / Q000737:chemistry; D006801:Humans; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction / Q000379:methods; D013942:Thymine Nucleotides / Q000737:chemistry*
chemical_list: D052157:Aptamers, Nucleotide; D001897:Boronic Acids; D034242:Cyclooctanes; D017931:DNA Primers; D005456:Fluorescent Dyes; D006573:Heterocyclic Compounds, 1-Ring; D013942:Thymine Nucleotides; C000598289:tetrazine dye 10; D004247:DNA; D004256:DNA Polymerase I; D016650:Fluorescein-5-isothiocyanate; C024157:thymidine 5'-triphosphate