ABSTRACT
We systematically investigated effects of molecular crowding with trimethylamine N-oxide (TMAO) as a zwitterionic and protective osmolyte and urea as a nonionic denaturing osmolyte on conformation and thermodynamics of the canonical DNA duplex and the non-canonical DNA G-quadruplex. It was found that TMAO and urea stabilized and destabilized, respectively, the G-quadruplex. On the other hand, these osmolytes generally destabilize the duplex; however, it was observed that osmolytes having the trimethylamine group stabilized the duplex at the lower concentrations because of a direct binding to a groove of the duplex. These results are useful not only to predict DNA structures and their thermodynamics under physiological environments in living cells, but also design of polymers and materials to regulate structure and stability of DNA sequences.
ABSTRACT
DNA sequences capable of forming triplexes induce DNA double-strand breaks that have attracted attention in genome editing technologies (e.g., CRISPR/Cas9 system, TALEN, and ZFN). Therefore, novel functional tools that stabilize triplex DNA structures must be further investigated to spark renewed interest. In this study, we investigated the unique character of cationic comb-type copolymers for the selective stabilization of triplex DNA. The melting temperature (Tm) of triplex DNA increased from 24.5 to 73.0 °C (ΔTm = 48.5 °C) by the addition of poly(allylamine)-graft-dextran (PAA-g-Dex) under physiological conditions (at pH 7.0), while PAA-g-Dex did not stabilize but rather destabilized the DNA duplex. On the other hand, poly(l-lysine)-graft-dextran (PLL-g-Dex) stabilized both the duplex and triplex structures at pH 7.0. Thermodynamic parameters evaluated by isothermal titration calorimetry (ITC) revealed that the binding constant (Ka) for the intermolecular triplex formation in the presence of PAA-g-Dex was 1.1 × 109 M-1 at 25 °C which is more than 10 times larger than that in the presence of PLL-g-Dex (8.6 × 107 M-1). Stabilizing activity and selectivity of cationic copolymers toward DNA assemblies were successfully controlled by selecting appropriate backbone structures of the copolymer. Various functional molecules that stabilize DNA duplexes have been developed and used in biological research. However, there are few cationic polymers that stabilize triplex DNA selectively. This study indicates that PAA-g-Dex has great potential to regulate the biological activities of triplex DNA.
Subject(s)
DNA/chemistry , Dextrans/pharmacology , Excipients/pharmacology , Polyamines/pharmacology , Polylysine/analogs & derivatives , Dextrans/chemistry , Excipients/chemistry , Models, Molecular , Nucleic Acid Conformation/drug effects , Nucleic Acid Denaturation/drug effects , Polyamines/chemistry , Polylysine/chemistry , Polylysine/pharmacology , ThermodynamicsABSTRACT
Although the B-form duplex is the canonical DNA structure, the A-form duplex plays critical roles in controlling gene expression. Here, reversible B-A transitions of DNA duplexes were induced by synthetic cationic and anionic polymers. Thermodynamic analysis demonstrated that the B-A transition was regulated by the dehydration of the DNA duplex caused by polymer binding.