Sequence-specific Recognition Of Double-stranded DNA Research Articles

Thermodynamic Swings: How Ideal Complex of Cas9-RNA/DNA Forms.

Most processes of the recognition and formation of specific complexes in living systems begin with collisions in solutions or quasi-solutions. Then, the thermodynamic regulation of complex formation and fine tuning of complexes come into play. Precise regulation is very important in all cellular processes, including genome editing using the CRISPR–Cas9 tool. The Cas9 endonuclease is an essential component of the CRISPR–Cas-based genome editing systems. The attainment of high-specificity and -efficiency Cas9 during targeted DNA cleavage is the main problem that limits the practical application of the CRISPR–Cas9 system. In this study, we analyzed the thermodynamics of interaction of a complex’s components of Cas9–RNA/DNA through experimental and computer simulation methods. We found that there is a small energetic preference during Cas9–RNA/DNA formation from the Cas9–RNA and DNA/DNA duplex. The small difference in binding energy is relevant for biological interactions and could be part of the sequence-specific recognition of double-stranded DNA by the CRISPR–Cas9 system.

Sequence-Specific Recognition of Double-Stranded DNA by Peptide Nucleic Acid Forming Double-Duplex Invasion Complex

Peptide nucleic acid (PNA) is an analog of natural nucleic acids, where the sugar-phosphate backbone of DNA is replaced by an electrostatically neutral N-(2-aminoethyl)glycine backbone. This unique peptide-based backbone enables PNAs to form a very stable duplex with the complementary nucleic acids via Watson–Crick base pairing since there is no electrostatic repulsion between PNA and DNA·RNA. With this high nucleic acid affinity, PNAs have been used in a wide range of fields, from biological applications such as gene targeting, to engineering applications such as probe and sensor developments. In addition to single-stranded DNA, PNA can also recognize double-stranded DNA (dsDNA) through the formation of a double-duplex invasion complex. This double-duplex invasion is hard to achieve with other artificial nucleic acids and is expected to be a promising method to recognize dsDNA in cellula or in vivo since the invasion does not require the prior denaturation of dsDNA. In this paper, we provide basic knowledge of PNA and mainly focus on the research of PNA invasion.

A colorimetric method for the sequence-specific recognition of double-stranded DNA on the surface of a silver-coated glass slide

In this study, a colorimetric method for sequence-specific recognition of double-stranded DNA (dsDNA) was established on the surface of a silver-coated glass slide. Oligo-1 was assembled on the surface of a silver-coated glass slide through an Ag–S bond, and Oligo-2 as reporter was used to bind with streptavidin-horseradish peroxidase (SA–HRP). They could bind with target dsDNA that was composed of Oligo-3 and Oligo-4 on the surface of a silver-coated glass slide through triplex formation. The bound HRP could be moved into the solution by DNase I and catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB). Therefore, the concentration of target dsDNA could be determined with the colour change of TMB. Under the optimum conditions, the absorbance was proportional to the concentration of target dsDNA over the range of 100 pmol/L to 2.0 nmol/L, with a detection limit of 13 pmol/L. In addition, this method showed good sequence selectivity, enabling it to be further developed for the detection of other polymerase chain reaction (PCR) products.

Sensitive Fluorescent Sensor for Recognition of HIV-1 dsDNA by Using Glucose Oxidase and Triplex DNA.

A sensitive fluorescent sensor for sequence-specific recognition of double-stranded DNA (dsDNA) was developed on the surface of silver-coated glass slide (SCGS). Oligonucleotide-1 (Oligo-1) was designed to assemble on the surface of SCGS and act as capture DNA, and oligonucleotide-2 (Oligo-2) was designed as signal DNA. Upon addition of target HIV-1 dsDNA (Oligo-3•Oligo-4), signal DNA could bind on the surface of silver-coated glass because of the formation of C•GoC in parallel triplex DNA structure. Biotin-labeled glucose oxidase (biotin-GOx) could bind to signal DNA through the specific interaction of biotin-streptavidin, thereby GOx was attached to the surface of SCGS, which was dependent on the concentration of target HIV-1 dsDNA. GOx could catalyze the oxidation of glucose and yield H2O2, and the HPPA can be oxidized into a fluorescent product in the presence of HRP. Therefore, the concentration of target HIV-1 dsDNA could be estimated with fluorescence intensity. Under the optimum conditions, the fluorescence intensity was proportional to the concentration of target HIV-1 dsDNA over the range of 10 pM to 1000 pM, the detection limit was 3 pM. Moreover, the sensor had good sequence selectivity and practicability and might be applied for the diagnosis of HIV disease in the future.

