Triple‐Stem DNA Probe: A New Conformationally Constrained Probe for SNP Typing

Abstract

The specificity of nucleic acid hybridization has been a subject of intensive investigation due largely to its significance in single-nucleotide polymorphism (SNP) analysis. SNPs represent the most abundant form of genetic variations, accounting for 80 to 90 % of the differences between two human genomes. SNP analysis is important in population-based genetic risk assessment, molecular diagnostics, pharmaceutical drug development, linkage analysis, and identity testing in forensic applications.[1] Allele-specific hybridization is a technique used in SNP typing among others.[2] In this approach, an allele-specific probe should hybridize to the perfectly matched target sequence while remaining unbound to a target containing a single base mispairing under the same hybridization conditions (Figure 1 A, left). The simplicity of the probe and the assay design is an important advantage of this method when compared with pyrosequencing, invasive cleavage, primer extension, or ligation techniques, which use extra proteinmediated steps for SNP discrimination. However, the low specificity of the probe-analyte hybridization is a major challenge in the application of hybridization approaches to SNP typing. Indeed, an oligonucleotide probe of a practical, useful length hybridizes with similar affinities to the perfectly matched nucleic acid and to a target containing a single noncomplementary base. [3] The formation of at least 15-20 nucleotide hybrids between the probe and the analyte is required to uniquely define a specific fragment in a genomesized nucleic acid. Hybrids of such length are too stable to be sensitive to a base mispairing, since a single mismatched unit results in an energetic penalty equivalent to only a small fraction of the total energy gain upon duplex formation (Figure 1 A, right). [3,4] It is possible to discriminate between two alleles if the energy of the probe-analyte dissociated state (DS) has a value between the energies of associated states (AS) for matched and mismatched duplexes. In this energy disposition, which is optimal for SNP typing, the fully matched complex is formed, while the mismatched hybrid is dissociated. This difference can be easily detected, thus constituting a means for SNP discrimination by hybridization techniques. In order to achieve these conditions, conventional techniques use destabilization of the probe-analyte AS (Figure 1 A, right, dashed bars) by employing buffers of low ionic strength, denaturing agents, Figure 1. Schemes and energy diagrams for the hybridization of oligonucleotide probes to a nucleic acid analyte. "DS" and "AS" represent probe-analyte dissociated and associated (hybrid) states, respectively. A) Hybridization of a linear oligonucleotide probe. The difference in the energy between matched and mismatched duplexes is much smaller than the energy gap between DS and AS. At high temperature or in denaturing buffers, the DS energy is higher than the energy of the fully matched hybrid, but lower than the energy of the mismatched hybrid. B) A triple-stem DNA pr...

Signaling-Probe Displacement Electrochemical Aptamer-based Sensor (SD-EAB) for Detection of Nanomolar Kanamycin A

“…The 21-base anti-kanamycin A aptamer used in this study possesses a higher affinity for kanamycin A (reported dissociation constant, K d = 78.8 nM) than for its structural analogue kanamycin B (K d = 84.5 nM) [15]. The small affinity differences among them can be enlarged by lowering the free energy of the aptamer via hybridizing with a short complimentary strand [42,43]. In another word, kanamycin B couldn't effectively bind with anti-kanamycin apatmer and induce the displacement of the signaling probe in SD-EAB while kanamycin A did.…”

An Elegant Biosensor Molecular Beacon Probe: Challenges and Recent Solutions

“…It hybridizes to a target analyte by consecutively unwinding the three short stems, which is more kinetically favorable than unwinding of one long stem. In this stepwise process, the high activation-energy barrier is divided into three lower barriers [31]. High probe selectivity toward mismatched analyte was maintained over a wide temperature range of 20 to 60°C.…”

“…This type of quenching occurs only for closely located fluorophore-quencher pairs and does not require overlap of fluorophore emission spectrum with quencher absorption spectrum. In addition, the secondary structure is a form of conformational constraint [29–31] that imparts extraordinary selectivity: the probe would hybridize to the target only if a significant energy gain is offered, thus rejecting mismatched targets. This property of MB probes is used to differentiate analytes with single-nucleotide differences, which is practically important for the analysis of single-nucleotide polymorphisms (SNPs) as well as point mutations.…”

A Differential Fluorescent Receptor for Nucleic Acid Analysis

“…In addition, it avoids long stems, which reduce the hybridization rates. [9] Therefore, DMB represents an attractive alternative to the conventional MB probe if a SNP-tolerant nucleic acid recognition is required. Since double-labeled fluorescent probes are relatively expensive, we designed an array of fluorescent sensors that utilizes a single DMB probe as a universal reporter.…”