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.

A sequence-specific plasmonic loop-mediated isothermal amplification assay with orthogonal color readouts enabled by CRISPR Cas12a.

Herein, we introduce a sequence-specific plasmonic loop-mediated isothermal amplification (LAMP) assay with dual, complementary color readouts enabled by CRISPR Cas12a. Using this assay, any double-stranded LAMP amplicon containing a 5'-TTN PAM sequence can be recognized by Cas12a through a specific CRISPR RNA. The signal transduction is achieved using two orthogonal plasmonic systems mediated by Cas12a.

Concentric DNA Amplifier That Streamlines In-Solution Biorecognition and On-Particle Biocatalysis.

Colloidal nanoparticle biosensors capable of on-particle biocatalysis are powerful tools for amplified detection of biomolecules. The development and practical uses of such concentric amplifiers can be complicated because of the on-particle biorecognition that involves varying interfacial factors at the biomolecule-nanoparticle interfaces. Herein, we reason that a nanoparticle biosensor equipped with an in-solution biorecognition element may be better fabricated, predicted, controlled, and performed. The in-solution biorecognition shall also be streamlined with the on-particle biocatalysis so that the overall analytical and kinetic performance is not compromised. As a testbed, we introduce a concentric DNA amplifier driven by an enzyme-powered three-dimensional DNA nanomachine, where a DNA walker can be instantly assembled onto a spherical nucleic acid (SNA) track through a polyadenosine anchor. As such, the free DNA walker can participate in reactions in a homogeneous solution before assembling to the SNA track. The instant and stable assembly enabled by both adsorption and complementary base pairing also ensures rapid on-particle biocatalysis. We demonstrate that the in-solution biorecognition effectively eliminates the binding hindrance encountered by the on-particle biorecognition and thus significantly reduced energy barriers for the detection of nucleic acids and proteins. Because of the in-solution biorecognition, our system can also be plugged readily into complex DNA strand displacement networks for rapid signal amplification.

Naked-Eye Detection of Grapevine Red-Blotch Viral Infection Using a Plasmonic CRISPR Cas12a Assay.

Herein, we described a novel plasmonic CRISPR Cas12a assay for the visual, colorimetric detection of grapevine viral infections. Our assay generates rapid and specific colorimetric signals for nucleic acid amplicons by combining the unique target-induced incriminate single-stranded DNase activity of Cas12a with plasmon coupling of DNA functionalized gold nanoparticles. The practical applicability of our plasmonic assay was successfully demonstrated through the detection of emerging red-blotch viral infections in grapevine samples collected from commercial vineyards.

Probing and Controlling Dynamic Interactions at Biomolecule-Nanoparticle Interfaces Using Stochastic DNA Walkers.

Herein, we report a bottom-up approach to assemble a series of stochastic DNA walkers capable of probing dynamic interactions occurring at the bio-nano interface. We systematically investigated the impact of varying interfacial factors, including intramolecular interactions, orientation, cooperativity, steric effect, multivalence, and binding hindrance on enzymatic behaviors at the interfaces of spherical nucleic acids. Our mechanistic study has revealed critical roles of various interfacial factors that significantly alter molecular binding and enzymatic behaviors from bulk solutions. The improved understanding of the bio-nano interface may facilitate better design and operation of nanoparticle-based biosensors and/or functional devices. We successfully demonstrate how improved understanding of the bio-nano interface help rationalize the design of amplifiable biosensors for nucleic acids and antibodies.

Simulation-guided engineering of an enzyme-powered three dimensional DNA nanomachine for discriminating single nucleotide variants.

Single nucleotide variants (SNVs) are important both clinically and biologically because of their profound biological consequences. Herein, we engineered a nicking endonuclease-powered three dimensional (3D) DNA nanomachine for discriminating SNVs with high sensitivity and specificity. Particularly, we performed a simulation-guided tuning of sequence designs to achieve the optimal trade-off between device efficiency and specificity. We also introduced an auxiliary probe, a molecular fuel capable of tuning the device in solution via noncovalent catalysis. Collectively, our device produced discrimination factors comparable with commonly used molecular probes but improved the assay sensitivity by ∼100 times. Our results also demonstrate that rationally designed DNA probes through computer simulation can be used to quantitatively improve the design and operation of complexed molecular devices and sensors.