Takuma Katayama

Thermodynamic and Kinetic Effects of Morpholino Modification on Pyrimidine Motif Triplex Nucleic Acid Formation under Physiological Condition

Due to instability of pyrimidine motif triplex nucleic acid under physiological pH and low magnesium ion concentration, stabilization of the triplex under the physiological condition is crucial in improving its therapeutic potential to artificially control gene expression in vivo. To this end, we investigated the thermodynamic and kinetic effects of morpholino (MOR) modification of triplex-forming oligonucleotide (TFO) on the triplex formation under the physiological condition. The thermodynamic analyses indicated that the MOR modification of TFO not only significantly increased the thermal stability of the triplex but also increased the binding constant for the triplex formation by nearly 2 orders of magnitude. The consideration of the observed thermodynamic parameters suggested that the increased rigidity of the MOR-modified TFO in the free state relative to the corresponding unmodified TFO may enable the significant increase in the binding constant. Kinetic data demonstrated that the observed increase in the binding constant resulted from the considerable increase in the association rate constant rather than the decrease in the dissociation rate constant. This information will be valuable for designing novel chemically modified TFO with higher binding affinity in the triplex formation under physiological conditions, leading to progress in therapeutic applications of the antigene strategy in vivo.

Triplex Formation-Based Artificial Transcription Factor to Regulate Target Gene Expression

A triplex formation-based artificial transcription factor to recognize any upstream sequence of target genes was developed to regulate the target gene expression. The artificial transcription factor contains a single-stranded RNA to bind with duplex DNA of the upstream sequence of the target gene to form triplex, and an effecter domain, such as activation or repression domain, of transcription factor. Reporter β -galactosidase activity in yeast was increased 1.5–2 times by introduction of the artificial transcription factor. The novel artificial transcription factor may be a useful tool to regulate the target gene expression and reveal unknown function of the target genes.

COMBINATION OF POLY(L-LYSINE)-GRAFT-DEXTRAN COPOLYMER AND 2′-O,4′-C-METHYLENE BRIDGED NUCLEIC ACID (2′,4′-BNA) MODIFICATION SYNERGISTICALLY STABILIZES PYRIMIDINE MOTIF TRIPLEX AT NEUTRAL PH

Triplex DNA has attracted considerable interest because of its possible biological function in vivo and its wide variety of potential applications, such as regulation of gene expression. A triplex is formed through the sequence-specific interaction of a single-stranded homopurine or homopyrimidine triplex-forming oligonucleotide (TFO) with the major groove of homopurine-homopyrimidine stretch in duplex DNA. In the pyrimidine motif triplex, a homopyrimidine TFO binds paral lel to the homopurine strand of the target duplex by Hoogsteen hydrogen bonding to form T A:T and C G:C triplets. Extreme instability of the pyrimidine motif triplex at physiological pH severely limits its utility for artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at neutral pH is, therefore, of great importance to improve its therapeutic potential. We have previously shown that poly(L-lysine)-graft-dextran (PLL-g-Dex) copolymer (Figure 1) and 2’-O,4’-C-methylene bridged nucleic acid (2’,4’-BNA) backbone modification of TFO (Figure 2) increased the thermal stability of the pyrimidine Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):635–638, (2005) Copyright D Taylor & Francis, Inc. ISSN: 1525-7770 print/ 1532-2335 online DOI: 10.1081/NCN-200060113

The interaction between the purine motif triplex and the triplex DNA-binding domain of Saccharomyces cerevisiae Stm1 protein

Saccharomyces cerevisiae Stm1 protein (273 amino acids) is a purine motif triplex DNA-binding protein. We have previously found that Stm(1-113) (amino acids 1-113) is the minimal domain to specifically bind with the purine motif triplex. Here, to reveal the triplex recognition mechanism of Stm(1-113), we have examined the interaction between Stm(1-113) and each of the purine motif triplexes with various lengths and base sequences. As the length of the target triplex was increased, the binding affinity of Stm(1-113) to the target triplex was increased. Stm(1-113) had the ability to bind to the purine motif triplexes with various base sequences. We conclude that Stm(1-113) may recognize the shape of the triplex rather than the base sequence of the triplex.