title: Rapid oligonucleotide-templated fluorogenic tetrazine ligations.
abstract: Template driven chemical ligation of fluorogenic probes represents a powerful method for DNA and RNA detection and imaging. Unfortunately, previous techniques have been hampered by requiring chemistry with sluggish kinetics and background side reactions. We have developed fluorescent DNA probes containing quenched fluorophore-tetrazine and methyl-cyclopropene groups that rapidly react by bioorthogonal cycloaddition in the presence of complementary DNA or RNA templates. Ligation increases fluorescence with negligible background signal in the absence of hybridization template. Reaction kinetics depend heavily on template length and linker structure. Using this technique, we demonstrate rapid discrimination between single template mismatches both in buffer and cell media. Fluorogenic bioorthogonal ligations offer a promising route towards the fast and robust fluorescent detection of specific DNA or RNA sequences.
authors.affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, USA.
authors.forename: Jolita
authors.initials: J
authors.lastname: Seckute
authors.forename: Jun
authors.initials: J
authors.lastname: Yang
authors.forename: Neal K
authors.initials: NK
authors.lastname: Devaraj
mesh_terms: D001483:Base Sequence; D003470:Culture Media / Q000737:chemistry; D061565:Cycloaddition Reaction; D003521:Cyclopropanes / Q000138:chemical synthesis / Q000737:chemistry*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000737:chemistry; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000379:methods; D017423:Sequence Analysis, RNA / Q000379:methods
chemical_list: D003470:Culture Media; D003521:Cyclopropanes; D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D016376:Oligonucleotides, Antisense