A helical amplification system composed of artificial nucleic acids

Herein we report an amplification system of helical excess triggered by nucleic acid hybridization for the first time. It is usually impossible to prepare achiral nanostructures composed of nucleic acids because of their intrinsic chirality. We used serinol nucleic acid (SNA) oligomers for the preparation of achiral nanowires because SNA oligomers with symmetrical sequences are achiral. Nanowire formation was confirmed by atomic force microscopy and size exclusion chromatography. When a chiral nucleic acid with a sequence complementary to SNA was added to the nanostructure, helicity was induced and a strong circular dichroism signal was observed. The SNA nanowire could amplify the helicity of chiral nucleic acids through nucleobase stacks. The SNA nanostructures have potential for use as platforms to detect chiral biomolecules under aqueous conditions because SNA can be readily functionalized and is water-soluble.

Herein we report an amplification system of helical excess triggered by nucleic acid hybridization for the first time.     

A minimal hybridization chain reaction (HCR) system using peptide nucleic acids

The HCR represents a powerful tool for amplification in DNA-based circuitry and sensing applications, yet requires the use of long DNA sequences to grant hairpin metastability. Here we describe a minimal HCR system based on peptide nucleic acids (PNAs). A system comprising a 5-mer stem and 5-mer loop/toehold hairpins was found to be suitable to achieve rapid amplification. These hairpins were shown to yield >10-fold amplification in 2 h and be suitable for the detection of a cancer biomarker on live cells. The use of γ-peg-modified PNA was found to be beneficial.

A minimal peptide nucleic acid (PNA) HCR system based on a 5-mer stem and 5-mer loop/toehold hairpins was developed. The system was applied to the detection of a cancer biomarker on the surface of living cells.     

DNA-based constitutional dynamic networks as functional modules for logic gates and computing circuit operations

A nucleic acid-based constitutional dynamic network (CDN) is introduced as a single computational module that, in the presence of different sets of inputs, operates a variety of logic gates including a half adder, 2 : 1 multiplexer and 1 : 2 demultiplexer, a ternary multiplication matrix and a cascaded logic circuit. The CDN-based computational module leads to four logically equivalent outputs for each of the logic operations. Beyond the significance of the four logically equivalent outputs in establishing reliable and robust readout signals of the computational module, each of the outputs may be fanned out, in the presence of different inputs, to a set of different logic circuits. In addition, the ability to intercommunicate constitutional dynamic networks (CDNs) and to construct DNA-based CDNs of higher complexity provides versatile means to design computing circuits of enhanced complexity.

A nucleic acid-based constitutional dynamic network (CDN) provides a single functional computational module for diverse input-guided logic operations and computing circuits.     

Simultaneous detection of small molecules,proteins and microRNAs using single molecule arrays

Biological samples such as blood, urine, cerebrospinal fluid and saliva contain a large variety of proteins, nucleic acids, and small molecules. These molecules can serve as potential biomarkers of disease and therefore, it is desirable to simultaneously detect multiple biomarkers in one sample. Current detection techniques suffer from various limitations including low analytical sensitivity and complex sample processing. In this work, we present an ultrasensitive method for simultaneous detection of small molecules, proteins and microRNAs using single molecule arrays (Simoa). Dye-encoded beads modified with specific capture probes were used to quantify each analyte. Multiplex competitive Simoa assays were established for simultaneous detection of cortisol and prostaglandin E2. In addition, competitive and sandwich immunoassays were combined with a direct nucleic acid hybridization assay for simultaneous detection of cortisol, interleukin 6 and microRNA 141. The multi-analyte Simoa assay shows high sensitivity and specificity, which provides a powerful tool for the analysis of many different samples.

The first example of multiplexed detection of proteins, nucleic acids, and small molecules using single molecule measurement methodology.     

