Poly(l-lysine)-graft-dextran Copolymer Promotes Pyrimidine Motif Triplex DNA Formation at Physiological pH

Abstract

Extreme instability of pyrimidine motif triplex DNA at physiological pH severely limits its use for artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is therefore of great importance in improving its therapeutic potential. To this end, isothermal titration calorimetry interaction analysis system and electrophoretic mobility shift assay have been used to explore the thermodynamic and kinetic effects of our previously reported triplex stabilizer, poly (L-lysine)-graft-dextran (PLL-g-Dex) copolymer, on pyrimidine motif triplex formation at physiological pH. Both the thermodynamic and kinetic analyses have clearly indicated that in the presence of the PLL-g-Dex copolymer, the binding constant of the pyrimidine motif triplex formation at physiological pH was about 100 times higher than that observed without any triplex stabilizer. Of importance, the triplex-promoting efficiency of the copolymer was more than 20 times higher than that of physiological concentrations of spermine, a putative intracellular triplex stabilizer. Kinetic data have also demonstrated that the observed copolymer-mediated promotion of the triplex formation at physiological pH resulted from the considerable increase in the association rate constant rather than the decrease in the dissociation rate constant. Our results certainly support the idea that the PLL-g-Dex copolymer could be a key material and may eventually lead to progress in therapeutic applications of the antigene strategy in vivo.In recent years, triplex DNA has attracted considerable interest because of its possible biological functions in vivo and its wide variety of potential applications, such as regulation of gene expression, site-specific cleavage of duplex DNA, mapping of genomic DNA, and gene-targeted mutagenesis (1-3). A triplex is usually formed through the sequence-specific interaction of a single-stranded homopurine or homopyrimidine triplex-forming oligonucleotide (TFO) 1 with the major groove of the homopurine-homopyrimidine stretch in duplex DNA (1-5).In the purine motif triplex, a homopurine TFO binds antiparallel to the homopurine strand of the target duplex by reverse Hoogsteen hydrogen bonding to form A⅐A:T (or T⅐A:T) and G⅐G:C triplets (1-5). On the other hand, in the pyrimidine motif triplex, a homopyrimidine TFO binds parallel to the homopurine strand of the target duplex by Hoogsteen hydrogen bonding to form T⅐A:T and C ϩ ⅐G:C triplets (1-5). Because the cytosine bases in a homopyrimidine TFO are to be protonated to bind with the guanine bases of the G:C duplex, the formation of the pyrimidine motif triplex needs an acidic pH condition and is thus extremely unstable at physiological pH (6 -10). The extreme instability of the pyrimidine motif triplex at physiological pH severely limits its use for artificial control of gene expression in vivo. Stabilization of the pyrimidine motif triplex at physiological pH is therefore of great importance in improving its therapeutic potential. Numerous efforts suc...

Sticky overhangs enhance siRNA-mediated gene silencing

“…The length of the single-stranded oligo was chosen such as to avoid loop formation with a single siRNA molecule. However, formation of nucleic acid networks bridged by antiparallel T⅐A helices and antiparallel/parallel T⅐A⅐T triple helices (26) are possible in the presence of a polycation. Other experiments showed that using either oligoribo or oligodeoxyribo sequences as added single-stranded oligonucleotides led to significant enhancement of silencing (data not shown).…”

Chemical Modification of Oligonucleotides: A Novel Approach Towards Gene Targeting

“…EMSA experiments for the triplex formation were performed essentially as described previously by a 15% native polyacrylamide gel electrophoresis [12,13,[19][20][21][22][23][24][25][26][27]. In a 9 µl of reaction mixture, 32 P-labeled Pur23A•Pyr23T duplex (~1 nM) (Figure 1c) was mixed with increasing concentrations of the specific TFO (Pyr15TM, Pyr15NC7-1, Pyr15NC7-2, Pyr15NC5-1, or Pyr15NC5-2) (Figure 1c) and the nonspecific oligonucleotide (Pyr15NS2M) (Figure 1c) in buffer [50 mM Tris-acetate (pH 7.0), 100 mM NaCl, and 10 mM MgCl2].…”

“…Isothermal titration experiments for the triplex formation were carried out on a VP ITC system (Microcal Inc., U.S.A.), essentially as described previously [12,13,[19][20][21][22][25][26][27]. The TFO (Figure 1c) and Pur23A•Pyr23T duplex (Figure 1c) solutions were prepared by extensive dialysis against 10 mM sodium cacodylate-cacodylic acid at pH 6.1 or pH 6.8 containing 200 mM NaCl and 20 mM MgCl2.…”

“…Kinetic experiments for the triplex formation were performed on a BIACORE J instrument (GE Healthcare, U.S.A.), in which a real-time biomolecular interaction was measured with a laser biosensor, essentially as described previously [12,13,[19][20][21][22][23][24][25]27]. The layer of a SA sensor tip with immobilized streptavidin was equilibrated with 10 mM sodium cacodylatecacodylic acid at pH 6.8 containing 200 mM NaCl and 20 mM MgCl2 at a flow rate of 30 µl/min.…”

Cationic Comb-type Copolymers Do Not Cause Collapse but Shrinkage of DNA Molecules

“…2 The copolymer with a high degree of grafting forms totally soluble DNA complexes without inducing collapse. 3 We have shown that PLL-g-Dex increases stability of DNA hybrids, 2 accelerates DNA hybridization, 4 and activates DNA strand-exchange reactions. 5 However, interaction of the copolymer with DNA is not thoroughly understood and the influence of the copolymer grafting degree on conformation of DNA has not been assessed.…”