Target dsDNA Research Articles

A DNA electrochemical biosensor based on homogeneous hybridization for the determination of Cryptococcus neoformans

The sensitive analysis of DNA is of extreme importance in the field of clinical diagnosis, targeted therapeutics, gene therapy, and a variety of biomedical studies. Even though significant achievements have been made in the detection of single-stranded DNA (ssDNA), facile strategy for the detection of double-stranded DNA (dsDNA) is still lacking. In this study, we have constructed an electrochemical DNA sensor based on homogeneous hybridization. Target dsDNA is thermal denatured and hybridized with thiolated capture probe and biotin-labeled reporter probe in solution phase. Excessive amount of the probes and precise control of hybridization temperature ensure the predominant formation of sandwich SH-dsDNA-biotin rather than reannealing product of the target dsDNA. A bovine serum albumin (BSA)-modified gold surface is designed to improve self-assembly capability of the sandwich SH-dsDNA-biotin structure in the presence of excessive SH-ssDNA probe. Thus, the target dsDNA can be successfully detected by using enzyme-linked amperometric amplification. By employing this strategy, dsDNA can be detected in a linear range from 5 pM to 1 nM with a detection limit down to 800 fM (S/N = 3). Cryptococcus neoformans in clinical real samples from cryptococcal meningitis patients can be discriminated successfully by the proposed method. Our research would make the electrochemical biosensor be an excellent candidate for pathogens detection in clinical diagnosis and prognosis.

A fluorometric lateral flow assay for visual detection of nucleic acids using a digital camera readout.

A fluorometric lateral flow assay has been developed for the detection of nucleic acids. The fluorophores phycoerythrin (PE) and fluorescein isothiocyanate (FITC) were used as labels, while a common digital camera and a colored vinyl-sheet, acting as a cut-off optical filter, are used for fluorescence imaging. After DNA amplification by polymerase chain reaction (PCR), the biotinylated PCR product is hybridized to its complementary probe that carries a poly(dA) tail at 3΄ edge and then applied to the lateral flow strip. The hybrids are captured to the test zone of the strip by immobilized poly(dT) sequences and detected by streptavidin-fluorescein and streptavidin-phycoerythrin conjugates, through streptavidin-biotin interaction. The assay is widely applicable, simple, cost-effective, and offers a large multiplexing potential. Its performance is comparable to assays based on the use of streptavidin-gold nanoparticles conjugates. As low as 7.8 fmol of a ssDNA and 12.5 fmol of an amplified dsDNA target were detectable. Graphical abstract Schematic presentation of a fluorometric lateral flow assay based on fluorescein and phycoerythrin fluorescent labels for the detection of single-stranded (ssDNA) and double-stranded DNA (dsDNA) sequences and using a digital camera readout. SA: streptavidin, BSA: Bovine Serum Albumin, B: biotin, FITC: fluorescein isothiocyanate, PE: phycoerythrin, TZ: test zone, CZ: control zone.

Structure of the DNA-binding domain of human myelin-gene regulatory factor reveals its potential protein-DNA recognition mode.

Myelin-gene regulatory factor (MYRF) is a membrane-bound transcription factors, which is responsible for the differentiation of oligodendrocytes and myelination of central nervous system. Followed by a self-cleavage by the intramolecular chaperone auto-processing (ICA) domain, DNA-binding domain (DBD) of MYRF is released from the endoplasmic reticulum (ER) and was then translocated to the nucleus to regulate gene expression. In present work, we have solved the crystal structure of the human MYRF-DBD to 1.85-Å resolution. It exhibits a typical s-type Ig-fold and packs as symmetric trimeric form in the crystal via hydrogen-bond networks in three regions. Accordingly, we identified a couple of key residues on MYRF-DBD, which might play important roles in DNA-binding, in particular Arg521 on its C-terminal tail. The R521A mutant of DBD showed only 17% affinity to dsDNA targets compared to wild-type DBD. Then we built a plausible protein-DNA binding model of MYRF-DBD, which will help to elucidate its mechanism in DNA-binding and transcriptional regulation.

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.