A bipedal DNA nanowalker fueled by catalytic assembly for imaging of base-excision repairing in living cells

DNA nanowalkers moving progressively along a prescribed DNA track are useful tools in biosensing, molecular theranostics and biosynthesis. However, stochastic DNA nanowalkers that can perform in living cells have been largely unexplored. We report the development of a novel stochastic bipedal DNA walker that, for the first time, realizes direct intracellular base excision repair (BER) fluorescence activation imaging. In our design, the bipedal walker DNA was generated by BER-related human apurinic/apyrimidinic endonuclease 1 (APE1)-mediated cleavage of DNA sequences at an abasic site in the intracellular environment, and it autonomously travelled on spherical nucleic acid (SNA) surfaces via catalyzed hairpin assembly (CHA). Our nanomachine outperforms the conventional single leg-based DNA walker with an improved sensitivity, kinetics and walking steps. Moreover, in contrast to the single leg-based DNA walker, the bipedal DNA walker is capable of monitoring the fluorescence signal of reduced APE1 activity, thus indicating amplified intracellular imaging. This bipedal DNA-propelled DNA walker presents a simple and modular amplification mechanism for intracellular biomarkers of interest, providing an invaluable platform for low-abundance biomarker discovery leading to the accurate identification and effective treatment of cancers.

The developed DNA bipedal walker represents improved sensitivity, kinetics and walking steps for intracellular fluorescence imaging of base-excision repairing.     

Amplified detection of nucleic acids and proteins using an isothermal proximity CRISPR Cas12a assay

Herein, we describe an isothermal proximity CRISPR Cas12a assay that harnesses the target-induced indiscrimitive single-stranded DNase activity of Cas12a for the quantitative profiling of gene expression at the mRNA level and detection of proteins with high sensitivity and specificity. The target recognition is achieved through proximity binding rather than recognition by CRISPR RNA (crRNA), which allows for flexible assay design. A binding-induced primer extension reaction is used to generate a predesigned CRISPR-targetable sequence as a barcode for further signal amplification. Through this dual amplification protocol, we were able to detect as low as 1 fM target nucleic acid and 100 fM target protein isothermally. The practical applicability of this assay was successfully demonstrated for the temporal profiling of interleukin-6 gene expression during allergen-mediated mast cell activation.

Herein, we develop an isothermal proximity CRISPR Cas12a assay that harnesses the target-induced collateral cleavage activity of Cas12a for the quantitative profiling of gene expression and detection of proteins with high sensitivity and specificity.     

Multiplexed droplet loop-mediated isothermal amplification with scorpion-shaped probes and fluorescence microscopic counting for digital quantification of virus RNAs

Highly sensitive digital nucleic acid techniques are of great significance for the prevention and control of epidemic diseases. Here we report the development of multiplexed droplet loop-mediated isothermal amplification (multiplexed dLAMP) with scorpion-shaped probes (SPs) and fluorescence microscopic counting for simultaneous quantification of multiple targets. A set of target-specific fluorescence-activable SPs are designed, which allows establishment of a novel multiplexed LAMP strategy for simultaneous detection of multiple cDNA targets. The digital multiplexed LAMP assay is thus developed by implementing the LAMP reaction using a droplet microfluidic chip coupled to a droplet counting microwell chip. The droplet counting system allows rapid and accurate counting of the numbers of total droplets and the positive droplets by collecting multi-color fluorescence images of the droplets in a microwell. The multiplexed dLAMP assay was successfully demonstrated for the quantification of HCV and HIV cDNA with high precision and detection limits as low as 4 copies per reaction. We also verified its potential for simultaneous digital assay of HCV and HIV RNA in clinical plasma samples. This multiplexed dLAMP technique can afford a useful platform for highly sensitive and specific detection of nucleic acids of viruses and other pathogens, enabling rapid diagnosis and prevention of infectious diseases.

The development of multiplexed dLAMP with scorpion-shaped probes and fluorescence microscopic counting affords simultaneous digital quantification of multiple virus RNAs.     

