Thermodynamic basis of the enhanced specificity of structured DNA probes

Bonnet, GrÃ©goire; Tyagi, Sanjay; Libchaber, Albert; Kramer, Fred Russell

doi:10.1073/pnas.96.11.6171

Cited by 647 publications

(542 citation statements)

References 19 publications

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“…(In an alternative MB design, different dyes rather than dye-quencher pairs can be used; excitation of the donor dye will lead to donor specific fluorescence in the absence, and FRET-induced acceptor emission in the presence of target mRNA.) MBs have several advantages such as high signal to background ratio (S/B), 5,22,23 and fast hybridization response to complementary target 24 which have been exploited for the detection of single nucleotide polymorphisms (SNPs). 20,21 However, a notable disadvantage is non-specific opening of the MB (opening of the MB in the absence of target), which gives rise to a false positive signal ( Figure 1c).…”

Section: Introductionmentioning

confidence: 99%

Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection

Martí

Jockusch

et al. 2007

Tetrahedron

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We report the design, synthesis and characterization of binary oligonucleotide probes for mRNA detection. The probes were designed to avoid common problems found in standard binary probes such as direct excitation of the acceptor fluorophore and overlap between the donor and acceptor emission spectra. Two different probes were constructed that contained an array of either two or three dyes and that were characterized using steady-state fluorescence spectroscopy, time-resolved fluorescence spectroscopy and fluorescence depolarization measurements. The three-dye binary probe (BP-3d) consists of a Fam fluorophore which acts as a donor, collecting light and transferring it as energy to Tamra, which subsequently transfers energy to Cy5 when the two probes are hybridized to mRNA. This design allows the use of 488 nm excitation, which avoids the direct excitation of Cy5 and at the same time provides a good fluorescence resonance energy transfer (FRET) efficiency. The two-dye binary probe system (BP-2d) was constructed of Alexa488 and Cy5 fluorophores. Although the overlap between the fluorescence of Alexa488 and the absorption of Cy5 is relatively low, FRET still occurs due to their close physical proximity when the probes are hybridized to mRNA. This framework also decreases the direct excitation of Cy5 and reduces the fluorescence overlap between the donor and the acceptor. Picosecond time-resolved spectroscopy showed a reduction in the fluorescence lifetime of donor fluorophores after the formation of the hybrid between the probes and target mRNA. Interestingly, BP-2d in the presence of mRNA shows a slow rise in the fluorescence decay of Cy5 due to a relatively low FRET rate, which together with the reduction in the Alexa488 lifetime provides a way to improve the signal to background ratio using time-resolved fluorescence spectra (TRES). In addition, fluorescence depolarization measurements showed complete depolarization of the acceptor dyes (Cy5) for both BP-3d (due to sequential FRET steps) and BP-2d (due to the relatively low FRET rate) in the presence of the mRNA target.

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Section: Introductionmentioning

confidence: 99%

Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection

Martí

Jockusch

et al. 2007

Tetrahedron

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“…The formation of pcPNA duplexes that we demonstrate here entails several significant consequences for the rational use and further development of pcPNAs. First, this process should result in enhanced sequence specificity of pcPNA-dsDNA complex formation via the ''stringency clamping'' effect of structurally constrained probes, as was the case for site-specific DNA recognition by different DNA clamps (47,48). Second, the pcPNA duplex formation causes the inhibition of the invasion process at elevated PNA concentration.…”

Section: Is Approximately Valid Even Atmentioning

confidence: 99%

Kinetics and mechanism of the DNA double helix invasion by pseudocomplementary peptide nucleic acids

Demidov

Protozanova

Izvolsky

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

102

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If adenines and thymines in two mutually complementary mixedbase peptide nucleic acid (PNA) oligomers are substituted with diaminopurines and thiouracils, respectively, so-called pseudocomplementary PNAs (pcPNAs) are created. Pairs of pcPNAs have recently demonstrated an ability to highly selectively target essentially any designated site on double-stranded DNA (dsDNA) by forming very stable PNA-DNA strand-displacement complexes via double duplex invasion (helix invasion). These properties of pcPNAs make them unique and very promising ligands capable of denying the access of DNA-binding proteins to dsDNA. To elucidate the sequence-unrestricted mechanism of sequence-specific dsDNA recognition by pcPNAs, we have studied the kinetics of formation of corresponding PNA-DNA complexes at various temperatures by the gel-shift assay. In parallel, the conditions for possible selfhybridization of pcPNA oligomers have been assayed by mixing curve (Job plot) and thermal melting experiments. The data indicate that, at physiological temperatures (Ϸ37°C), the equilibrium is shifted toward the pairing of corresponding pcPNAs with each other. This finding explains a linear concentration dependence, within the submicromolar range, of the pcPNA invasion rate into dsDNA at 37°C. At elevated temperatures (>50°C), the rather unstable pcPNA duplexes dissociate, yielding the expected quadratic dependence for the rate of pcPNA invasion on the PNA concentration. The polycationic character of pcPNA pairs, carrying the duplicated number of protonated terminal PNA residues commonly used to increase the PNA solubility and binding affinity, also explains the self-inhibition of pcPNA invasion observed at higher PNA concentrations. Melting of pcPNA duplexes occurs with the integral transition enthalpies ranged from ؊235 to ؊280 kJ⅐mol ؊1 , contributing to an anomalously high activation energy of Ϸ150 kJ⅐mol ؊1 found for the helix invasion of pcPNAs carrying four different nucleobases. A simplified kinetic model for pcPNAs helix invasion is proposed that interprets all unusual features of pcPNAs binding to dsDNA. Our findings have important implications for rational use of pcPNAs.double-stranded DNA ͉ pseudocomplementary PNA ͉ sequence-selective recognition P eptide nucleic acids (PNAs) and their derivatives are of significant biomedical and biotechnological interest as prospective biomolecular tools for highly selective manipulation of nucleic acids (1-8). Recently, a new modification of PNAs has been introduced for sequence-unrestricted targeting of doublestranded DNA (dsDNA). Along with ordinary guanines and cytosines, these PNAs, dubbed pseudocomplementary PNAs (pcPNAs; refs. 9 and 10), carry 2,6-diaminopurines (D) and 2-thiouracils ( s U) instead of adenines and thymines, respectively.Model building revealed a steric clash between the bulky thio group of s U (or similarly modified thymine) and one of the two amino groups of D within the s U-D pair (9, 11). This clashing effect must severely destabilize the pcPNA-pcPNA duplexes whereas bindin...

