Person:

Dai, Mingjie

Programmed self-assembly of strands of nucleic acid has proved highly effective for creating a wide range of structures with desired shapes. A particularly successful implementation is DNA origami, in which a long scaffold strand is folded by hundreds of short auxiliary strands into a complex shape. Modular strategies are in principle simpler and more versatile and have been used to assemble DNA or RNA tiles into periodic and algorithmic5 two-dimensional lattices, extended ribbons and tubes, three-dimensional crystals, polyhedra and simple finite two-dimensional shapes. But creating finite yet complex shapes from a large number of uniquely addressable tiles remains challenging. Here we solve this problem with the simplest tile form, a 'single-stranded tile' (SST) that consists of a 42-base strand of DNA composed entirely of concatenated sticky ends and that binds to four local neighbours during self-assembly. Although ribbons and tubes with controlled circumferences have been created using the SST approach, we extend it to assemble complex two-dimensional shapes and tubes from hundreds (in some cases more than one thousand) distinct tiles. Our main design feature is a self-assembled rectangle that serves as a molecular canvas, with each of its constituent SST strands—folded into a 3 nm-by-7 nm tile and attached to four neighbouring tiles—acting as a pixel. A desired shape, drawn on the canvas, is then produced by one-pot annealing of all those strands that correspond to pixels covered by the target shape; the remaining strands are excluded. We implement the strategy with a master strand collection that corresponds to a 310-pixel canvas, and then use appropriate strand subsets to construct 107 distinct and complex two-dimensional shapes, thereby establishing SST assembly as a simple, modular and robust framework for constructing nanostructures with prescribed shapes from short synthetic DNA strands.

Recent advances in fluorescence super-resolution microscopy have allowed sub-cellular features and synthetic nanostructures down to ~15 nm in size to be imaged. However, direct optical observation of individual molecular targets (~5 nm) in a densely packed biomolecular cluster remains a challenge. Here, we show that such discrete molecular imaging is possible using DNA-PAINT (points accumulation for imaging in nanoscale topography) - a super-resolution fluorescence microscopy technique that exploits programmable transient oligonucleotide hybridisation - on synthetic DNA nanostructures. We examined the effects of high photon count, high blinking statistics, and appropriate blinking duty cycle on imaging quality, and developed a software-based drift correction method that achieves <1 nm residual drift (r.m.s.) over hours. This allowed us to image a densely packed triangular lattice pattern with ~5 nm point-to-point distance, and analyse DNA origami structural offset with angstrom-level precision (2 Å) from single-molecule studies. By combining the approach with multiplexed Exchange-PAINT imaging, we further demonstrated an optical nano-display with 5×5 nm pixel size and three distinct colours, and with <1 nm cross-channel registration accuracy. This method opens up possibilities for direct and quantitative optical observation of individual biomolecular features in crowded environments.

Current super-resolution techniques offer unprecedented spatial resolution, but quantitative counting of spatially unresolvable molecules remains challenging. Here, we use the programmable and specific binding of dye-labeled DNA probes to count integer numbers of targets. This method, called quantitative Points Accumulation In Nanoscale Topography (qPAINT), avoids the challenging task of analyzing the environmentally sensitive hard-to-predict photophysics of dyes, and enables robust counting by analyzing the predictable binding kinetics of dye-labeled DNA probes. We benchmarked qPAINT in vitro and in situ by counting strands on DNA nanostructures, Nup98 protein clusters in the nuclear pore complex, Bruchpilot proteins in Drosophila, and finally the number of fluorescence in situ hybridization probes on single mRNA targets in fixed cells. We achieved high accuracy (~98–99 %), high precision (~80–95 %), and multiplexed detection over a large dynamic range.

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.

Self-folding of an information-carrying polymer into a defined structure is foundational to biology and offers attractive potential as a synthetic strategy. Although multicomponent self-assembly has produced complex synthetic nanostructures, unimolecular folding has seen limited progress. We describe a framework to design and synthesize a single DNA or RNA strand to self-fold into a complex yet unknotted structure that approximates an arbitrary user-prescribed shape. We experimentally construct diverse multikilobase single-stranded structures, including a ~10,000-nucleotide (nt) DNA structure and a ~6000-nt RNA structure. We demonstrate facile replication of the strand in vitro and in living cells. The work here thus establishes unimolecular folding as a general strategy for constructing complex and replicable nucleic acid nanostructures, and expands the design space and material scalability for bottom-up nanotechnology.

Super-resolution fluorescence microscopy is a powerful tool for biological research, but obtaining multiplexed images for a large number of distinct target species remains challenging. Here we use the transient binding of short fluorescently labeled oligonucleotides (DNA-PAINT, a variation of point accumulation for imaging in nanoscale topography) for simple and easy-to-implement multiplexed super-resolution imaging that achieves sub-10-nm spatial resolution in vitro on synthetic DNA structures. We also report a multiplexing approach (Exchange-PAINT) that allows sequential imaging of multiple targets using only a single dye and a single laser source. We experimentally demonstrate ten-color super-resolution imaging in vitro on synthetic DNA structures as well as four-color two-dimensional (2D) imaging and three-color 3D imaging of proteins in fixed cells.

Many genome-processing reactions, including transcription, replication and repair, generate DNA rotation. Methods that directly measure DNA rotation, such as rotor bead tracking angular optical trapping and magnetic tweezers, have helped to unravel the action mechanisms of a range of genome-processing enzymes that includes RNA polymerase (RNAP), gyrase2, a viral DNA packaging motor and DNA recombination enzymes8. Despite the potential of rotation measurements to transform our understanding of genome-processing reactions, measuring DNA rotation remains a difficult task. The time resolution of existing methods is insufficient for tracking the rotation induced by many enzymes under physiological conditions, and the measurement throughput is typically low. Here we introduce origami-rotor-based imaging and tracking (ORBIT), a method that uses fluorescently labelled DNA origami rotors to track DNA rotation at the single-molecule level with a time resolution of milliseconds. We used ORBIT to track the DNA rotations that result from unwinding by the RecBCD complex, a helicase that is involved in DNA repair9, as well as from transcription by RNAP. We characterized a series of events that occur during RecBCD-induced DNA unwinding—including initiation, processive translocation, pausing and backtracking—and revealed an initiation mechanism that involves reversible ATP-independent DNA unwinding and engagement of the RecB motor. During transcription by RNAP, we directly observed rotational steps that correspond to the unwinding of single base pairs. We envisage that ORBIT will enable studies of a wide range of interactions between proteins and DNA.