Optimisation by selection for oligonucleotide-based synthetic molecular machinery

Thesis

Nucleic acid nanotechnology has emerged as a promising frontier for artificial molecular machines, owing to the intrinsic programmability of nucleic acids. However, its potential is constrained by the slow kinetics of strand displacement reactions, which typically occur over minutes. In contrast, ATP hydrolysis-powered biological motors operate on millisecond timescales. Inspired by the efficient chemomechanical cycles of nature, this thesis aims to develop nucleic acid-based motors that allosterically bind to their fuel, catalyse its chemical conversion, and autonomously repeat this cycle until fuel depletion.

The primary strategy used was in vitro selection to develop nucleic acid-based mechanocatalysts that couple the chemical reactions of small-molecule fuels with the mechanical cycles of nucleic acid nanostructures. GTP was chosen as the fuel and covalently attached to a library containing an integrated triphosphate-specific aptamer. After eight rounds of selection and high-throughput sequencing, ten enriched sequences were identified. The most active sequence exhibited activity comparable to that of RNA 5′pyrophosphohydrolase, which converts the 5′triphosphate of RNA into a 5′monophosphate. Truncation experiments identified a minimal 77-nucleotide ribozyme that retained catalytic activity. Kinetic analysis revealed an observed rate constant of k_obs = 0.08min⁻¹. However, under multiple-turnover conditions (with free GTP), no significant catalytic activity was observed. Another strategy attempted to replace the adenine-specific aptamer in a catalyst-aptamer conjugate with a triphosphate-recognising aptamer. However, this design was hindered by difficulties in synthesising the organic catalytic centre.

This work explores a new approach to enhancing nucleic acid-based molecular machines. Although sustained catalytic activity was not achieved under multiple-turnover conditions, this thesis provides insight into the feasibility and challenges of developing enzyme-like autonomous molecular machines and informs future developments.

Authors: Zhang, Q

• DOI: 10.5287/ora-ozao6bbpo
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: ribozyme; in vitro selection; molecular machines; functional nucleic acids; DNA nanotechnology
• Subjects: Nucleic acids; Nanotechnology; Biochemistry; Biophysics