Hairpin probe for sequence-specific recognition of double-stranded DNA on simian virus 40

Simian virus 40(SV40) is a polyomavirus and can induce a series of different tumors. The recognition of SV40 genome is crucial to tumor diagnosis and gene therapy. Herein, a sensitive and selective colorimetric method for sequence-specific recognition of homopyrimidine·homopurine duplex DNA(dsDNA) of SV40(4424—4440, gp6) was established with a hairpin probe based upon the formation of triplex DNA. Hairpin probe 5′-CCC TAC CCA TTT TTT CTT CTC TTT CCT GGG TAG GGC GGG TTG GG-3′(HP) containing G-rich sequence and 17-bp trip-lex-forming sequence was used as the signal probe, which was stem-loop structure alone and exhibited low catalytic activity. Upon its binding to the target duplex of SV40, hairpin probe transferred from stem-loop structure to parallel triplex DNA, accompanied by the recovery of catalytic activity of DNAzyme and a sharp increase of absorbance. Under optimum conditions, the absorbance was increased proportionally to the concentration of dsDNA over the range from 500 pmol/L to 40.0 nmol/L with a detection limit of 433 pmol/L. Moreover, satisfied results were obtained when the assay was used to recognize the mismatched sequences.

Fluorescence recognition of double-stranded DNA based on the quenching of gold nanoparticles to a fluorophore labeled DNA probe.

An ultrasensitive fluorescent platform for sequence-specific recognition of double-stranded DNA (dsDNA) based on the quenching of gold nanoparticles (AuNPs) to a fluorophore labeled DNA probe was developed. The target dsDNA could hybridize with the loop portion of the molecular beacon (MB) to form a triplex DNA structure and opened the "stem-loop" structure of the MB; such triplex DNA was used as an assistant probe (AP). Meanwhile, a fluorophore labeled DNA-AuNP probe that contained a specific enzyme cleavage site was introduced and its fluorescence signal was efficiently quenched due to the vicinity of the fluorophore to the AuNP surface. Such a DNA-AuNP probe could hybridize with the 5' stem portion of the MB in the AP to form duplex DNA strands that contained a specific enzyme cleavage site for the nicking enzyme assisted cleavage reaction, and resulted in the release of the fluorophore from the AuNP surface and the recovery of the fluorescence signal. Because the AP remains intact during such a cleavage process, it could be reused to hybridize with the next DNA-AuNP probe and trigger the nicking nuclease assisted signal amplification. Under optimal conditions, a low detection limit of 3.8 pM was obtained for dsDNA detection, and the assay has high sequence specificity for dsDNA detection.

Enhanced electrochemical recognition of double-stranded DNA by using hybridization chain reaction and positively charged gold nanoparticles.

Enhanced sequence-specific recognition of double-stranded DNA (dsDNA) was realized by using hybridization chain reaction (HCR) and positively charged gold nanoparticles ((+)AuNPs) dual signal amplification. To construct such a sensor, capture probe was initially assembled onto gold electrode surface. Upon addition of dsDNA, sandwiched DNA complex was formed between the capture probe and the detection probe, then another exposed part of the detection probe opened two alternating DNA hairpins (H1 and H2) in turn and initiated HCR to form a double-helix. Meantime, (+)AuNPs were electrostatically adsorbed onto such double-helix to amplify the electrochemical signal. Upon optimal conditions, the electronic signals of ferrocene (Fc) that modified on H1 and H2 increased linearly with increasing dsDNA concentration over the range from 15 pM to 1.0 nM, with a detection limit of 2.6 pM. Moreover, the proposed method showed good sequence specificity for dsDNA recognition.

Electrochemical molecular beacon biosensor for sequence-specific recognition of double-stranded DNA

Direct recognition of double-stranded DNA (dsDNA) was crucial to disease diagnosis and gene therapy, because DNA in its natural state is double stranded. Here, a novel sensor for the sequence-specific recognition of dsDNA was developed based on the structure change of ferrocene (Fc) redox probe modified molecular beacon (MB). For constructing such a sensor, gold nanoparticles (AuNPs) were initially electrochemical-deposited onto glass carbon electrode (GCE) surface to immobilize thiolated MB in their folded states with Au–S bond. Hybridization of MB with target dsDNA induced the formation of parallel triplex DNA and opened the stem-loop structure of it, which resulted in the redox probe (Fc) away from the electrode and triggered the decrease of current signals. Under optimal conditions, dsDNA detection could be realized in the range from 350pM to 25nM, with a detection limit of 275pM. Moreover, the proposed method has good sequence-specificity for target dsDNA compared with single base pair mismatch and two base pairs mismatches.

Sequence-specific recognition of double-stranded DNA with molecular beacon with the aid of Ag+ under neutral pH environment

A sensitive fluorescent sensor for sequence-specific recognition of dsDNA was established with a molecular beacon (MB) based upon the formation of parallel triplex DNA in the presence of Ag(+) under neutral pH environment.

