title: Chemical modification of triplex-forming oligonucleotide to promote pyrimidine motif triplex formation at physiological pH.
abstract: Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use in wide variety of potential applications, such as artificial regulation of gene expression, mapping of genomic DNA, and gene-targeted mutagenesis in vivo. Stabilization of pyrimidine motif triplex at physiological pH is, therefore, crucial for improving its potential in various triplex-formation-based strategies in vivo. To this end, we investigated the effect of 3'-amino-2'-O,4'-C-methylene bridged nucleic acid modification of triplex-forming oligonucleotide (TFO), in which 2'-O and 4'-C of the sugar moiety were bridged with the methylene chain and 3'-O was replaced by 3'-NH, on pyrimidine motif triplex formation at physiological pH. The modification not only significantly increased the thermal stability of the triplex but also increased the binding constant of triplex formation about 15-fold. The increased magnitude of the binding constant was not significantly changed when the number and position of the modification in TFO changed. The consideration of the observed thermodynamic parameters suggested that the increased rigidity of the modified TFO in the free state resulting from the bridging of different positions of the sugar moiety with an alkyl chain and the increased hydration of the modified TFO in the free state caused by the introduction of polar nitrogen atoms may significantly increase the binding constant at physiological pH. The study on the TFO viability in human serum showed that the modification significantly increased the resistance of TFO against nuclease degradation. This study presents an effective approach for designing novel chemically modified TFOs with higher binding affinity of triplex formation at physiological pH and higher nuclease resistance under physiological condition, which may eventually lead to progress in various triplex-formation-based strategies in vivo.
authors.affiliation: Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan. htorigoe@rs.kagu.tus.ac.jp
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Torigoe
authors.forename: Osamu
authors.initials: O
authors.lastname: Nakagawa
authors.forename: Takeshi
authors.initials: T
authors.lastname: Imanishi
authors.forename: Satoshi
authors.initials: S
authors.lastname: Obika
authors.forename: Kiyomi
authors.initials: K
authors.lastname: Sasaki
mesh_terms: D001483:Base Sequence; D001952:Bridged-Ring Compounds / Q000737:chemistry; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry*; D053837:DNA Cleavage; D003851:Deoxyribonucleases / Q000097:blood; D054306:Dideoxynucleotides / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000737:chemistry*; D011743:Pyrimidines / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D044366:Transition Temperature
chemical_list: D001952:Bridged-Ring Compounds; D054306:Dideoxynucleotides; D009841:Oligonucleotides; D011743:Pyrimidines; C051802:triplex DNA; D004247:DNA; D003851:Deoxyribonucleases