No abstract

While single-molecule sensing offers the ultimate detection limit, its throughput is often restricted as sensing events are carried out one at a time in most cases. 2D and 3D DNA origami nanostructures are used as expanded single-molecule platforms in a new mechanochemical sensing strategy. As a proof of concept, six sensing probes are incorporated in a 7-tile DNA origami nanoassembly, wherein binding of a target molecule to any of these probes leads to mechanochemical rearrangement of the origami nanostructure, which is monitored in real time by optical tweezers. Using these platforms, 10 pM platelet-derived growth factor (PDGF) are detected within 10 minutes, while demonstrating multiplex sensing of the PDGF and a target DNA in the same solution. By tapping into the rapid development of versatile DNA origami nanostructures, this mechanochemical platform is anticipated to offer a long sought solution for single-molecule sensing with improved throughput.

Conspectus Methods for detecting and quantifying disease biomarkers in biofluids with high specificity and sensitivity play a pivotal role in enabling clinical diagnostics, including point-of-care tests. The most widely used molecular biomarkers include proteins, nucleic acids, hormones, metabolites, and other small molecules. While numerous methods have been developed for analyzing biomarkers, most techniques are challenging to implement for clinical use due to insufficient analytical performance, high cost, and/or other practical shortcomings. For instance, the detection of cell-free nucleic acid (cfNA) biomarkers by digital PCR and next-generation sequencing (NGS) requires time-consuming nucleic acid extraction steps, often introduces enzymatic amplification bias, and can be costly when high specificity is required. While several amplification-free methods for detecting cfNAs have been reported, these techniques generally suffer from low specificity and sensitivity. Meanwhile, the quantification of protein biomarkers is generally performed using immunoassays such as enzyme-linked immunosorbent assay (ELISA); the analytical performance of these methods is often limited by the availability of antibodies with high affinity and specificity as well as the significant nonspecific binding of antibodies to assay surfaces. To address the drawbacks of existing biomarker detection methods and establish a universal diagnostics platform capable of detecting different types of analytes, we have developed an amplification-free approach, named single-molecule recognition through equilibrium Poisson sampling (SiMREPS), for the detection of diverse biomarkers with arbitrarily high specificity and single-molecule sensitivity. SiMREPS utilizes the transient, reversible binding of fluorescent detection probes to immobilized target molecules to generate kinetic fingerprints that are detected by single-molecule fluorescence microscopy. The analysis of these kinetic fingerprints enables nearly perfect discrimination between specific binding to target molecules and any nonspecific binding. Early proof-of-concept studies demonstrated the in vitro detection of miRNAs with a limit of detection (LOD) of approximately 1 fM and >500-fold selectivity for single-nucleotide polymorphisms. The SiMREPS approach was subsequently expanded to the detection of rare mutant DNA alleles from biofluids at mutant allele fractions of as low as 1 in 1 million, corresponding to a specificity of >99.99999%. Recently, SiMREPS was generalized to protein quantification using dynamically binding antibody probes, permitting LODs in the low-femtomolar to attomolar range. Finally, SiMREPS has been demonstrated to be suitable for the in situ detection of miRNAs in cultured cells, the quantification of small-molecule toxins and drugs, and the monitoring of telomerase activity at the single-molecule level. In this Account, we discuss the principles of SiMREPS for the highly specific and sensitive detection of molecular analytes, including considerations for assay desi...

Targeting DNA G-quadruplexes using small-molecule ligands has shown to modulate biological functions mediated by G-quadruplexes inside cells. Given >716 000 G-quadruplex hosting sites in human genome, the specific binding of ligands to quadruplex becomes problematic. Here, we innovated a polyvalency based mechanism to specifically target multiple telomeric G-quadruplexes. We synthesized a tetrameric telomestatin derivative and evaluated its complex polyvalent binding with multiple G-quadruplexes by single-molecule mechanical unfolding in laser tweezers. We found telomestatin tetramer binds to multimeric telomeric G-quadruplexes >40 times stronger than monomeric quadruplexes, which can be ascribed to the polyvalency induced unstacking of binding units (or PIU binding) for G-quadruplexes. While stacking of telomestatin units in the tetramer imparts steric hindrance for the ligand to access stand-alone G-quadruplexes, the stacking disassembles to accommodate the potent polyvalent binding between the tetramer ligand and multimeric G-quadruplexes. We anticipate this adaptive PIU binding offers a generic mechanism to selectively target polymeric biomolecules prevalent inside cells.