2′-Fluoro ribonucleic acid modified DNA dual-probe sensing strategy for enzyme-amplified electrochemical detection of double-strand DNA of PML/RARα related fusion gene

In the study, a novel sensing strategy based on dual-probe mode, which involved two groups of 2′-fluoro ribonucleic acid (2′-F RNA) modified probes, was designed for the detection of synthetic target double-strand DNA (dsDNA) of PML/RARα fusion genes in APL. And each pair of probes contained a thiolated capture probe (C1 or C2) immobilized on one of electrode surfaces in the dual-channel electrochemical biosensor and a biotinylated reporter probe (R1 or R2). The two groups of 2′-F RNA modified probes were separately complementary with the corresponding strand (Sa or Sb) from target dsDNA in order to prevent renaturation of target dsDNA. Through flanking target dsDNA, two “sandwitch” complexes (C1/Sa/R1 and C2/Sb/R2) were separately shaped by capture probes (C1 and C2) and free reporter probes (R1 and R2) in hybridization solution on the surfaces of different electrodes after the thermal denaturation. The biotin-modified enzyme which produced the measurable electrochemical current signal was localized to the surface by affinity binding between biotin with streptavidin. Under the optimal condition, the biosensor was able to detect 84 fM target dsDNA and showed a good specificity in PBS hybridization solution. Otherwise, the investigations of the specificity and sensitivity of the biosensor were carried out further in the mixed hybridization solution containing different kinds of mismatch sequences as interference background. It can be seen that under a certain interference background, the method still exhibited excellent selectivity and specificity for the discrimination between the fully-complementary and the mismatch sequences. The results of our research laid a good basis of further detection research in practical samples.

Polymer-mineral scaffold augments in vivo equine multipotent stromal cell osteogenesis

BackgroundUse of bioscaffolds to direct osteogenic differentiation of adult multipotent stromal cells (MSCs) without exogenous proteins is a contemporary approach to bone regeneration. Identification of in vivo osteogenic contributions of exogenous MSCs on bioscaffolds after long-term implantation is vital to understanding cell persistence and effect duration.MethodsThis study was designed to quantify in vivo equine MSC osteogenesis on synthetic polymer scaffolds with distinct mineral combinations 9 weeks after implantation in a murine model. Cryopreserved, passage (P)1, equine bone marrow-derived MSCs (BMSC) and adipose tissue-derived MSCs (ASC) were culture expanded to P3 and immunophenotyped with flow cytometry. They were then loaded by spinner flask on to scaffolds composed of tricalcium phosphate (TCP)/hydroxyapatite (HA) (40:60; HT), polyethylene glycol (PEG)/poly-l-lactic acid (PLLA) (60:40; GA), or PEG/PLLA/TCP/HA (36:24:24:16; GT). Scaffolds with and without cells were maintained in static culture for up to 21 days or implanted subcutaneously in athymic mice that were radiographed every 3 weeks up to 9 weeks. In vitro cell viability and proliferation were determined. Explant composition (double-stranded (ds)DNA, collagen, sulfated glycosaminoglycan (sGAG), protein), equine and murine osteogenic target gene expression, microcomputed tomography (μCT) mineralization, and light microscopic structure were assessed.ResultsThe ASC and BMSC number increased significantly in HT constructs between 7 and 21 days of culture, and BMSCs increased similarly in GT constructs. Radiographic opacity increased with time in GT-BMSC constructs. Extracellular matrix (ECM) components and dsDNA increased significantly in GT compared to HT constructs. Equine and murine osteogenic gene expression was highest in BMSC constructs with mineral-containing scaffolds. The HT constructs with either cell type had the highest mineral deposition based on μCT. Regardless of composition, scaffolds with cells had more ECM than those without, and osteoid was apparent in all BMSC constructs.ConclusionsIn this study, both exogenous and host MSCs appear to contribute to in vivo osteogenesis. Addition of mineral to polymer scaffolds enhances equine MSC osteogenesis over polymer alone, but pure mineral scaffold provides superior osteogenic support. These results emphasize the need for bioscaffolds that provide customized osteogenic direction of both exo- and endogenous MSCs for the best regenerative potential.

A novel and convenient chemiluminescence sensing of DNA: Nanometer interface effect and DNA action mechanism

Ultra high sensitive and simple detection of DNA is crucial to early diagnosis of diseases and biodefense application. Herein, we report an amplified chemiluminescence (CL) biosensing platform for ultralow concentration DNA detection. It is based on the distinguishing ability of positively charged gold nanoparticles ((+)AuNPs) between ss-DNA and ds-DNA. (+)AuNPs’ effect on luminol CL reaction is used to amplify the response signal of target DNA. Moreover, the three key experimental procedures (DNA hybridization, ss-DNA action on (+)AuNPs and CL detection) in the proposed strategy are independent so that the three experiments can occur in the own optimal condition, respectively. (+)AuNPs’ direct effect produced by different structure DNAs (ss-DNA and ds-DNA) can make CL signal to be significantly amplified. The proposed biosensing has high sensitivity for detecting target DNA. The detection limit of target DNA was 0.62 fM, which was lower than that of the reported DNA assays based on other signal amplification. Also, the present method is more convenient. Such direct signal amplification from nanometer interface effect will provide a promising alternative avenue for the construction of high sensitive CL sensing platform. It is especially noteworthy that the action mechanisms of AuNPs and DNA can be clarified by this research, which will be important theoretical support for the design of DNA biosensors.

