DNA-polymer conjugates via the graft-through polymerisation of native DNA in water

Abstract

The direct, graft-through, ring-opening metathesis polymerisation (ROMP) of unprotected DNA macromonomers is reported. By tuning the polymerisation conditions, good control is achieved, enabling the rapid and efficient synthesis of DNA-containing bottlebrush copolymers, without the need for protection of the DNA bases.

Fig. 1. Synthesis of DNA-containing bottlebrush polymers via a macroinitiator approach in water. A water-soluble macroinitiator is first synthesised by ROMP using Grubbs’ third generation catalyst (G3), and then transferred to an aqueous solution for chain extension. Use of a norbornene-terminated DNA strand allows direct access to the desired DNA-polymer conjugates without the requirement for protection.

Fig. 2. Effect of the presence of the nucleobase guanine (G) on macroinitiator chain extension, measured by SEC. A clear loss of control was observed when guanine was present (blue trace). Recovery of polymerisation control was possible by addition of succinimide (brown trace). Eluent: DMF + 5 mM NH₄BF₄, PMMA standards.

Fig. 3. DMF SEC analysis of DNA-BBPs with UV-vis detection at 559 nm. Each DNA-BBP (dash line) is compared with the analogous DNA-free control polymer (solid line). (a) DNA-BBP1 and BBP1. (b) DNA-BBP2 and BBP2. All plots have been normalised to the BBP peak.

Fig. 4. Denaturing PAGE analysis of (a) DNA-BBP1 and (b) DNA-BBP2 visualised by TAMRA fluorescence. In both cases the lanes are as follows: 1 = *DNA-NH2; 2 = DNA-free BBP (BBP1 or BBP2 as appropriate); 3 = *DNA-NH2 + DNA-free BBP mixed after polymerisation; 4 = *DNA-NH2 + DNA-free BBP mixed during polymerisation; 5 = DNA-BBP (DNA-BBP1 or DNA-BBP2). Densitometric plots are included to the right of each gel, with the following band densities recorded: (a) 41% DNA-BBP1, 59% free DNA. (b) 15% DNA-BBP2, 85% free DNA.

Fig. 5. PEG chain collapse measured by changes in the fluorescence of an environment-sensitive ACM dye. The ACM monomer (black circles) and DNA-BBP2˙ (blue dots) show a decrease in fluorescence as water content increases, due to quenching of the ACM by the protic solvent. By contrast, DNA-BBP1˙ (red dots) recovers its fluorescence at high water contents, indicating chain collapse.