While single‐molecule sensing offers the ultimate detection limit, its throughput is often restricted as sensing events are carried out one at a time in most cases. 2D and 3D DNA origami nanostructures are used as expanded single‐molecule platforms in a new mechanochemical sensing strategy. As a proof of concept, six sensing probes are incorporated in a 7‐tile DNA origami nanoassembly, wherein binding of a target molecule to any of these probes leads to mechanochemical rearrangement of the origami nanostructure, which is monitored in real time by optical tweezers. Using these platforms, 10 pM platelet‐derived growth factor (PDGF) are detected within 10 minutes, while demonstrating multiplex sensing of the PDGF and a target DNA in the same solution. By tapping into the rapid development of versatile DNA origami nanostructures, this mechanochemical platform is anticipated to offer a long sought solution for single‐molecule sensing with improved throughput.

The formation of biologically significant tetraplex DNA species, such as G-quadruplexes and i-motifs, is affected by chemical (ions and pH) and mechanical [superhelicity (σ) and molecular crowding] factors. Because of the extremely challenging experimental conditions, the relative importance of these factors on tetraplex folding is unknown. In this work, we quantitatively evaluated the chemical and mechanical effects on the population dynamics of DNA tetraplexes in the insulin-linked polymorphic region using magneto-optical tweezers. By mechanically unfolding individual tetraplexes, we found that ions and pH have the largest effects on the formation of the G-quadruplex and i-motif, respectively. Interestingly, superhelicity has the second largest effect followed by molecular crowding conditions. While chemical effects are specific to tetraplex species, mechanical factors have generic influences. The predominant effect of chemical factors can be attributed to the fact that they directly change the stability of a specific tetraplex, whereas the mechanical factors, superhelicity in particular, reduce the stability of the competing species by changing the kinetics of the melting and annealing of the duplex DNA template in a nonspecific manner. The substantial dependence of tetraplexes on superhelicity provides strong support that DNA tetraplexes can serve as topological sensors to modulate fundamental cellular processes such as transcription.

While single-molecule sensing has offered ultimate mass sensitivity at the precision of individual molecules, it requires longer time to detect analytes at lower concentrations when analyte binding to single-molecule probes becomes diffusion-limited. Here, we solved this accuracy problem in the concentration sensitivity determination by using single-molecule DNA homopolymers, in which up to 473 identical sensing elements (DNA hairpins) were introduced by rolling circle amplification. Surprisingly, the DNA homopolymers containing as few as 10 tandem hairpins displayed ensemble unfolding/refolding transitions, which were exploited to recognize microRNAs (miRNAs) that populated unfolded hairpins. Within 20 mins, femtomolar detection limit for miRNAs was observed, 6 orders of magnitude better than standalone hairpins. By incorporating different hairpin probes in an alternating DNA copolymer, multiplex recognition of different miRNAs was demonstrated. These DNA co-polymers represent new materials for innovative sensing strategies that combine the single-molecule precision with the accuracy of ensemble assays to determine concentration sensitivities.

The separate arrangement of target recognition and signal transduction in conventional biosensors often compromises the real-time response and can introduce additional noise. To address these issues, we combined analyte recognition and signal reporting by mechanochemical coupling in a single-molecule DNA template. We incorporated a DNA hairpin as a mechanophore in the template, which, under a specific force, undergoes stochastic transitions between folded and unfolded hairpin structures (mechanoescence). Reminiscent of a tuning fork that vibrates at a fixed frequency, the device was classified as a molecular tuning fork (MTF). By monitoring the lifetime of the folded and unfolded hairpins with equal populations, we were able to differentiate between the mono- and bivalent binding modes during individual antibody-antigen binding events. We anticipate these mechanospectroscopic concepts and methods will be instrumental for the development of novel bioanalyses.