CoLAMP: CRISPR-based one-pot loop-mediated isothermal amplification enables at-home diagnosis of SARS-CoV-2 RNA with nearly eliminated contamination utilizing amplicons depletion strategy.

Rapid point-of-care diagnostics, essential in settings such as airport on-site testing and home-based screening, displayed important implications for infectious disease control during the SARS-CoV-2 outbreak. However, the deployment of simple and sensitive assays in real-life scenarios still faces the concern of aerosol contamination. Here, we report an amplicon-depleting CRISPR-based one-pot loop-mediated isothermal amplification (CoLAMP) assay for point-of-care diagnosis of SARS-CoV-2 RNA. In this work, AapCas12b sgRNA is designed to recognize the activator sequence sited in the loop region of the LAMP product, which is crucial for exponential amplification. By destroying the aerosol-prone amplifiable products at the end of each amplification reaction, our design can significantly reduce the amplicons contamination that causes false positive results in point-of-care diagnostics. For at-home self-testing, we designed a low-cost sample-to-result device for fluorescence-based visual interpretation. As well, a commercial portable electrochemical platform was deployed as a proof-of-concept of ready-to-use point-of-care diagnostic systems. The field deployable CoLAMP assay can detect as low as 0.5 copies/µL of SARS-CoV-2 RNA in clinical nasopharyngeal swab samples within 40 min without the need for specialists for its operation.

Integrating Magnetic-Bead-Based Sample Extraction and Molecular Barcoding for the One-Step Pooled RT-qPCR Assay of Viral Pathogens without Retesting.

Pooling multiple samples prior to real-time reverse-transcription polymerase chain reaction (RT-PCR) analysis has been proposed as a strategy to minimize expenses and boost test throughput during the COVID-19 pandemic. Nevertheless, the traditional pooling approach cannot be effectively deployed in high-prevalence settings due to the need for secondary tests in the case of a positive pool. In this study, we present a pooling test platform with high adaptability and simplicity that allows sample-specific detection of multiple-tagged samples in a single run without the need for retesting. This was accomplished by labeling distinct samples with predefined ID-Primers and identifying tagged pooled samples using one-step RT-PCR followed by melting curve analysis with rationally designed universal fluorescence- and quencher-tagged oligo probes. Using magnetic beads (MBs), nucleic acid targets from different individuals can be tagged and extracted concurrently and then pooled before RT, eliminating the need for extra RNA extraction and separate RT and enzyme digestion steps in the recently developed barcoding strategies. Pools of six samples (positive and negative) were successfully identified by melting temperature values under two fluorescent channels, with a detection sensitivity of 5 copies/µL. We validated the reproducibility of this assay by running it on 40 clinical samples with a hypothetical infection rate of 15%. In addition, to aid the scenario of large-scale pooling tests, we constructed a melting curve autoreadout system (MCARS) for statistical analysis of melting curve plots to eliminate error-prone manual result readout. Our results suggest that this strategy could be a simple and adaptable tool for alleviating existing bottlenecks in diagnostic pooling testing.

Efficient large-scale screening of viral pathogens by fragment length identification of pooled nucleic acid samples (FLIPNAS).

The necessity for the large-scale screening of viral pathogens has been amply demonstrated during the COVID-19 pandemic. During this time, SARS-CoV-2 nucleic acid pooled testing, such as Dorfman-based group testing, was widely adopted in response to the sudden increased demand for detection. However, the current approach still necessitates the individual retesting of positive pools. Here, we established an efficient method termed the fragment-length identification of pooled nucleic acid samples (FLIPNAS), where all subsamples (n = 8) can be uniquely labelled and tested in a single-time detection among pools of samples. We used a novel and simple design of unique primers (UPs) to generate amplicons of unique lengths after reverse transcription and polymerase chain reaction to reach this aim. As a result, the unique lengths of the amplicons can be recognized and traced back to the corresponding UPs and specific samples. Our results demonstrated that FLIPNAS could recognize one to eight positive subsamples in a single test without retesting positive pools. The system also showed sufficient sensitivity for the mass monitoring of SARS-CoV-2 and no cross-reactivity against three common respiratory diseases. Moreover, the FLIPNAS results of 40 samples with a positive ratio of 7.8% were in 100% agreement with their individual detection results using the gold standard. Collectively, this study shows that the efficiency of nucleic acid pooling detection can be further improved by FLIPNAS, which can speed up testing and mitigate the urgent demand for resources.

