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Sensitive SNP Dual‐Probe Assays Based on Pyrene‐Functionalized 2′‐Amino‐LNA: Lessons To Be Learned
Author(s) -
Umemoto Tadashi,
Hrdlicka Patrick J.,
Babu B. Ravindra,
Wengel Jesper
Publication year - 2007
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200700408
Subject(s) - pyrene , chemistry , fluorescence , nucleic acid , ternary complex , stereochemistry , hybridization probe , base pair , dna , covalent bond , nucleotide , excimer , biophysics , biochemistry , biology , organic chemistry , physics , optics , gene , enzyme
A homogenous fluorescence dual‐probe assay containing 2′‐ N ‐(pyren‐1‐ylmethyl)‐2′‐amino‐LNA (locked nucleic acid) building blocks has been developed for effective mismatch‐sensitive nucleic acid detection. The pyrene units, which are connected to the rigid bicyclic furanose derivative of 2′‐amino‐LNA through a short linker, are positioned at the 3′ and 5′ ends of a dual‐probe system. Whereas hybridization with complementary DNA/RNA results in very strong excimer signals, as the pyrene units are in close proximity to one another in the ternary complex, exposure to most singly mismatched DNA/RNA targets results in significantly lower excimer emission intensity. The mechanism that underlies this excellent optical discrimination of singly mismatched targets is clarified by comparison of the thermal‐denaturation profiles and fluorescence properties of the dual probe and a covalently linked analogue. Optical discrimination of singly mismatched targets arises from a decrease in excimer emission intensity due to a failure to form a ternary complex (a decrease in thermal stability) and/or local mismatch‐induced changes in the helix geometry, depending on the position of the mismatched base pair. The devised dual‐probe assay constitutes a simple and sensitive system for the detection of single‐nucleotide polymorphism and highlights that conformational restriction combined with the use of short probes conveys favorable properties to dual‐probe constructs.