Sequence-selective Hybridization Research Articles

Sequence-selective dynamic covalent assembly of information-bearing oligomers

Relatively robust dynamic covalent interactions have been employed extensively to mediate molecular self-assembly reactions; however, these assembly processes often do not converge to a thermodynamic equilibrium, instead yielding mixtures of kinetically-trapped species. Here, we report a dynamic covalent self-assembly process that mitigates kinetic trapping such that multiple unique oligomers bearing covalently coreactive pendant groups are able to undergo simultaneous, sequence-selective hybridization with their complementary strands to afford biomimetic, in-registry molecular ladders with covalent rungs. Analogous to the thermal cycling commonly employed for nucleic acid melting and annealing, this is achieved by raising and lowering the concentration of a multi-role reagent to effect quantitative dissociation and subsequently catalyze covalent bond rearrangement, affording selective assembly of the oligomeric sequences. The hybridization specificity afforded by this process further enabled information encoded in oligomers to be retrieved through selective hybridization with complementary, mass-labeled sequences.

Temperature-mediated molecular ladder self-assembly employing Diels–Alder cycloaddition

Thermal annealing of sequence-defined, maleimide- and furan-bearing oligomers enables sequence-selective hybridization to afford molecular ladders incorporating Diels–Alder adduct-based rungs.

ZNA probe immobilized single-use electrodes for impedimetric detection of nucleic acid hybridization related to single nucleotide mutation

The development of a low-cost and disposable biosensing technologies has received a great interest of healthcare for the sensitive and reliable detection of single nucleotide mutation related to single nucleotide polymorphisms (SNPs). In the present study, an impedimetric biosensing platform based on zip nucleic acids (ZNA) was developed for the sensitive detection of Factor V Leiden (FV Leiden) mutation. After optimization of experimental parameters, the sequence selective hybridization between ZNA probe and target related to FV Leiden mutation was evaluated via electrochemical impedance spectroscopy technique (EIS) by measuring changes at the charge transfer resistance, Rct. Sensitive and selective impedimetric analysis was performed using carbon nanofiber (CNF) modified screen printed electrodes (SPE) and multi-channel screen printed array of electrodes (MULTIx8 CNF-SPE) resulting in a relatively shorter time in comparison to conventional methods. The selectivity of ZNA probe to mutation-free DNA sequences was also investigated. The applicability of single-use ZNA biosensor was also tested in synthetic PCR samples containing a single base mutation.

Indicator-free electrochemical biosensor for microRNA detection based on carbon nanofibers modified screen printed electrodes

Carbon nanofibers (CNFs) are fiber-structured nano-sized carbon materials, which have excellent mechanical, thermal and electrical properties. CNFs possess a wide range of application especially in biorecognition area. In this study, we developed an indicator-free voltammetric biosensor for detection of miRNA-34a target RNA by using carbon nanofiber enriched disposable screen printed electrodes (CNF-SPEs). The sensitive and selective recognition of miRNAs has been an attractive topic due to they have been defined as small RNAs and play major roles in regulating the translation and degradation of messenger RNAs. The characterization of CNF-SPEs in contrast to unmodified ones was firstly performed by scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS) techniques. The amino linked DNA probe, that was the complementary sequence of miRNA-34a, was immobilized onto the surface of CNF-SPEs. After measuring the guanine oxidation signal measured by differential pulse voltammetry (DPV), the detection of sequence-selective hybridization was then investigated between probe and miRNA-34a RNA target, or other miRNA sequences; miRNA-15a, or miRNA-660, which were chosen as noncomplementary sequences (NC). The detection limit was corresponded to 10.98μg/mL (54 pmol in 35μL sample) in the linear concentration range of target miRNA-34a from 25 to 100μg/mL by using CNF-SPEs. The features of this present study was comprehensively discussed by means of the unique characteristics of single-use CNF-SPEs and importance of miRNA monitoring.

Single-Nanoparticle Near-Infrared Surface Plasmon Resonance Microscopy for Real-Time Measurements of DNA Hybridization Adsorption

A novel 814 nm near-infrared surface plasmon resonance (SPR) microscope is used for the real-time detection of the sequence-selective hybridization adsorption of single DNA-functionalized gold nanoparticles. The objective-coupled, high numerical aperture SPR microscope is capable of imaging in situ the adsorption of single polystyrene and gold particles with diameters ranging from 450 to 20 nm onto a 90 μm × 70 μm area of a gold thin film with a time resolution of approximately 1-3 s. Initial real-time SPR imaging (SPRI) measurements were performed to detect the accumulation of 40 nm gold nanoparticles for 10 min onto a gold thin film functionalized with a 100% complementary DNA surface at concentrations from 5 pM to 100 fM by counting individual particle binding events. A 100% noncomplementary DNA surface exhibited virtually no nanoparticle adsorption. In contrast, in a second set of SPRI measurements, two component complementary/noncomplementary mixed DNA monolayers that contained a very small percentage of complementary sequences ranging from 0.1 to 0.001%, showed both permanent and transient hybridization adsorption of the gold nanoparticles that could be tracked both temporally and spatially with the SPR microscope. These experiments demonstrate that SPR imaging measurements of single biofunctionalized nanoparticles can be incorporated into bioaffinity biosensing methods at subpicomolar concentrations.