Prediction of Aptamer-Small-Molecule Interactions Using Metastable States from Multiple Independent Molecular Dynamics Simulations.

Understanding aptamer-ligand interactions is necessary to rationally design aptamer-based systems. Commonly used in silico tools have proven to be accurate to predict RNA and DNA oligonucleotide tertiary structures. However, given the complexity of nucleic acids, the most thermodynamically stable conformation is not necessarily the one with the highest affinity for a specific ligand. Because many metastable states may coexist, it remains challenging to predict binding sites through molecular docking simulations using available computational pipelines. In this study, we used independent simulations to broaden the conformational diversity sampled from DNA initial models of distinct stability and assessed the binding affinity of selected metastable representative structures. In our results, utilizing multiple metastable conformations for molecular docking analysis helped identify structures favorable for ligand binding and accurately predict the binding sites. Our workflow was able to correctly identify the binding sites of the characterized adenosine monophosphate and l-argininamide aptamers. Additionally, we demonstrated that our pipeline can be used to aid the design of competition assays that are conducive to aptasensing strategies using an uncharacterized aflatoxin B1 aptamer. We foresee that this approach may help rationally design effective and truncated aptamer sequences interacting with protein biomarkers or small molecules of interest for drug design and sensor applications.

Disposable and low-cost pen-like sensor incorporating nucleic-acid amplification based lateral-flow assay for at-home tests of communicable pathogens.

Rapid at-home test is a good alternative to the gold standard quantitative polymerase chain reaction (qPCR) for early identification and management of infected individuals in pandemic. However, the currently available at-home rapid antigen kits and nucleic acid tests (NATs) are prone to false results. Although some CRISPR-mediated NATs enhanced accuracy, long turnaround time (ca. 1 h) and aerosol contamination due to additional open-lid reaction hinder its applicability for self-tests. Moreover, the accuracy of at-home NATs is also impacted by interference of sample matrix due to lack of sample purification. Here we report a Fast, Low-cost, Aerosol contamination-free and Sensitive molecular assay for at-Home tests of communicable pathogens (FLASH) incorporating oLAMP, a recently reported isothermal and target-specific NATs by our group, and a visible lateral-flow readout. The integrated platform enabled sample-to-result SARS-CoV-2 RNA detection in 20-30 min achieving a sensitivity of 0.5 copies/µL in a blinded experiment with a high accuracy comparable with the qPCR. Its prototype consists of two disposable pen-like instruments for single-step sample preparation and contamination-free NATs, respectively. The simplified workflow of the FLASH enabled detection to be readily conducted by untrained users for at-home tests. All in all, the FLASH prototype demonstrates itself to be a promising home-use assay platform for effective mitigation of the pandemic.

Unique Barcoded Primer-Assisted Sample-Specific Pooled Testing (Uni-Pool) for Large-Scale Screening of Viral Pathogens.

