2'-O,4'-C-aminomethylene-bridged nucleic acid modification with enhancement of nuclease resistance promotes pyrimidine motif triplex nucleic acid formation at physiological pH.
Due to the instability of pyrimidine motif triplex DNA at physiological pH, triplex stabilization at physiological pH is crucial in improving its potential in various triplex-formation-based strategies in vivo, such as gene expression regulation, genomic DNA mapping, and gene-targeted mutagenesis. To this end, we investigated the thermodynamic and kinetic effects of our previously reported chemical modification, 2'-O,4'-C-aminomethylene-bridged nucleic acid (2',4'-BNA(NC)) modification of triplex-forming oligonucleotide (TFO), on triplex formation at physiological pH. The thermodynamic analyses indicated that the 2',4'-BNA(NC) modification of TFO increased the binding constant of the triplex formation at physiological pH by more than 10-fold. The number and position of the 2',4'-BNA(NC) modification in TFO did not significantly affect the magnitude of the increase in the binding constant. The consideration of the observed thermodynamic parameters suggested that the increased rigidity and the increased degree of hydration of the 2',4'-BNA(NC)-modified TFO in the free state relative to the unmodified TFO may enable the significant increase in the binding constant. Kinetic data demonstrated that the observed increase in the binding constant by the 2',4'-BNA(NC) modification resulted mainly from the considerable decrease in the dissociation rate constant. The TFO stability in human serum showed that the 2',4'-BNA(NC) modification significantly increased the nuclease resistance of TFO. Our results support the idea that the 2',4'-BNA(NC) modification of TFO could be a key chemical modification to achieve higher binding affinity and higher nuclease resistance in the triplex formation under physiological conditions, and may lead to progress in various triplex-formation-based strategies in vivo.
Departmentof Applied Chemistry, Faculty of Science, Tokyo University of Science, Tokyo, Japan. htorigoe@rs.kagu.tus.ac.jp
Hidetaka
H
Torigoe
S M Abdur
SM
Rahman
Hiroko
H
Takuma
Norihiro
N
Sato
Takeshi
T
Imanishi
Satoshi
S
Obika
Kiyomi
K
Sasaki
D001952:Bridged-Ring Compounds / Q000737:chemistry*; D004720:Endonucleases / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides / Q000097:blood / Q000737:chemistry*; D011743:Pyrimidines / Q000737:chemistry*; D012689:Sequence Homology, Nucleic Acid; D013816:Thermodynamics
C527205:2',4'-BNA(NC); D001952:Bridged-Ring Compounds; D009711:Nucleotides; D009838:Oligodeoxyribonucleotides; D011743:Pyrimidines; D004720:Endonucleases