Bioorthogonal regulation of DNA circuits for smart intracellular microRNA imaging

Catalytic DNA circuits represent a versatile toolbox for tracking intracellular biomarkers yet are constrained with low anti-interference capacity originating from their severe off-site activation. Herein, by introducing an unprecedented endogenous DNA repairing enzyme-powered pre-selection strategy, we develop a sequential and specific on-site activated catalytic DNA circuit for achieving the cancer cell-selective imaging of microRNA with high anti-interference capacity. Initially, the circuitry reactant is firmly caged by an elongated stabilizing duplex segment with a recognition/cleavage site of a cell-specific DNA repairing enzyme, which can prevent undesired signal leakage prior to its exposure to target cells. Then, the intrinsic DNA repairing enzyme of target cells can liberate the DNA probe for efficient intracellular microRNA imaging via the multiply guaranteed molecular recognition/activation procedures. This bioorthogonal regulated DNA circuit presents a modular and programmable amplification strategy for highly reliable assays of intracellular biomarkers, and provides a pivotal molecular toolbox for living systems.

An on-site bioorthogonal regulated DNA circuit was developed by introducing an endogenous DNA repairing enzyme-mediated sequential activation strategy to achieve cancer cell-selective microRNA imaging with high anti-interference ability.     

A serological aptamer-assisted proximity ligation assay for COVID-19 diagnosis and seeking neutralizing aptamers

Rapid and accurate diagnosis of COVID-19 plays an essential role in the current epidemic prevention and control. Despite the promise of nucleic acid and antibody tests, there is still a great challenge to reduce the misdiagnosis, especially for asymptomatic individuals. Here we report a generalizable method for highly specific and ultrasensitive detection of serum COVID-19-associated antigens based on an aptamer-assisted proximity ligation assay. The sensor is based on binding two aptamer probes to the same protein target that brings the ligation DNA region into close proximity, thereby initiating ligation-dependent qPCR amplification. Using this system, serum nucleocapsid protein has been detected quantitatively by converting protein recognition into a detectable qPCR signal using a simple, homogeneous and fast detection workflow in ∼2 hours. In addition, this system has also been transformed into a universal platform for measuring specific interactions between spike S1 and its receptor ACE2, and more importantly demonstrated the feasibility for screening and investigation of potential neutralizing aptamers. Since in vitro selection can obtain aptamers selective for many COVID-19-associated antigens, the method demonstrated here will serve as an important tool for the diagnosis and therapeutics of COVID-19.

A versatile aptamer-assisted proximity ligation system improves diagnosis of COVID-19, and allows the evaluation of potential neutralizing aptamers.     

Mechanism of inhibition of SARS-CoV-2 Mpro by N3 peptidyl Michael acceptor explained by QM/MM simulations and design of new derivatives with tunable chemical reactivity

The SARS-CoV-2 main protease (M^pro) is essential for replication of the virus responsible for the COVID-19 pandemic, and one of the main targets for drug design. Here, we simulate the inhibition process of SARS-CoV-2 M^pro with a known Michael acceptor (peptidyl) inhibitor, N3. The free energy landscape for the mechanism of the formation of the covalent enzyme-inhibitor product is computed with QM/MM molecular dynamics methods. The simulations show a two-step mechanism, and give structures and calculated barriers in good agreement with experiment. Using these results and information from our previous investigation on the proteolysis reaction of SARS-CoV-2 M^pro, we design two new, synthetically accessible N3-analogues as potential inhibitors, in which the recognition and warhead motifs are modified. QM/MM modelling of the mechanism of inhibition of M^pro by these novel compounds indicates that both may be promising candidates as drug leads against COVID-19, one as an irreversible inhibitor and one as a potential reversible inhibitor.

QM/MM simulations identify the mechanism of reaction of N3, a covalent peptidyl inhibitor of SARS-CoV-2 main protease. Modelling of two novel proposed compounds, B1 and B2, suggests that reversibility of covalent inhibition could be tailored.     