Pooled testing has been widely adopted recently to facilitate large-scale community testing during the COVID-19 pandemic. This strategy allows to collect and screen multiple specimen samples in a single test, thus immensely saving the assay time and consumable expenses. Nevertheless, when the outcome of a pooled testing is positive, it necessitates repetitive retesting steps for each sample which can pose a serious challenge during a rising infection wave of increasing prevalence. In this work, we develop a unique barcoded primer-assisted sample-specific pooled testing strategy (Uni-Pool) where the key genetic sequences of the viral pathogen in a crude sample are extracted and amplified with concurrent tagging of sample-specific identifiers. This new process improves the existing pooled testing by eliminating the need for retesting and allowing the test results-positive or negative-for all samples in the pool to be revealed by multiplex melting curve analysis right after real-time polymerase chain reaction. It significantly reduces the total assay time for large-scale screening without compromising the specificity and detection sensitivity caused by the sample dilution of pooling. Our method was able to successfully differentiate five samples, positive and negative, in one pool with negligible cross-reactivity among the positive and negative samples. A pooling of 40 simulated samples containing severe acute respiratory syndrome coronavirus-2 pseudovirus of different loads (min: 10 copies/µL; max: 103 copies/µL) spiked into artificial saliva was demonstrated in eight randomized pools. The outcome of five samples in one pool with a hypothetical infection prevalence of 15% in 40 samples was successfully tested and validated by a typical Dorman-based pooling.

Rational design of allosterically regulated toehold mediated strand displacement circuits for sensitive and on-site detection of small molecule metabolites.

Development of small molecule biosensors enables rapid and de-centralized small molecule detection that meets the demands of routine health monitoring and rapid diagnosis. Among them, allosteric transcription factor (aTF)-based biosensors have shown potential in modular design of small molecule detection platforms due to their ligand-regulated DNA binding activity. Here, we expand the capabilities of a biosensor that leverages the aTF-based regulation of toehold-mediated strand displacement (TMSD) circuits for uric acid (UA) detection in non-invasive salivary samples by utilizing the UA-responsive aTF HucR. The impact of the low ligand affinity of the native HucR was addressed by engineering a two-pass TMSD circuit with in silico rational design. This combined strategy achieved enrichment of the output signal and overcame the negative impact of the matrix effect on the sensitivity and overall response of the biosensor when using real samples, which enabled semi-quantitative detection in the normal salivary UA levels. As well, enhancements provided by the two-pass design halved the turnaround time to less than 15 minutes. To sum up, the two-cycle DNA circuit design enabled aTF-based simple, rapid and one-step non-invasive salivary UA detection, showing its potential in metabolite detection for health monitoring.

An Insight into Tunable Innate Piezoelectricity of Silk for Green Bioelectronics.

Commercially available bioelectronics account for significant percentage of e-waste, especially battery waste, that demand immediate intervention due to rising environmental concerns. Consumers are becoming increasingly aware and cautious of their contribution to carbon footprint on a regular basis. It has become imperative to adopt sustainability in every aspect of production of bioelectronics taking into consideration the growing market for wearable healthcare monitoring system. Green electronics is a relatively new concept gaining tremendous attention within the scientific and industrial community with the ultimate goal of employing organic, biodegradable, and self-sustainable system to replace the conventional inorganic battery-powered electronics. Silk is a green material that has been extensively explored for its use in functional electronics due to its tunable biodegradability and flexibility. Nevertheless, an intriguing property of Silk is its innate piezoelectricity. This review highlights the importance of crystal orientation and structure of Silk Fibroin to display piezoelectric response and documents possible strategies for its enhancement. It also provides insight into the possibility of using piezoelectric Silk as a piezoelectric sensor, actuator, and energy harvester to form self-powered hybrid systems for autonomous bioelectronics.

Detection of rare variant alleles using the AsCas12a double-stranded DNA trans-cleavage activity.

The sensitive and accurate detection of rare mutations has profound clinical implications; however, current methods require expensive instrumentation and are laborious and time-consuming. Thus, there is a need for a probe-based alternative that can effectively discriminate single-base mutations. Recently, several groups have shown the potential of the CRISPR/Cas12a system for sensitive and selective DNA detection but its application on single nucleotide variants (SNVs) detection is limited by the requirement of a protospacer adjacent motif (PAM) directly upstream to the SNV site and the amplification of non-specific signals due to the rapid and indiscriminate trans cleavage activity. Here, we report an ultra-selective Cas12a-based system that eliminates the need for the PAM sequence in the target with lower noise from the wild-type sequence by using its non-canonical double-stranded trans-cleavage activity. We show that our strategy can allow the detection of an EGFR gene mutation in sub-femtomolar concentrations up to 0.1% variant allele frequency using either fluorescence or electrochemical readouts.

