Nanoscale Organization and Optical Observation of Biomolecules With DNA Nanotechnology

Abstract

Understanding biomolecular information at the single-molecule level requires tools for manipulating and observing individual biomolecules at the nanoscale. Programmable DNA nanotechnology provides an ideal interface to bridge engineering principles with biomolecular compatibility, especially with high-information-content, programmable molecular interactions. In my dissertation research, I have focused on two specific topics that both harness the programmable and high-information-content nature of complementary DNA interactions, to arrange and observe biomolecules at the single-molecule level, and with high spatial precision.

First, I studied the capability of using self-assembled DNA nanostructure to pattern biomolecules with high precision and tunable spatial arrangement. Previous efforts in DNA nanostructure synthesis with complex patterning have mostly focused on rigid tile-based or DNA origami approaches, which did not provide a modular and scalable method. With my colleagues, I have designed and assembled complex and programmable two-dimensional nano-patterns with a simple and robust synthesis method, based on flexible single-stranded DNA tiles (SST). This method allowed for a modular, scalable, and synthetically economic way of synthesis and biomolecule patterning at the nanoscale, for potential use of studying molecular interactions and construction of novel biomolecular devices.

Next, I investigated the capability of using programmable transient DNA hybridisation for optical super-resolution imaging of single biomolecular targets. Recent advances in fluorescence super-resolution microscopy have circumvented the conventional diffraction limit and shown images of sub-cellular features and synthetic nanostructures down to ~15 nm in size, but observation of individual molecular targets remains difficult, and has only been shown with multiple labelling and tens of nanometres target separation. In particular, direct optical observation of individual molecular targets in a densely packed (~5 nm spacing) biomolecular cluster has not been demonstrated. I called this concept "discrete molecular imaging (DMI) and tackled this challenge as part of my dissertation work by adopting the DNA-PAINT method, which utilised programmable transient DNA hybridisation for localisation-based super-resolution microscopy. I proposed systematic characterisation and optimisation of four technical requirements to achieve DMI with DNA-PAINT. I examined the effects of high photon count, high blinking statistics and appropriate blinking duty cycle on imaging quality, and reported a novel software-based drift correction method that achieves <1 nm residual drift (r.m.s.) over hours. With this method, I reported fluorescence imaging of a densely packed triangular lattice pattern with ~5 nm point-to-point distance, and analysed DNA origami structural offset with angstrom-level precision (<2 A) from single-molecule studies. Combined with multiplexed exchange-PAINT imaging, I further demonstrated an optical nano-display with 5x5 nm pixel size and three distinct colours, and with <1 nm cross-channel registration accuracy.

After this study, I further extended the capability of single-molecule observation from nanostructures to cellular environments. Super-resolution imaging of single molecular targets have been difficult in cellular context, due to high levels of fluorescence background and potential crosstalk between multiple fluorophores. In my dissertation work, I proposed a data analysis framework that exploits the repetitive blinking that is typical of DNA-PAINT, and performs temporal analysis on single-target blinking time traces to detect single targets in noisy environment. With my colleagues, I first studied the possibility of kinetic trace profiling and accurate blinking on-time for kinetic multiplexing, then applied the method to detect single-copy mRNA targets in situ. As a proof of principle, I demonstrated specific and sensitive single-target detection with this method in fixed cells.

Taken together, the two branches of nanotechnology that have tried to develop during my dissertation, a modular and versatile synthesis method for nanoscale molecular organisation and templating, as well as an imaging method capable of visualising and interrogating singly-labelled molecular targets, from a complementary package of nanoscale research tools towards enabling a thorough molecular characterisation of biology.