Operating Cooperatively (OC) Sensor for Highly Specific Recognition of Nucleic Acids

Molecular Beacon (MB) probes have been extensively used for nucleic acid analysis because of their ability to produce fluorescent signal in solution instantly after hybridization. The indirect binding of MB probe to a target analyte offers several advantages, including: improved genotyping accuracy and the possibility to analyse folded nucleic acids. Here we report on a new design for MB-based sensor, called \u27Operating Cooperatively\u27 (OC), which takes advantage of indirect binding of MB probe to a target analyte. The sensor consists of two unmodified DNA strands, which hybridize to a universal MB probe and a nucleic acid analyte to form a fluorescent complex. OC sensors were designed to analyze two human SNPs and E.coli 16S rRNA. High specificity of the approach was demonstrated by the detection of true analyte in over 100 times excess amount of single base substituted analytes. Taking into account the flexibility in the design and the simplicity in optimization, we conclude that OC sensors may become versatile and efficient tools for instant DNA and RNA analysis in homogeneous solution

Binary Probes For Nucleic Acid Analysis

The advances in the development of binary probe (BPs) and their improved selectivity in comparison with other hybridization-based techniques were studied. The first BP, which used Förster resonance energy transfer (FRET), was suggested in 1988. A commonly adopted BP architecture employs the different affinity mode BPs. In this design one strand with a longer analyte binding arm binds tightly to the position abutting to the single-nucleotide polymorphisms (SNP) site. A second shorter analyte binding arm interrogates the SNP site by forming stable hybrid only with the perfectly matched sequence. The design of BPs employs self-assembly of more than two nucleic acid components. The same principle is adopted by DNA nanotechnology, which deals with constructing objects and functionally active assemblies from DNA molecules. Newly designed constructs based on aptamers, DNA junctions, and DNA enzymes offer an opportunity to utilize DNA probes that avoid direct covalent attachment with organic dyes

Nucleic Acid Analysis Using Multifunctional Hybridization Sensors

Hybridization of nucleic acid probes remains one of the most common strategies for sensing of [...

An Elegant Biosensor Molecular Beacon Probe: Challenges and Recent Solutions

Molecular beacon (MB) probes are fluorophore- and quencher-labeled short synthetic DNAs folded in a stem-loop shape. Since the first report by Tyagi and Kramer, it has become a widely accepted tool for nucleic acid analysis and triggered a cascade of related developments in the field of molecular sensing. The unprecedented success of MB probes stems from their ability to detect specific DNA or RNA sequences immediately after hybridization with no need to wash out the unbound probe (instantaneous format). Importantly, the hairpin structure of the probe is responsible for both the low fluorescent background and improved selectivity. Furthermore, the signal is generated in a reversible manner; thus, if the analyte is removed, the signal is reduced to the background. This paper highlights the advantages of MB probes and discusses the approaches that address the challenges in MB probe design. Variations of MB-based assays tackle the problem of stem invasion, improve SNP genotyping and signal-to-noise ratio, as well as address the challenges of detecting folded RNA and DNA

A Universal Split Spinach Aptamer (Ussa) For Nucleic Acid Analysis And Dna Computation

We demonstrate how a single universal spinach aptamer (USSA) probe can be used to detect multiple (potentially any) nucleic acid sequences. USSA can be used for cost-efficient and highly selective analysis of even folded DNA and RNA analytes, as well as for the readout of outputs of DNA logic circuits

Split Spinach Aptamer For Highly Selective Recognition Of Dna And Rna At Ambient Temperatures

Split spinach aptamer (SSA) probes for fluorescent analysis of nucleic acids were designed and tested. In SSA design, two RNA or RNA/DNA strands hybridized to a specific nucleic acid analyte and formed a binding site for low-fluorescent 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) dye, which resulted in up to a 270-fold increase in fluorescence. The major advantage of the SSA over state-of-the art fluorescent probes is high selectivity: it produces only background fluorescence in the presence of a single-base-mismatched analyte, even at room temperature. SSA is therefore a promising tool for label-free analysis of nucleic acids at ambient temperatures

Snp Analysis Using A Molecular Beacon-Based Operating Cooperatively (Oc) Sensor

Analysis of single-nucleotide polymorphisms (SNPs) is important for diagnosis of infectious and genetic diseases, for environment and population studies, as well as in forensic applications. Herein is a detailed description to design an operating cooperatively (OC) sensor for highly specific SNP analysis. OC sensors use two unmodified DNA adaptor strands and a molecular beacon probe to detect a nucleic acid targets with exceptional specificity towards SNPs. Genotyping can be accomplished at room temperature in a homogenous assay. The approach is easily adaptable for any nucleic acid target, and has been successfully used for analysis of targets with complex secondary structures. Additionally, OC sensors are an easy-to-design and cost-effective method for SNP analysis and nucleic acid detection. © 2013 Springer Science+Business Media, New York