A Janus 3D DNA nanomachine for simultaneous and sensitive fluorescence detection and imaging of dual microRNAs in cancer cells

Herein, a Janus three-dimensional (3D) DNA nanomachine was constructed for the simultaneous and sensitive fluorescence detection and imaging of dual microRNAs (miRNAs) in cancer cells, which could effectively eliminate signal interference in a homogeneous nanoparticle-based 3D DNA nanostructure caused by the proximity of the two different signal probes to achieve accurate co-location in the same position of living cancer cells. In this system, the Janus nanoparticles were synthesized as the carrier for immobilizing two different oligonucleotides on two different functionalized hemispheres of the nanoparticles to form a Janus 3D DNA nanostructure, which could convert trace amounts of miRNA-21 and miRNA-155 targets into massive FAM and Cy5-labeled duplexes to induce two remarkable fluorescence emissions by the catalytic hairpin assembly (CHA) and 3D DNA walker cascade nucleic acid amplification strategy, realizing sensitive detection and imaging of miRNA targets in cancer cells. Impressively, in comparison with current miRNA imaging methods based on nanoparticle assemblies, the proposed strategy could efficiently eliminate “false positive” results obtained in single type miRNA detection and distinctly increase the immobilization concentration of two different signal probes using Janus nanoparticles as the carrier to further enhance fluorescence intensity, resulting in accurate co-location in the same position of living cells. Meanwhile, the proposed fluorescence imaging technology makes it possible to visualize low concentrations of miRNAs with tiny change associated with some cancers, which could significantly improve the accuracy and precision compared to those of the conventional fluorescence in situ hybridization (FISH) approach. Therefore, it could serve as persuasive evidence for supplying accurate information to better understand biological processes and investigate mechanisms of various biomolecules and subcellular organelles, resulting in the further validation of their function in tumor proliferation and differentiation. This strategy provided an innovative approach to design new generations of nanomachines with ultimate applications in bioanalysis and clinical diagnoses.

A Janus three-dimensional DNA nanomachine was constructed for the simultaneous and sensitive fluorescent detection and imaging of dual microRNAs in the cancer cells.     

Revealing the molecular mechanisms of proteolysis of SARS-CoV-2 Mpro by QM/MM computational methods

SARS-CoV-2 M^pro is one of the enzymes essential for the replication process of the virus responsible for the COVID-19 pandemic. This work is focused on exploring its proteolysis reaction by means of QM/MM methods. The resulting free energy landscape of the process provides valuable information on the species appearing along the reaction path and suggests that the mechanism of action of this enzyme, taking place in four steps, slightly differs from that of other cysteine proteases. Our predictions, which are in agreement with some recently published experimental data, can be used to guide the design of COVID-19 antiviral compounds with clinical potential.

The molecular mechanism of the proteolysis reaction catalyzed by SARS-CoV-2 M^pro, one of the enzymes essential for the replication process of the virus responsible for the COVID-19 pandemic, is described using computational QM/MM methods.     

Design and enhanced gene silencing activity of spherical 2′-fluoroarabinose nucleic acids (FANA-SNAs)

Drug delivery vectors for nucleic acid therapeutics (NATs) face significant barriers for translation into the clinic. Spherical nucleic acids (SNAs) – nanoparticles with an exterior shell made up of DNA strands and a hydrophobic interior – have recently shown great potential as vehicles to improve the biodistribution and efficacy of NATs. To date, SNA design has not taken advantage of the powerful chemical modifications available to NATs. Here, we modify SNAs with 2′-deoxy-2′-fluoro-d-arabinonucleic acid (FANA-SNA), and show increased stability, enhanced gene silencing potency and unaided uptake (gymnosis) as compared to free FANA. By varying the spacer region between the nucleic acid strand and the attached hydrophobic polymer, we show that a cleavable DNA based spacer is essential for maximum activity. This design feature will be important when implementing functionalized nucleic acids into nanostructures for gene silencing. The modularity of the FANA-SNA was demonstrated by silencing two different targets. Transfection-free delivery was superior for the modified SNA compared to the free FANA oligonucleotide.

Optimizing FANA modified spherical nucleic acids (FANA-SNAs) for highly efficient delivery of nucleic acid therapeutics.     

A chiroptical approach for the absolute stereochemical determination of P-stereogenic centers

A simple chiroptical solution for the absolute stereochemical determination for asymmetric phosphorus V stereocenters is presented. Strong coordination of the phosphorus oxide with the Zn-metallo center of the racemic host Zn-MAPOL 2 leads to an induced axial chirality of the host, yielding a strong ECCD signal. A mnemonic is proposed to correlate the asymmetry of the guest molecule with the observed ECCD signal.

