Europium Cryptate Labeled Deoxyuridine-Triphosphate Analog: Synthesis and Enzymatic Incorporation

Abstract

The synthesis of an europium tris-bipyridine cryptate labeled 2'-deoxyuridine-5 '-triphosphate analog (K-11-dUTP) is described. This labeled triphosphate was incorporated into DNA through enzymatic reactions with terminal transferase and DNA polymerases. The enzymatic reactions were monitored by TRACE (Time Resolved Amplification of Cryptate Emission), a homogeneous method using Fluorescence Resonance Energy Transfer (FRET) from an europium cryptate as donor to a modified allophycocyanine as acceptor.

Time Gating Improves Sensitivity in Energy Transfer Assays with Terbium Chelate/Dark Quencher Oligonucleotide Probes

“…In most cases, conjugation of lanthanide chelates to oligonucleotides has been accomplished post oligonucleotide synthesis, with the notable exceptions of a chelate−phosphoramidite and a europium cryptate−dUTP . For sensitive, sequence-specific binding assays, Förster resonance energy transfer (FET or FRET) and, in the case of lanthanides, luminescence resonance energy transfer (LRET) are used to quench the luminescence of the unbound probe.…”

A homogeneous time-resolved fluorescence detection of telomerase activity

“…1 gives a representation of the TRAP-HTRF assay format. K-dU units may be statistically incorporated on the biotinylated primer during the telomerization and amplification steps, K-11-dUTP being a substrate for both reverse transcriptase and polymerase [16,17]. The HTRF Ò detection used is based on the technology developed by Mathis [22].…”

“…This allows us to develop simple homogeneous assays based on a ''mix and measure'' format particularly well adapted to high throughput. Since a deoxyuridine analogue labeled with europium cryptate (symbolized as K-11-dUTP) was shown to be efficiently incorporated by Taq DNA polymerase and reverse transcriptase [16][17][18], the incorporation of K-11-dUTP in a TRAP assay was thus tested. We present herein the application of HTRF Ò to the detection of telomerase activity and the screening of inhibitors.…”

Nucleoside Triphosphates — Building Blocks for the Modification of Nucleic Acids

“…This convenient strategy, which completely bypasses the traditional multi-step procedures, has successfully been applied for the generation of dNTPs bearing a vast array of functional groups ranging from functional tags [ 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 ] and bile acids [ 67 ] to amino acids [ 68 , 69 ]. Finally, amide bond formation reactions were extensively used to connect dNTPs equipped with amine residues to side-chains bearing carboxylic acid groups, so as to yield triphosphates adorned with amino acid-like functionalities [ 14 ] or metal complexes [ 70 , 71 ].…”