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“…This conformational difference is consistent with the fluorescence measurements based on molecular beacons. [21] This distinct diversity should be ascribed to the cooperative effects of the weaker affinity of hairpin DNAs with single-base mismatched targets and the less stable duplex structures formed by hairpin DNAs and mismatched targets. It is because of the weaker ability of opening the stem by mutant-type targets that not every target binding attempt will completely undergo hairpin opening and duplex formation, resulting in a lower frequency of occurrence in the conformational transitions and simultaneously leaving more opportunities for hairpin folding and unfolding.…”

Section: Resultsmentioning

confidence: 99%

Single Nucleotide Polymorphism Genotyping in Single‐Molecule Electronic Circuits

et al. 2017

Advanced Science

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among individual genomes and understanding the relationship between genetic variations and their biological functions on a genomic scale have attracted extensive attention from geneticists in the postgenomic era. [1] Single nucleotide polymorphisms (SNPs) are prevalent and abundant genetic mutations. Because of their involvement in the emergence of numerous inherited diseases, SNPs have been used as genetic markers for mapping disease loci, [2] and studying candidate gene association, [3] revealing fundamental information for clinical diagnosis and drug discovery for related genetic diseases. [4] Therefore, various genotyping techniques have been developed for SNP detection, such as allele-specific real-time polymerase chain reaction (PCR) assays, [5] hybridization methods based on artificial DNA probes (molecular beacons, peptide nucleic acids (PNAs), and locked nucleic acids (LNAs)), [6] enzyme-assisted primer extension or chain ligation reaction genotyping by using DNA polymerase or ligase, [7] and enzyme mismatch cleavages. [8] However, most of these methods require pre-or post-treatments such as complex and costly PCR or rolling circle amplification steps to generate large amounts of sample collection or for signal enhancement, significantly restricting their applications. [9] To establish high-throughout, simple, and accurate genotyping techniques, with the ultimate goal of detecting SNPs at the single-molecule/single-event level, it is necessary to develop next-generation SNP-genotyping technology based on single-molecule analysis, which is promising for reducing the number of PCR amplification steps and lowering costs. [10] Molecular beacons (MBs), [6a,11] which are derived from hairpin-loop-structured oligonucleotides containing a fluorophore and quencher at different ends of the strand, have been widely used for biological detection both in vitro and in vivo, such as polymorphism analysis, clinical diagnosis, genotyping, and allele discrimination. [12] Because of their enhanced specificity and sensitivity, MBs are common and effective DNA probes for SNP genotyping. A single-base mismatch can be easily distinguished by measuring the differences in fluorescence intensity induced by the mismatched site after binding to well-matched and single-base mismatched targets due to the dynamic diversities of MB hybridization with different targets. Comparing the thermal stability of probe targets and investigating an optimized temperature range to maximize signal Establishing low-cost, high-throughput, simple, and accurate single nucleotide polymorphism (SNP) genotyping techniques is beneficial for understanding the intrinsic relationship between individual genetic variations and their biological functions on a genomic scale. Here, a straightforward and reliable single-molecule approach is demonstrated for precise SNP authentication by directly measuring the fluctuations in electrical signals in an electronic circuit, which is fabricated from a high-gain field-effect silicon nanowire decorated with a single...

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Thermodynamic basis of the enhanced specificity of structured DNA probes

Cited by 647 publications

References 19 publications

Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection

Design and characterization of two-dye and three-dye binary fluorescent probes for mRNA detection

Kinetics and mechanism of the DNA double helix invasion by pseudocomplementary peptide nucleic acids

Single Nucleotide Polymorphism Genotyping in Single‐Molecule Electronic Circuits

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