Allosteric Regulation of DNA Circuits Enables Minimal and Rapid Biosensors of Small Molecules.

Detection of environmental pollutants is crucial to safeguard ecological and public health. Here, we report a modular biosensing approach for the detection of contaminants based on the regulation of a minimal DNA signal amplifier and transducer circuit by allosteric transcription factors and their cognate ligands. We leverage the competition between allosteric proteins and an endonuclease to modulate cascade toehold-mediated strand displacement reactions, which are triggered in the presence of specific effectors and sustained by the endonuclease. We built two optical biosensors for the detection of tetracyclines and macrolides in water using repressors TetR and MphR, respectively. We demonstrate that our minimal, fast, and single-step biosensors can successfully detect antibiotics in nanomolar levels and apply them to report the presence of spiked-in antibiotics in water samples in a matter of minutes, suggesting great potential for monitoring of water contaminants.

Oligonucleotide hybridization with magnetic separation assay for multiple SNP phasing.

Since humans have two copies of each gene, multiple mutations in different loci may or may not be found on the same strand of DNA (i.e., inherited from one parent). When a person is heterozygous at more than one position, the placement of these mutations, also called the haplotype phase, (i.e., cis for the same strand and trans for different strands) can result in the expression of different amount and type of proteins. In this work, we described an enzyme-free method to phase two single nucleotide polymorphisms (SNPs) using two fluorophore/quencher-labelled probes, where one of which was biotinylated. The fluorescence signal was obtained twice: first, after the addition of the labelled probes and second, after the addition of the magnetic beads. The first signal was shown to be proportional to the total number of SNP A and SNP B present in the target analyte, while the second signal showed a marked decrease of the fluorescence signal from the non-biotinylated probe when the SNPs were in trans, showing that the probe immobilized on the magnetic bead selectively captures targets with SNPs in a cis configuration. We then mimic the nature of the human genome which consists of two haplotype copies of each gene, and showed that 250 nM of the 10 possible pairs of haplotypes could be differentiated using a combination of fluorescence microscopy and fluorescence detection.

Toehold probe-based interrogation for haplotype phasing of long nucleic acid strands.

The arrangement of multiple single nucleotide polymorphisms (SNPs) in a gene, called a haplotype phase, is increasingly recognized as critical for accurate determination of disease risk and severity. However, conventional toehold-mediated strand displacement reactions are only able to interrogate SNPs, but not phase them since it is not known whether two SNPs in the same copy of the gene (cis) or in different copies of the same gene (trans) will give the same readout. While the rational introduction of an enzyme enables haplotype phasing, the complicated and stable secondary structure of long, single-stranded DNA sequences at room temperature limits its use. Complex nucleic acid structures make the hybridization of the probes difficult. Thus, we designed a molecular method to reveal the relative positions of SNPs located 1.4 kb apart in two copies of a gene by employing a competitive toehold probes and sink strategy at an elevated temperature. As such, we have successfully differentiated 20 nM of the 10 possible diplotypes in a long DNA target with two SNP sites located 1.4 kb apart within an hour without any additional amplification step. This offers a promising technology for accurate and fast haplotype phasing of SNPs that are over multiple kilobases away from each other.

Organic Electrochemical Transistor Arrays for In Vitro Electrophysiology Monitoring of 2D and 3D Cardiac Tissues.

Here, a multichannel organic electrochemical transistor (OECT) array is reported for electrophysiological monitoring and mapping of action potential propagation of a wide range of cardiac cells, including cell lines, primary cell lines, and human-sourced stem cell derivatives in 2D and 3D structures. The results suggest that the ability to exploit this OECT-based platform to map 2D action potential propagation provides a viable strategy to better characterize cardiac cells in response to various chronotropic drugs. The effects of chronotropic agents Isoproterenol and Verapamil on cardiac tissues validate the utility of OECT for drug screening capability, and a preliminary demonstration of a 64-channel OECT array to monitor the cardiac action potentials for better spatial resolution is presented. The study demonstrates that OECT will be a viable and versatile platform for applications in medical and pharmacological industries.

