Sensitive SNP dual-probe assays based on pyrene-functionalized 2'-amino-LNA: lessons to be learned.
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.
Umemoto
Tadashi
T
Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, 5230 Odense M, Denmark.
Hrdlicka
Patrick J
PJ
Babu
B Ravindra
BR
Wengel
Jesper
J
D001483:Base Sequence; D001681:Biological Assay / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009696:Nucleic Acids / Q000032:analysis*; D015345:Oligonucleotide Probes / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide; D011721:Pyrenes / Q000737:chemistry*; D013936:Thymidine / Q000031:analogs & derivatives* / Q000737:chemistry
C518188:2'-N-(pyren-1-yl)methyl-2'-amino-LNA monomer; D005456:Fluorescent Dyes; D009696:Nucleic Acids; D015345:Oligonucleotide Probes; D011721:Pyrenes; D013936:Thymidine