Viscoelastic and Thermoreversible Networks Crosslinked by Non-covalent Interactions Between "Clickable" Nucleic Acids Oligomers and DNA

Abstract

An approach to efficient and scalable production of oligonucleotide-based gel networks is presented. Specifically, a new class of xenonucleic acid (XNA) synthesized through a scalable and efficient thiol-ene polymerization mechanism, "Clickable" Nucleic Acids (CNAs), were conjugated to a multifunctional poly(ethylene glycol), PEG. In the presence of complementary single stranded DNA (ssDNA), the macromolecular conjugate assembled into a crosslinked 3D gel capable of achieving storage moduli on the order of 1 kPa. Binding studies between the PEG-CNA macromolecule and complementary ssDNA indicate that crosslinking is due to the CNA/DNA interaction. Gel formation was specific to the base sequence and length of the ssDNA crosslinker. The gels were fully thermoreversible, completely melting at temperatures above 60°C and re-forming upon cooling over multiple cycles and with no apparent hysteresis. Shear stress relaxation experiments revealed that relaxation dynamics are dependent on crosslinker length, which is hypothesized to be an effect of the polydisperse CNA chains. Arrhenius analysis of characteristic relaxation times was only possible for shorter crosslinker lengths, and the activation energy for these gels was determined to be 110 ± 20 kJ/mol. Overall, the present work demonstrates that CNA is capable of participating in stimuli-responsive interactions that would be expected from XNAs, and that these interactions support 3D gels that have potential uses in biological and materials science applications.

Figure 1 -

a) Synthesis of 8PEG-T was achieved through a copolymerization technique. The solid product was obtained after dialysis and deprotection. b) Comparison between a DNA monomer and CNA monomer. The CNA repeat unit maintains the 6-atom spacing allowing for the hybridization to complementary nucleic acids.

Figure 2.

a) Microscale thermophoresis titration experiment showing binding between 8PEG-T and a Cy-5 labelled A20 ssDNA. Solvent was 20% H₂O in DMSO. A20 DNA was supplied at 50 nM. Data points are averages of technical replicates, n=3. b) Circular dichroism of A20 ssDNA only and A20 bound to 8PEG-T. The profile of the 8PEG-T alone was subtracted out of the spectra showing binding (red curve). Spectra are averages of technical replicates, n=3.

Figure 3.

a) Gels only form when the ssDNA crosslinker is complementary and sufficiently long enough to bridge two adjacent 8PEG-T molecules b) The CNA/DNA gel’s modulus was tracked as a function of time, rising to over 1 kPa in around 10 min. Rheology done at 0.1% strain and 1 Hz. c) Hypothesized mechanism of gelation including cycles and the potential for multiple CNA oligomers to bind to one ssDNA crosslinker

Figure 4 –

a) Melting and reconstitution of the CNA/DNA gel. Gels were made at 5% w/v 8PEG-T, with an A40 ssDNA, at 1:1 A:T, in 20% H₂O in DMSO. Rheology was performed at 5% strain at 1 Hz. b) Thermal cycles of CNA/DNA gels showing complete reversibility.

Figure 5 –

a) Circular dichroism of the melting transition of 8PEG-T and A20. The signal at 290 nm was tracked as a function of temperature and fit to a sigmoidal decay curve yielding a melting point of 24.5°C. Data points are averages of technical replicates, n=3. b) CNA/DNA gels were prepared at either 5% w/v or 3% w/v 8PEG-T and subjected to thermally induced melting. The gels were found to melt quicker and at a lower temperature at 3% w/v than at 5% w/v.

Figure 6 –

(a) Oscillation sweep of 5% w/v CNA/DNA gel. (b) Frequency sweep of 5% w/v CNA/DNA gel. Gels were made with a 1:1 A:T ratio, in 20% H₂O in DMSO. Plots are representative of 3 replicates.

Figure 7 –

Representative stress relaxation plots for (a) A40 crosslinker and (b) A20 crosslinker. Relaxation profiles overlap regardless of the temperature with A40 as the crosslinker, but exhibit temperature dependent relaxation with the A20 crosslinker. Rheology was performed with a 10% immediate initial strain in both cases.