A simple chiroptical solution for the absolute stereochemical determination for asymmetric phosphorus V stereocenters is presented.     

TMTHSI,a superior 7-membered ring alkyne containing reagent for strain-promoted azide–alkyne cycloaddition reactions

We describe the development of TMTH-SulfoxImine (TMTHSI) as a superior click reagent. This reagent combines a great reactivity, with small size and low hydrophobicity and compares outstandingly with existing click reagents. TMTHSI can be conveniently functionalized with a variety of linkers allowing attachment of a diversity of small molecules and (peptide, nucleic acid) biologics.

TMTHSI was developed as new reagent for strain-promoted azide–alkyne cycloaddition reactions, enabling connection of a diversity of small to large molecular constructs.     

A biocompatible stapling reaction for in situ generation of constrained peptides

Constrained peptides are promising next-generation therapeutics. Peptide stapling is a particularly attractive technique to generate constrained macrocycles with improved biological activity and metabolic stability. We introduce a biocompatible two-component stapling approach based on the reagent 2,6-dicyanopyridine and a pseudo-cysteine amino acid. Stapling can proceed either directly on-resin during solid-phase synthesis or following isolation of the linear peptide. The stapling reaction is orthogonal to natural amino acid side chains and completes in aqueous solution at physiological pH, enabling its direct use in biochemical assays. We performed a small screening campaign of short peptides targeting the Zika virus protease NS2B-NS3, allowing the direct comparison of linear with in situ stapled peptides. A stapled screening hit showed over 28-fold stronger inhibition than its linear analogue, demonstrating the successful identification of constrained peptide inhibitors.

A synthetically straightforward and biocompatible peptide-stapling strategy that can be used directly in biochemical assays to identify constrained enzyme inhibitors.     

Machine-enabled inverse design of inorganic solid materials: promises and challenges

Developing high-performance advanced materials requires a deeper insight and search into the chemical space. Until recently, exploration of materials space using chemical intuitions built upon existing materials has been the general strategy, but this direct design approach is often time and resource consuming and poses a significant bottleneck to solve the materials challenges of future sustainability in a timely manner. To accelerate this conventional design process, inverse design, which outputs materials with pre-defined target properties, has emerged as a significant materials informatics platform in recent years by leveraging hidden knowledge obtained from materials data. Here, we summarize the latest progress in machine-enabled inverse materials design categorized into three strategies: high-throughput virtual screening, global optimization, and generative models. We analyze challenges for each approach and discuss gaps to be bridged for further accelerated and rational data-driven materials design.

The grand challenge of materials science, discovery of novel materials with target properties, can be greatly accelerated by machine-learned inverse design strategies.     

A pyrone remodeling strategy to access diverse heterocycles: application to the synthesis of fascaplysin natural products

The synthesis of diverse N-fused heterocycles, including the pyrido[1,2-a]indole scaffold, using an efficient pyrone remodeling strategy is described. The pyrido[1,2-a]indole core was demonstrated to be a versatile scaffold that can be site-selectively functionalized. The utility of this novel annulation strategy was showcased in a concise formal synthesis of three fascaplysin congeners.

The synthesis of diverse N-fused heterocycles, including the pyrido[1,2-a]indole scaffold, using an efficient pyrone remodeling strategy is described.     

Post translational modification-assisted cancer immunotherapy for effective breast cancer treatment

Post translational modifications (PTM) such as phosphorylation are often correlated with tumorigenesis and malignancy in breast cancer. Herein, we report a PTM-assisted strategy as a simplified version of a personalized cancer vaccine for enhanced cancer immunotherapy. Titanium modified dendritic mesoporous silica nanoparticles (TiDMSN) are applied to assist the specific enrichment of phosphorylated tumor antigens released upon immunogenic cell death. This strategy significantly improved the tumor inhibition efficacy in a bilateral breast cancer model and the expansion of both CD8⁺ and CD4⁺ T cells in the distant tumor site. The nanotechnology based PTM-assisted strategy provides a simple and generalizable methodology for effective personalized cancer immunotherapy.

The nano-enabled post-translational modification assisted strategy for effective cancer immunotherapy.