G-Quadruplex Induction by the Hairpin Pyrrole-Imidazole Polyamide Dimer.

The G-quadruplex (G4) is one type of higher-order structure of nucleic acids and is thought to play important roles in various biological events such as regulation of transcription and inhibition of DNA replication. Pyrrole-imidazole polyamides (PIPs) are programmable small molecules that can sequence-specifically bind with high affinity to the minor groove of double-stranded DNA (dsDNA). Herein, we designed head-to-head hairpin PIP dimers and their target dsDNA in a model G4-forming sequence. Using an electrophoresis mobility shift assay and transcription arrest assay, we found that PIP dimers could induce the structural change to G4 DNA from dsDNA through the recognition by one PIP dimer molecule of two duplex-binding sites flanking both ends of the G4-forming sequence. This induction ability was dependent on linker length. This is the first study to induce G4 formation using PIPs, which are known to be dsDNA binders. The results reported here suggest that selective G4 induction in native sequences may be achieved with PIP dimers by applying the same design strategy.

Structural basis of DNA duplex distortion induced by thiazole-containing hairpin polyamides.

This manuscript reports the molecular basis for double-stranded DNA (dsDNA) binding of hairpin polyamides incorporating a 5-alkyl thiazole (Nt) unit. Hairpin polyamides containing an N-terminal Nt unit induce higher melting stabilisation of target dsDNA sequences relative to an archetypical hairpin polyamide incorporating an N-terminal imidazole (Im) unit. However, modification of the N-terminus from Im to Nt-building blocks results in an increase in dsDNA binding affinity but lower G-selectivity. A general G-selectivity trend is observed for Nt-containing polyamide analogues. G-selectivity increases as the steric bulk in the Nt 5-position increases. Solution-based NMR structural studies reveal differences in the modulation of the target DNA duplex of Nt-containing hairpin polyamides relative to the Im-containing archetype. A structural hallmark of an Nt polyamide•dsDNA complex is a more significant degree of major groove compression of the target dsDNA sequence relative to the Im-containing hairpin polyamide.

Cryo-EM Structures Reveal Mechanism and Inhibition of DNA Targeting by a CRISPR-Cas Surveillance Complex

Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. Using cryoelectron microscopy (cryo-EM), we show that the binding of target double-stranded DNA (dsDNA) to a type I-F CRISPR system yersinia (Csy) surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally encoded anti-CRISPR suppressors that inhibit dsDNA binding, reveals mechanistic details underlying target recognition and inhibition.

Electrochemically mediated in situ growth of electroactive polymers for highly sensitive detection of double-stranded DNA without sequence-preference

The ability to directly detect double-stranded DNA (dsDNA) without sequence-preference continues to be a major challenge. Herein, we report an electrochemical method for the direct, highly sensitive detection of dsDNA based on the strand replacement of dsDNA by peptide nucleic acid (PNA) and the in situ growth of electroactive polymers through the surface-initiated electrochemically mediated atom transfer radical polymerization (SI-eATRP). Thiolated PNA molecules are firstly self-assembled onto gold electrode surface for the specific recognition of target dsDNA (dsDNA-T), which in turn leads to the formation of a high density of PNA/DNA heteroduplexes on the electrode surface for the subsequent attachment of ATRP initiators via the phosphate-Zr4+-carboxylate chemistry. By applying a negative potential to the electrode, the air-stable CuII deactivators can be reduced into the CuI activators so as to trigger the surface-initiated polymerization for the in situ growth of electroactive polymers. Due to the strand replacement of dsDNA by PNA, dsDNA can be directly detected without sequence-preference. Besides, the growth of polymers enables the modification of numerous electroactive probes, thereby greatly improving the electrochemical signal. Under optimal conditions, a good linearity between the electrochemical signal and the logarithm of dsDNA-T concentration over the range from 1.0 fM to 1.0nM, with a detection limit of 0.47 fM, can be obtained. Results indicate that it is highly selective, and holds high anti-interference capability in the presence of human serum samples. Therefore, this method offers great promises in providing a universal and efficient solution for the direct detection of dsDNA.

Sequence-specific recognition of HIV-1 DNA based upon nicking-assisted strand displacement amplification and triplex DNA

Nucleic acid amplification test is a reliable method for primary human immunodeficiency virus (HIV) infection diagnosis. Herein, a novel fluorescent method for sequence-specific recognition of DNA fragment of HIV-1 was established based upon nicking-assisted strand displacement amplification (SDA) and triplex DNA. In the presence of target dsDNA, nicking-assisted SDA process generated a lot of ssDNA, which hybridized with molecular beacon to produce signal. The fluorescence intensity was proportional to the concentration of target dsDNA within the range from 5 to 1000 pmol/L, with a detection limit of 1.4 pmol/L. Moreover, it successfully distinguished target dsDNA from the nucleic acid extractive of human blood. Thus this method has the merit of high sensitivity, and it is suitable for sequence-specific recognition of target dsDNA in complex matrices, which made it a potential application in diagnosis of acquired immunodeficiency syndrome (AIDS) in the future.