Thiol-free oligonucleotide surface modification of gold nanoparticles for nanostructure assembly.

Gold nanoparticles (AuNPs) decorated with thiol-modified DNA (HS-DNA) strands are an extensively studied, easily adjustable, and highly controllable material for constructing 3D nanostructures with various shapes and functions. However, few reproducible and robust methods involving DNA templates as a key reagent are available for obtaining 3D nanoparticle assemblies. It is still challenging to strictly control the number and location of DNA strands on the AuNP surface. Here, we introduce an efficient approach for the surface modification of AuNPs using unmodified DNA oligonucleotides by building DNA cages that trap the nanoparticles. This enables us to vary the process of nanostructure assembly and create anisotropic nanoparticles that are necessary for directed structure construction. This developed method simplifies the production process in comparison with conventional HS-DNA modification protocols and helps to precisely control the density and position of functional DNA strands designed for further hybridization with other AuNP conjugates.

Kinetically-enhanced DNA detection via multiple-pass exonuclease III-aided target recycling.

One of the promising approaches to address the challenge of detecting dilute nucleic acid analytes is exonuclease III-aided target recycling. In this strategy, the target DNA self-assembles with the reactant DNA probes and displays itself as a reactant and product at the same time. This provides an autonomous mechanism to release and reuse the analyte from each round of reactions for repetitive cycles, which amplifies the signal without amplifying the analyte itself. However, for very low amounts of the analyte, it takes a considerably long time before a detectable signal is generated. Thus, in this paper, we report a kinetically-enhanced target recycling strategy by designing two more target recycling sub-reactions that are triggered by the byproducts of the first reaction involving the target analyte. In this manner, concentrations of up to 0.5 pM of target DNA can be detected in 15 minutes.

Nucleic Acid Self-Assembly Circuitry Aided by Exonuclease III for Discrimination of Single Nucleotide Variants.

Robust and rapid discrimination of one base mutations in nucleic acid sequences is important in clinical applications. Here, we report a hybridization-based assay exploiting nucleic acid self-assembly circuitry and enzyme exonuclease III (Exo III) for the differentiation of single nucleotide variants (SNVs). This one-step approach combines the merits of discrimination power of competitive DNA hybridization probes (probe + sink) with catalytic amplification assisted by Exo III. The phosphorothioate bonds modified on a wild-type (WT) specific sink inhibit the Exo III digestion; thus, subsequent catalytic amplification magnifies only the intended SNV targets. The integrated assay exhibits improved SNV discrimination rather than hybridization probes relying solely on competition or amplification and enables SNV detection at 1% abundance. Two frequent cancer-driver mutation sequences (EGFR-L861Q, NRAS-Q61K) were tested. Our strategy allows simple sequence design and can easily adapt to multianalyte SNV detections.

Conditional Displacement Hybridization Assay for Multiple SNP Phasing.

The two chromosomal copies of the human genome are highly polymorphic, and the allelic content on each strand can dictate a person's biological outcomes. While many of the current diagnostic tools are able to detect the presence of multiple mutations at the same time, most cannot determine the phase of these mutations unless long-range PCR or sequencing techniques are used or if templates are compartmentalized into single copies prior to amplification. Here, an enzyme-coupled hybridization assay, named conditional displacement hybridization assay (CDHA), is described for the concurrent and rapid determination of the presence and phase of SNP variants. In this approach, short DNA probes were utilized to first quantify the amount of SNPs on the templates using a two-channel fluorescence measurement. The hybrids formed between the probes and the templates then set up the right condition for the subsequent enzymatic displacement and quenching of a fluorophore-labeled strand, which happens only if both SNPs are present on the same strand. The drop in the fluorescence signal thereby indicates the phase of the two SNPs. As a proof of concept, we tested the assay on four variants of an arbitrary sequence-with or without mutation on two sites 100 nts apart. The assay described herein was able to determine the haplotype phase of the samples in less than 1 h. This method promises a direct, cost-effective, and laboratory-based test to extract further genetic information to determine and/or predict diseases and traits dependent on SNP phasing.

