A Fluorogenic, Nucleic Acid Directed “Click” Reaction

Abstract

A fluorogenic, nucleic acid directed, Cu(+)-catalyzed chemical reaction was developed that allows sequence-specific detection of nucleic acids at concentrations as low as 20 nM through monitoring of the increase of the fluorescence intensity. A single nucleotide mismatch in the template strand leads to the complete inhibition of this reaction. We observed that a Cu(+) ion stabilized with a water-soluble ligand is a significantly more efficient catalyst than free Cu(+).

Gas-phase Dissociation of homo-DNA Oligonucleotides

“…Due to the spatial proximity of the reactive groups after hybridization, a chemical reaction is triggered, upon which, for example, a profluorophore is converted into a fluorophore. This technique allows for facile sensing of the hybridized oligonucleotide by means of fluorescence spectroscopy [7,8]. A similar principle was applied by Erben et al who reported a DNA templatetriggered, sequence-specific transfer of an aminoacyl group from one phosphorothioate modified PNA-peptide hybrid to another [9].…”

Reactions Templated by Nucleic Acids: More Ways to Translate Oligonucleotide‐Based Instructions into Emerging Function

“…Using DNA fragments derivatized with the alkyne profluorophore and an azide at the 3' and 5' ends, respectively, the reaction proceeded in a sequence-specific templated fashion at template concentrations as low as 20 nm. [48] The use of excess tris(3-hydroxypropyltriazolylmethyl)amine ligand helped to prevent the copper-induced degradation of reac- tants, but also interfered with the fluorescence readout. Reliable results were obtained after thermal denaturation or copper removal with sodium sulfide.…”

Reaktionen an Nucleinsäuretemplaten: mehr Methoden zur Übersetzung Oligonucleotid‐basierter Informationen in neue Funktionen

“…Unter Verwendung von DNA‐Fragmenten, die mit dem Alkin‐Profluorophor und einem Azid an den 3′‐ bzw. 5′‐Enden derivatisiert waren, lief die Reaktion sequenzspezifisch in Templat‐gestützter Weise bei geringen Templatkonzentrationen von 20 n M ab 48. Ein Überschuss an Tris(3‐hydroxypropyltriazolylmethyl)amin‐Ligand half, den Kupfer‐induzierten Abbau der Reaktanten zu verhindern, störte jedoch die Fluoreszenzdetektion.…”