Ruiping Wu’s research while affiliated with First Affiliated Hospital of China Medical University and other places

Through controlling the ssDNA product length of rolling circle amplification with AcyNTP, here we develop a nanopore signal enhancement strategy (STSS), which can successfully transfer the short oligonucleotide targets into...

Nanopore sensing is highly promising in single molecular analysis but their broad applications have been challenged by the limited strategies that can transduce a target‐of‐interest into a specific and anti‐false/inference signal, especially for solid‐state nanopores with relatively lower resolution and higher noise. Here we report a high‐resolution signal‐production concept named target‐induced duplex polymerization strategy (DPS). Through linking the same or different duplex substrates (DSs) with a special linker (L) and an optional structure tag (ST), the DPS can generate target‐specific DS polymers with highly controllable duration times, duration intervals and even distinguished secondary tagging currents. Experimentally, DPS mono‐polymerization of single DS and co‐polymerization of multiple DSs has verified the duration time of a DPS product is the sum of those for each DS monomer. Tetrahedron‐DNA structures with different sizes are used as the STs to provide needle‐like secondary peaks for further resolution enhancement and multiplex assay. With these examples DPS represents a general, programmable and advanced strategy that may simultaneously provide size‐amplification, concentration amplification, and signal‐specificity for molecular recognition. It is also promisingly in various applications regarding to single molecular investigation, such as polymerization degree, structure/side chain conformation, programmable multiplex decoding and information index.

Nanopore sensing is highly promising in single molecular analysis but their broad applications have been challenged by the limited strategies that can transduce a target‐of‐interest into a specific and anti‐false/inference signal, especially for solid‐state nanopores with relatively lower resolution and higher noise. Here we report a high‐resolution signal‐production concept named target‐induced duplex polymerization strategy (DPS). Through linking the same or different duplex substrates (DSs) with a special linker (L) and an optional structure tag (ST), the DPS can generate target‐specific DS polymers with highly controllable duration times, duration intervals and even distinguished secondary tagging currents. Experimentally, DPS mono‐polymerization of single DS and co‐polymerization of multiple DSs has verified the duration time of a DPS product is the sum of those for each DS monomer. Tetrahedron‐DNA structures with different sizes are used as the STs to provide needle‐like secondary peaks for further resolution enhancement and multiplex assay. With these examples DPS represents a general, programmable and advanced strategy that may simultaneously provide size‐amplification, concentration amplification, and signal‐specificity for molecular recognition. It is also promisingly in various applications regarding to single molecular investigation, such as polymerization degree, structure/side chain conformation, programmable multiplex decoding and information index.

In this paper, we propose a "reciprocal strategy" that, on the one hand, explores the ability of solid-state nanopores in a homogeneous high-fidelity characterization of nucleic acid assembly and, on the other hand, the formed nucleic acid assembly with a large size serves as an amplifier to provide a highly distinguished and anti-interference signal for molecular sensing. Four-hairpin hybridization chain reaction (HCR) with G-rich tail tags is taken as the proof-of-concept demonstration. G-rich tail tags are commonly used to form G-quadruplex signal probes on the side chain of HCR duplex concatemers. When such G-tailed HCR concatemers translocate the nanopore, abnormal, much higher nanopore signals over normal duplexes can be observed. Combined with atomic force microscopy, we reveal the G-rich tail may easily induce the "intermolecular interaction" between HCR concatemers to form "branched assembly structure (BAS)". To the best of our knowledge, this is the first evidence for the formation BAS of the G tailed HCR concatemers in a homogeneous solution. Systematic nanopore measurements further suggest the formation of these BASs is closely related to the types of salt ions, the amount of G, the concentration of substrate hairpins, the reaction time, and so forth. Under optimized conditions, these BASs can be grown to just the right size without being too large to block the pores, while producing a current 14 times that of conventional double-stranded chains. Here, these very abnormal large current blockages have, in turn, been taken as an anti-interference signal indicator for small targets in order to defend the high noises resulting from co-existing big species (e.g., enzymes or other long double-stranded DNA).

While the solid‐state nanopore shows increasing potential during sensitive and label‐free single molecular analysis, target concentration and signal amplification method is in urgent need. In this article, a solution via designing a model nucleic acid circuit reaction that can produce “Y” shape‐structure three‐way DNA oligomers with controllable size and polymerization degree is proposed. Such a so‐called lego‐like three‐way catalytic hairpin assembly (LK‐3W‐CHA) can provide both concentration amplification (via CHA circuit) and programmable size control (via lego‐like building mode) to enhance spatiotemporal resolution in single molecular sensing of solid‐state nanopore. Oligomers containing 1–4 DNA three‐way junctions (Y monomers, Y1–Y4) are designed in proof‐of‐concept experiments and applications. When the oligomers are applied to direct translocation measurements, Y2‐Y4 can significantly increase the signal resolution and stability than that of Y1. Meanwhile, Y1 to Y4 can be used as the tags on the long DNA carrier to provide very legible secondary signals for specific identification, multiple assays, and information storage. Compared with other possible tags, Y1–Y4 provides higher signal density and amplitude, and quasi‐linear “inner reference” for each other, which may provide more systematic, reliable, and controllable experimental results.