Kinetically modulated specificity against single-base mutants in nucleic acid recycling circuitry using the destabilization motif.

Signal amplification in nucleic acid sensing improves detection sensitivity, but difficulties remain in sustaining specificity over time, particularly under excess amounts of single-base mutants. Here, we report simple, self-refining target recycling circuitry, which cumulates differentiation between on and off targets by 2-step cyclic interaction with the sensing probe. In the reaction, the analyte recycles only if the protective strand of the sensing probe is removed. The dissociation kinetics of such interaction was modulated by reacting it with different lengths of assistant strands. When shorter assistant strands are used, the destabilization motif of the sensing probe has to spontaneously dissociate before another assistant strand approaches and fully displaces it. This sets up a high kinetic barrier sensitive to the subtle reaction energy differences imposed by the single-base mutants, and substantially improved specificity. As a proof of concept, a microRNA 21 DNA analogue was chosen as our target analyte together with its 14 point mutants (substitution, insertion, or deletion) for specificity measurements. The experimental results corroborate that our system amplifies signals in a comparable manner to the traditional one-layer recycling approach but with negligible system leakage. With the use of shortened assistant strands, up to 100 fold increase in the discrimination factor against the single-base mutants is observed. Specificity is sustainable or even increased over long period measurements (i.e. 4 days). More importantly, target differentiation is successfully demonstrated even in excess amounts of spurious analogs (100×) and low target frequency mixtures (i.e. 0.1%), which mimic the lean conditions practically encountered. Explicit mechanisms of the system specificity are elucidated through analytical calculations and free energy level diagrams. The modularity of the destabilization motif herein promises detection of different nucleic acid based targets and integration into other signal amplification approaches for specificity enhancement.

B12-dependent photoresponsive protein hydrogels for controlled stem cell/protein release.

Thanks to the precise control over their structural and functional properties, genetically engineered protein-based hydrogels have emerged as a promising candidate for biomedical applications. Given the growing demand for creating stimuli-responsive "smart" hydrogels, here we show the synthesis of entirely protein-based photoresponsive hydrogels by covalently polymerizing the adenosylcobalamin (AdoB12)-dependent photoreceptor C-terminal adenosylcobalamin binding domain (CarHC) proteins using genetically encoded SpyTag-SpyCatcher chemistry under mild physiological conditions. The resulting hydrogel composed of physically self-assembled CarHC polymers exhibited a rapid gel-sol transition on light exposure, which enabled the facile release/recovery of 3T3 fibroblasts and human mesenchymal stem cells (hMSCs) from 3D cultures while maintaining their viability. A covalently cross-linked CarHC hydrogel was also designed to encapsulate and release bulky globular proteins, such as mCherry, in a light-dependent manner. The direct assembly of stimuli-responsive proteins into hydrogels represents a versatile strategy for designing dynamically tunable materials.

Integrating DNA strand displacement circuitry to the nonlinear hybridization chain reaction.

Programmable and modular attributes of DNA molecules allow one to develop versatile sensing platforms that can be operated isothermally and enzyme-free. In this work, we present an approach to integrate upstream DNA strand displacement circuits that can be turned on by a sequence-specific microRNA analyte with a downstream nonlinear hybridization chain reaction for a cascading hyperbranched nucleic acid assembly. This system provides a two-step amplification strategy for highly sensitive detection of the miRNA analyte, conducive for multiplexed detection. Multiple miRNA analytes were tested with our integrated circuitry using the same downstream signal amplification setting, showing the decoupling of nonlinear self-assembly with the analyte sequence. Compared with the reported methods, our signal amplification approach provides an additional control module for higher-order DNA self-assembly and could be developed into a promising platform for the detection of critical nucleic-acid based biomarkers.