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A new fluorescent sensing approach for detection of single-nucleotide polymorphisms (SNPs) is proposed based on the ligase reaction and gold nanoparticle (AuNPs)-quenched fluorescent oligonucleotides. The design exploits the strong fluorescence quenching of AuNPs for organic dyes and the difference in noncovalent interactions of the nanoparticles with single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA), where ssDNA can be adsorbed onto the surface of AuNPs while dsDNA cannot be. In the assay, two half primer DNA probes, one being labeled with a dye and the other being phosphorylated, were first incubated with a target DNA template. In the presence of DNA ligase, the two captured ssDNAs are linked for the perfectly matched DNA target to form a stable duplex, but the duplex could not be formed by the single-base mismatched DNA template. After addition of AuNPs, the fluorescence of dye-tagged DNA probe will be efficiently quenched unless the perfectly matched DNA target is present. To demonstrate the feasibility of this design, the performance of SNP detection using two different DNA ligases, T4 DNA ligase and Escherichia coli DNA ligase, were investigated. In the case of T4 DNA ligase, the signal enhancement of the dye-tagged DNA for perfectly matched DNA target is 4.6-fold higher than that for the single-base mismatched DNA. While in the presence of E. coli DNA ligase, the value raises to be 30.2, suggesting excellent capability for SNP discrimination. 10.1021/ac101503t

Conformationally constraint nucleic acid probes were usually designed by forming an intramolecular duplex based on Watson--Crick hydrogen bonds. The disadvantages of these approaches are the inflexibility and instability in complex environment of the Watson--Crick-based duplex. We report that this hydrogen bonding pattern can be replaced by metal-ligation between specific metal ions and the natural bases. To demonstrate the feasibility of this principle, two linear oligonucleotides and silver ions were examined as models for DNA hybridization assay and adenosine triphosphate detection. The both nucleic acids contain target binding sequences in the middle and cytosine (C)-rich sequences at the lateral portions. The strong interaction between [Ag.sup.+] ions and cytosines forms stable C-[Ag.sup.+]-C structures, which promises the oligonucleotides to form conformationally constraint formations. In the presence of its target, interaction between the loop sequences and the target unfolds the C-[Ag.sup.+]-C structures, and the corresponding probes unfolding can be detected by a change in their fluorescence emission. We discuss the thermodynamic and kinetic opportunities that are provided by using [Ag.sup.+] ion complexes instead of traditional Watson-Crick-based duplex. In particular, the intrinsic feature of the metalligation motif facilitates the design of functional nucleic acids probes by independently varying the concentration of [Ag.sup.+] ions in the medium. 10.1021/ac101114w

To construct efficient oligonucleotide probes, specific nucleic acid is designed as a conformationally constrained form based on the formation of a Watson-Crick-based duplex. However, instability of Watson-Crick hydrogen bonds in a complex biological environment usually leads to high background signal from the probe itself and false positive signal caused by nonspecific binding. To solve this problem, we propose a way to restrict the labeled-dyes in a hydrophobic cavity of cyclodextrin. This bounding, which acts like extra base pairs to form the Watson--Crick duplex, achieves variation of level of space proximity of the two labels and thus the degree of conformational constraint. To demonstrate the feasibility of the design, a stem-containing oligonucleotide probe (P1) for DNA hybridization assay and a stemless one (P2) for protein detection were examined as models. Both olignnucleotides were doubly labeled with pyrene at the 5'-and 3'-ends, respectively. It is the cyclodextrin/pyrene inclusion interaction that allows modulating the degree of conformational constraints of P1 and P2 and thus their background signals and selectivity. Under the optimal conditions, the ratio of signal-to-background of P1/[gamma]-CD induced by 1.0 equiv target DNA is near 174, which is 4-fold higher than that in the absence of [gamma]-CD. In addition, the usage of [gamma]-CD shifts the melting temperature of P1 from 57 to 68[degrees]C, which is reasonable for improving target-binding selectivity. This approach is simple in design, avoiding any variation of the stem's length and sequences. Furthermore, the strategy is generalizable which is suited for not only the stem-containing probe but also the linear probe with comparable sensitivity and selectivity to conventional structured DNA probes. 10.1021/ac1004713

