Determination of affinity constants of locked nucleic acid (LNA) and DNA duplex formation using label free sensor technology.
Locked nucleic acid (LNA) is a nucleic acid analogue containing 2'-O,4'-C-methylene-beta-D-ribofuranosyl nucleotides, which have a bicyclic furanose unit locked in a RNA mimicking sugar conformation. Oligonucleotides containing LNA monomers show an enhanced thermal stability and robustness against nuclease mediated cleavage. Therefore special tailored LNA is a versatile tool for gene array analysis and single nucleotide polymorphism (SNP) analysis. The higher melting temperatures result from a higher affinity between the LNA and its complementary base. This was verified by the determination of the affinity constants of the duplex formation of 3 oligonucleotides: DNA, L-DNA, in which all thymidines are substituted by LNA, and a fully modified LNA, to their complementary DNA strand. Affinity constants were calculated to be 1.5 x 10(9), 4.0 x 10(9) and >10(12) L mol(-1). This was done using the label free and time resolved sensing technology reflectometric interference spectroscopy (RIfS), in an assay format similar to a titration called binding inhibition assay.
Möhrle
Bernd P
BP
Eberhard-Karls-University of Tübingen, Institute of Physical and Theoretical Chemistry, Auf der Morgenstelle 8, D-72076 Tübingen, Germany. bernd.moehrle@ipc.uni-tuebingen.de
Kumpf
Michael
M
Gauglitz
Günter
G
D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D009696:Nucleic Acids / Q000737:chemistry*; D020411:Oligonucleotide Array Sequence Analysis; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D013816:Thermodynamics
D009692:Nucleic Acid Heteroduplexes; D009696:Nucleic Acids; D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; C477371:locked nucleic acid