As an important enzyme-free amplifier, the hybridization chain reaction (HCR) uses an ssDNA to trigger cycled displacement interactions between substrate hairpins and finally form elongated dsDNA concatamer mixtures. In many cases, to provide a signal probe or advanced function, additional oligonucleotides (named hairpin tails) have to be extended upon classic HCR hairpin substrates, but by doing so the HCR assembly efficiency and signal-to-noise ratio (SNR) may get seriously reduced. In this Article, a rational and general model that may guide the study on HCR functionalization and signaling efficiency is provided. We rationally design a four-hairpin model HCR system (4H-HCR) in which one or more hairpin substrates are appended with additional tails as a signaling probe. After HCR assembly, two adjacent tails are supposedly integrating into a full G-quadruplex structure to provide the evidence or signal for the assembly. A systematic study has been applied to reveal the relationship between the “tail-design” with assembly efficiency and SNR. A clear design rule-set guiding the optimized assembly and signal has been provided for traditional electrophoresis and G-quadruplex-enhanced fluorescence signal. Importantly, solid-state nanopore single molecular detection has been innovatively introduced and recommended as an “antirisk” and “mutual benefit” readout to traditional G-quadruplex signaling. Nanopore detection can provide a clear signal distinguished before and after the HCR reaction, especially when the traditional G-quadruplex-enhanced signal only provides low SNR. The G-quadruplex, in turn, may enhance the nanopore signal amplitude via increasing the diameter of the HCR products.

Solid-state nanopores show special potential as a new single-molecular characterization for nucleic acid assemblies and molecular machines. However, direct recognition of small dimensional species is still quite difficult due the lower resolution compared with biological pores. We recently reported a very efficient noise-reduction and resolution-enhancement mechanism via introducing high-dielectric additives (e.g., formamide) into conical glass nanopore (CGN) test buffer. Based on this advance, here, for the first time, we apply a bare CGN to directly recognize small dimensional assemblies induced by small molecules. Cocaine and its split aptamer (Capt assembly) are chosen as the model set. By introducing 20% formamide into CGN test buffer, high cocaine-specific distinguishing of the 113 nt Capt assembly has been realized without any covalent label or additional signaling strategies. The signal-to-background discrimination is much enhanced compared with control characterizations such as gel electrophoresis and fluorescence resonance energy transfer (FRET). As a further innovation, we verify that low-noise CGN can also enhance the resolution of small conformational/size changes happening on the side chain of large dimensional substrates. Long duplex concatamers generated from the hybridization chain reaction (HCR) are selected as the model substrates. In the presence of cocaine, low-noise CGN has sensitively captured the current changes when the 26 nt aptamer segment is assembled on the side chain of HCR duplexes. This paper proves that the introduction of the low-noise mechanism has significantly improved the resolution of the solid-state nanopore at smaller and finer scales and thus may direct extensive and deeper research in the field of CGN-based analysis at both single-molecular and statistical levels, such as molecular recognition, assembly characterization, structure identification, information storage, and target index.

Solid-state nanopores have shown special high potential in a label-free molecular assay, structure identification, and target-index at the single-molecular level, even though frustrating electrical baseline noise is still one of the major factors that limit the spatial resolution and signaling reliability of solid-state nanopores, especially in small target detection. Here we develop a significant and easy-operating noise-reduction approach via mixing organic solvents with high dielectric constants into a traditional aqueous electrolyte. The strategy is generally effective for pores made of different materials, such as the most commonly used conical glass (CGN) or SiN x . While the mechanism should be multisourced, MD simulations suggest the noise reduction may partially arise from the even ionic distribution caused by the addition of higher dielectric species. Among all solvents experimentally tested, the two with the highest dielectric constants, formamide and methylformamide, exhibit the best noise reduction effect for target detection of CGN. The power spectral density at the low-frequency limit is reduced by nearly 3 orders with the addition of 20% formamide. Our work qualifies the reliability of solid-state nanopores into much subtler scales of detection, such as dsDNAs under 100 bp. As a practical example, bare CGN is innovatively employed to perform in-situ tracking of trigger-responsive DNA machine forming oligomers.

Glass capillary-based nanopore is exploited for single-molecule conformational sensing of multi-arm DNA concatemers during translocation. Both translocation frequency and orientation preference were found related with the number of arms of the DNA concatemers.

Recent advances have proven solid-state nanopore is a powerful analysis platform that enables label-free and separation-free single-molecule analysis. However, the relatively low resolution still limits its application because many chemicals or targets with small sizes could not be recognized in a label-free condition. In this paper we provide a possible solution that uses solid-state nanopore for small species signaling via the transition of huge DNA assembly products. DNAzyme responding to metal ions, and hybridized chain reaction (HCR) generating nanopore detectable dsDNA concatamers are used as the transition model set. By the two-step DNAzyme-HCR transition, Pb(II) that is too tiny to be sensed is successfully recognized by the nanopore. And the whole process happens in completely homogeneous solution without any chemical modification. During condition optimization we also discuss one possible application challenge that may affect the HCR signal-background distinction. Solid-state nanopore provides a potential solution to this challenge due to its ability to profile product length or even 3D structure information.