AND-Logic Cascade Rolling Circle Amplification for Optomagnetic Detection of Dual Target SARS-CoV-2 Sequences.

DNA logic operations are accurate and specific molecular strategies that are appreciated in target multiplexing and intelligent diagnostics. However, most of the reported DNA logic operation-based assays lack amplifiers prior to logic operation, resulting in detection limits at the subpicomolar to nanomolar level. Herein, a homogeneous and isothermal AND-logic cascade amplification strategy is demonstrated for optomagnetic biosensing of two different DNA inputs corresponding to a variant of concern sequence (containing spike L452R) and a highly conserved sequence from SARS-CoV-2. With an "amplifiers-before-operator" configuration, two input sequences are recognized by different padlock probes for amplification reactions, which generate amplicons used, respectively, as primers and templates for secondary amplification, achieving the AND-logic operation. Cascade amplification products can hybridize with detection probes grafted onto magnetic nanoparticles (MNPs), leading to hydrodynamic size increases and/or aggregation of MNPs. Real-time optomagnetic MNP analysis offers a detection limit of 8.6 fM with a dynamic detection range spanning more than 3 orders of magnitude. The accuracy, stability, and specificity of the system are validated by testing samples containing serum, salmon sperm, a single-nucleotide variant, and biases of the inputs. Clinical samples are tested with both quantitative reverse transcription-PCR and our approach, showing highly consistent measurement results.

On-Particle Hyperbranched Rolling Circle Amplification-Scaffolded Magnetic Nanoactuator Assembly for Ferromagnetic Resonance Detection of MicroRNA.

Inspired by natural molecular machines, scientists are devoted to designing nanomachines that can navigate in aqueous solutions, sense their microenvironment, actuate, and respond. Among different strategies, magnetically driven nanoactuators can easily be operated remotely in liquids and thus are valuable in biosensing. Here we report a magnetic nanoactuator swarm with rotating-magnetic-field-controlled conformational changes for reaction acceleration and target quantification. By grafting nucleic acid amplification primers, magnetic nanoparticle (MNP) actuators can assemble and be fixed with a flexible DNA scaffold generated by surface-localized hyperbranched rolling circle amplification in response to the presence of a target microRNA, osa-miR156. Net magnetic anisotropy changes of the system induced by the MNP assembly can be measured by ferromagnetic resonance spectroscopy as shifts in the resonance field. With a total assay time of ca. 120 min, the proposed biosensor offers a limit of detection of 6 fM with a dynamic detection range spanning 5 orders of magnitude. The specificity of the system is validated by testing different microRNAs and salmon sperm DNA. Endogenous microRNAs extracted from Oryza sativa leaves are tested with both quantitative reverse transcription-PCR and our approach, showing comparable performances with a Pearson correlation coefficient >0.9 (n = 20).

Moderate Constraint Facilitates Association and Force-Dependent Dissociation of HA-CD44 Complex.

Binding of cell surface glycoprotein CD44 to hyaluronic acid (HA) is a key event for mediating cell adhesion, motility, metastasis, inflammatory responses and tumor development, but the regulation mechanism and its molecular basis under diverse mechanical constraints remain unclear. We herein investigated interaction of CD44 HABD (HA binding site domain) to HA through free and steered molecular dynamics (MD) simulations as well as atomic force microscope (AFM) measurement using different constraints on HA. The middle, two ends or both of the constrained HA chains were fixed for MD simulations, while one and two biotin-avidin linkage or physical absorption were used to immobilize HA on substrates for AFM experiments, to model HA chains with low, moderate and high HA flexibilities, respectively. We found that binding of CD44 to moderate fixed HA was possessed of a better thermo-stability, a lower mechanical strength and a higher dissociation probability, while higher adhesive frequency, smaller rupture force and shorter lifetime were assigned to CD44 on the two biotin-immobilized HA rather than one biotin-immobilized or physically absorbed HA on substrates, suggesting a moderate HA flexibility requirement in favor of association and force-induced dissociation of CD44-HA complex. Tensile-induced convex conformation of HA chain was responsible for reduction of complex mechano-stability and did inversely a shrunken CD44 HABD under stretching; transition from catch bond to slip bond governed CD44-HA interaction. This study uncovered the regulation mechanism and its molecular basis for CD44-HA affinity under diverse mechano-microenvironments and provided a new insight into CD44-HA interaction-mediated cell inflammatory responses and tumor development.