Dynamic‐Light‐Scattering‐Based Sequence‐Specific Recognition of Double‐Stranded DNA with Oligonucleotide‐Functionalized Gold Nanoparticles

An ultrasensitive and simple dynamic-light-scattering (DLS) assay for the sequence-specific recognition of double-stranded DNA (dsDNA) was developed based on detection of the average diameter change of Au nanoparticle (AuNP) probes modified with oligonucleotides 5'-TTTCTCTTCCTT- CTCTTC-(T)(12)-SH-3' (Oligo 1) and 5'-TTCTTTCTTTTCTTTTTC-(T)(12)- SH-3' (Oligo 2). The target dsDNA was composed of two complementary oligonucleotides: 5'-AAAGAGAAGGAAGAGAAGAAGAAAGAAAAGAAAAAG-3' (Oligo 3) and 3'-TTTCTCTTCCTTCTCTTCTTCTTTCTTTTCTTTTTC-5' (Oligo 4). Hybridization of the two AuNPs-Oligo probes with the target dsDNA induced aggregation of the target dsDNA by forming triplex DNA, which accordingly increased the average diameter. This diameter change could then be detected by DLS. The average diameter was proportional to the target dsDNA concentration over the range from 593 fM to 40 pM, with a detection limit of 593 fM. Moreover, the assay had good sequence specificity for the target dsDNA.

A novel fluorescent biosensor for sequence-specific recognition of double-stranded DNA with the platform of graphene oxide

A fluorescent biosensor for sequence-specific recognition of double-stranded DNA (dsDNA) was developed based upon the DNA hybridization between dye-labeled single-stranded DNA (ssDNA) and double-stranded DNA. The fluorescence of FAM-labeled single-stranded DNA was quenched when it adsorbed on the surface of graphene oxide (GO). Upon addition of the target dsDNA, a homopyrimidine·homopurine part of dsDNA on the Simian virus 40 (SV40) (4424-4440, gp6), hybridization occurred between the dye-labeled DNA and the target dsDNA, which induced the dye-labeled DNA desorbed from the surface of GO, and turned on the fluorescence of the dye. Under the optimum conditions, the enhanced fluorescence intensity was proportional to the concentration of target dsDNA in the range 40.0-260 nM, and the detection limit was found to be 14.3 nM alongside the good sequence selectivity.

Sequence-Specific Recognition of Double-Stranded DNA by Synthetic Minor Groove Binder Conjugates. toward the Construction of Artificial Site-Specific Deoxyribonucleases

Bis-conjugates of hairpin N-methylpyrrole/N-methylimidazole oligocarboxamide minor groove binders (MGB) possessing enhanced affinity and sequence-specificity for dsDNA were synthesized. Two hairpin MGBs were connected by their N-termini via an aminodiacetate linker. The binding of bis-MGB conjugates to the target DNA was studied by gel mobility retardation, footprinting, and circular dichroism; their affinity and binding mode in the DNA minor groove were determined. In order to functionalize the bis-MGB conjugates, DNA-cleaving agents such as phenanthroline or bipyridine were attached. Effective site-specific cleavage of target DNA in the presence of Cu 2+ ions was observed.

Performing DNA computation with RecA-mediated triple-stranded DNA structure

A new method of DNA computing using an unusual triple-stranded DNA structure mediated by RecA protein is introduced and a minor 3-variable instance of the satisfiability (SAT) problem is solved. Separations were performed by using biotinylated oligodeoxynucleotide probes coated with RecA protein. During the computation, sequence-specific recognition of double-stranded DNA by homologous oligodeoxynucleotides was fulfilled in the presence of ATPγS. Captured by the magnetic particles coated with streptavidin, the desirable double-stranded DNA strands were retained and extracted. It is suggested that the methods employed here may help decrease the errors in DNA computation and solve some larger size NP- complete problems.

A novel DNA computing model based on RecA-mediated triple-stranded DNA structure

Abstract The field of DNA computing emerged in 1994 after Adleman's paper was published. Henceforth, a few scholars solved some noted NP-complete problems in this way. And all these methods of DNA computing are based on conventional Watson-Click hydrogen bond of doublehelical DNA molecule. In this paper, we show that the triple-stranded DNA structure mediated by RecA protein can be used for solving computational problems. Sequence-specific recognition of double-stranded DNA by oligonucleotide-directed triple helix (triplex) formation is used to carry out the algorithm. We present procedure for the 3-vertex-colorability problems. In our proposed procedure, it is suggested that it is possible to solve more complicated problems with more variables by this model. *Supported by National Natural Science Foundation of China (Grant Nos. 60533010, 60373089, 60674106), Science Foundation of Shaanxi Bureau of Education (Grant No. 06JK202), Program for New Century Excellent Talents in University (No. NCET-05-0612) an...

Sequence-specific recognition of double-stranded DNA by cooperative strand invasion

A remarkable feature of peptide nucleic acid (PNA) is its ability to recognize some sequences within duplex DNA by strand invasion. In order to improve binding properties of PNA triplex invasion, we tested the effect of cooperativity on the sequence specificity. A PNA targeting six bases within duplex DNA stringently recognized 12 base-pair homopurine site at a single base level.