A highly sensitive and specific colorimetry-based rolling circle amplification (RCA) assay method for single-nucleotide polymorphism genotyping has been developed. A circular template is generated by ligation upon the recognition of a point mutation on DNA targets. An RCA amplification is then initiated using the circular template in the presence of Phi29 polymerase. The RCA product can be digested by a restricting endonuclease, and the cleaved DNA fragments can mediate the aggregation of gold nanoparticle-tagged DNA probes. This causes a calorimetric change of the solution as the indicator of the mutation occurrence, which can be detected using UV--vis spectroscopy or viewed by naked eyes. On the basis of the high amplification efficiency of Phi29 polymerase, a mutated target of ~70 fM can be detected in this assay. In addition, the protection of the circle template using phosphorothioated nucleotides allows the digestion reaction to be performed simultaneously in RCA. Moreover, DNA ligase offers high fidelity in distinguishing the mismatched bases at the ligation site, resulting in positive detection of mutant targets even when the ratio of the wild-type to the mutant is 10 000:1. The developed RCA-based colorimetric detection scheme was demonstrated for SNP typing of [beta]-thalassemia gene at position -28 in genomic DNA. 10.1021/ac100336n

Small RNA molecules are effective regulators of gene expression, and the expression signature of one subgroup of small RNA, the microRNA (miRNA), has been linked to disease development and progression. Therefore, detection of small RNA in biological samples will greatly improve the understanding of their functions and render effective tools to researchers for cellular process control and disease prevention. To solve the challenges in detecting the low-abundance and short strand-length of small RNA molecules, we designed a ligation-assisted binding assay and applied the cation exchange-based fluorescence amplification (CXFluoAmp) method developed in our group for detection. Nonfluorescent, ionic nanocrystals (NCs) of CdSe were conjugated to detection probes and immobilized onto the array surface via ligation with the target small RNA, miR21, which bound to the capture probe complimentarily. Each binding event induced by one target miR21 molecule was then amplified by the release of thousands of [Cd.sup.2+] from one NC. The free [Cd.sup.2+] immediately turned on the fluorescence of thousands of fluorogenic Rhod-5N molecules. With such a powerful signal amplification strategy, our assay achieved a limit of detection (LOD) of 35 fM and signals were detectible with analyte concentrations spanning over 7 orders of magnitude. We also identified the differential expression of miR21 in total RNA extracts from healthy breast tissue and diseased cells. Furthermore, our detection scheme demonstrated good specificity in small RNA detection, because significant signal intensity could be observed from small RNAs with one or two nucleotides difference in sequences. Thus, our assay has great application potential for disease diagnosis relying on miRNA biomarkers, or in small RNA expression profiling for now target discovery and functional study. 10.1021/ac901983s

A new strategy for molecular beacon binding readout is proposed by using separation of the molecular recognition element and signal reporter. The signal transduction of the target binding event is based on displacing interaction between the target DNA and a competitor, the signal transducer. The target-free capture DNA is first interacted with the competitor, forming an assembled complex. In the presence of a target DNA that the affinity is stronger than that of the competitor, hybridization between capture DNA and the target disassembles the assembled complex and releases the free competitor to change the readout of the signal reporter. To demonstrate the feasibility of the design, a thymine-rich oligonucleotide was examined as a model system. [Hg.sup.2+] was selected as the competitor, and mercaptoacetic acid-coated CdTe/ZnS quantum dots served as the fluorescent reporter. Selective binding of [Hg.sup.2+] between the two thymine bases of the capture DNA forms a hairpin-structure. Hybridization between the capture DNA and target DNA destroys the hairpin-structure, releasing [Hg.sup.2+] ions to quench the quantum dots fluorescence. Under the optimal conditions, fluorescence intensity of the quantum dots against the concentration of perfect cDNA was linear over the concentration range of 0.1-1.6 [micro]M, with a limit of detection of 25 nM. This new assay method is simple in design, avoiding any oligonucleotide labeling. Furthermore, this strategy is generalizable since any target binding can in principle release the signal transducer and be detected with separated signal reporter. 10.1021/ac901906w