Sequence-specific fluorometric recognition of HIV-1 ds-DNA with zwitterionic zinc(II)-carboxylate polymers

Four water-stable zwitterionic zinc-carboxylate polymers are prepared by reacting N-carboxymethyl-(3,5-dicarboxy)-pyridinium bromide (H3CmdcpBr) with zinc(II) nitrate in the presence of NaOH, through adjusting the solvents and ancillary ligands. With H2O as the solvent and the absence of an ancillary ligand, a two-dimensional (2D) polymer network [Zn(Cmdcp)(H2O)]n (1) is formed. In a mixed H2O/DMF solvent and with the presence of chelating ligands 2,2′-bipyridine (bipy), 1,10-phenanthroline (phen) and 2-(4-pyridyl)benzimidazole (pbz), a one-dimensional (1D) polymer of {[Zn2(Cmdcp)(bipy)2(H2O)5](NO3)2·3H2O}n (2), a mononuclear ionic species of [Zn(phen)(H2O)4][Cmdcp] (3), and a 2D polymer of {[Zn(Cmdcp)(pbz)][pbz]·7H2O}n (4) are accordingly formed. Compounds 1–4 are characterized by IR, elemental analyses and single crystal X-ray crystallography. Compound 2 strongly adsorbs single-stranded DNA (ss-DNA) probe (denoted as P-DNA) labeled with carboxyfluorescein (FAM) and quenches its fluorescence via a photo-induced electron transfer process. If, however, a double-stranded DNA (ds-DNA) of the human immunodeficiency virus 1 (HIV-1 ds-DNA) is further present, the P-DNA interacts with the major groove in HIV-1 ds-DNA via Hoogsteen hydrogen bonding to form a rigid triplex structure. This results in partial or complete fluorescence recovery depending on the concentration of HIV-1 ds-DNA. The findings are applied in fluorometric sensing of HIV-1 ds-DNA. The calibration plot is linear in the 0–60nM target DNA concentration range, with a 7.4nM detection limit (at a signal-to-noise ratio of 3). The assay is highly specific and not interfered by one base pair mutated for complementary target HIV-1 ds-DNA, complementary ss-DNA, single-base pair mutated for complementary ss-DNA, non-specific ss-DNA sequences, and higher-order dimeric G-quadruplexes.

Assembly of Francisella novicida Cpf1 endonuclease in complex with guide RNA and target DNA.

Bacteria and archaea use the CRISPR-Cas system as an adaptive response against infection by foreign nucleic acids. Owing to its remarkable flexibility, this mechanism has been harnessed and adopted as a powerful tool for genome editing. The CRISPR-Cas system includes two classes that are subdivided into six types and 19 subtypes according to conservation of the cas gene and loci organization. Recently, a new protein with endonuclease activity belonging to class 2 type V has been identified. This endonuclease, termed Cpf1, in complex with a single CRISPR RNA (crRNA) is able to recognize and cleave a target DNA preceded by a 5'-TTN-3' protospacer-adjacent motif (PAM) complementary to the RNA guide. To obtain structural insight into the inner workings of Cpf1, the crystallization of an active complex containing the full extent of the crRNA and a 31-nucleotide dsDNA target was attempted. The gene encoding Cpf1 from Francisella novicida was cloned, overexpressed and purified. The crRNA was transcribed and purified in vitro. Finally, the ternary FnCpf1-crRNA-DNA complex was assembled and purified by preparative electrophoresis before crystallization. Crystals belonging to space group C2221, with unit-cell parameters a = 85.2, b = 137.6, c = 320.5 Å, were obtained and subjected to preliminary diffraction experiments.

Structure Basis for Directional R-loop Formation and Substrate Handover Mechanisms in Type I CRISPR-Cas System

Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two cryo-EM snapshots of the Thermobifida fusca type I-E Cascade: (1) unwinding 11bp of dsDNA at the seed-sequence region to scout for sequence complementarity, and (2) further unwinding of the entire protospacer to form a full R-loop. These structures provide the much-needed temporal and spatial resolution to resolve key mechanistic steps leading to Cas3 recruitment. In the early steps, PAM recognition causes severe DNA bending, leading to spontaneous DNA unwinding to form a seed-bubble. The full R-loop formation triggers conformational changes in Cascade, licensing Cas3 to bind. The same process also generates a bulge in the non-target DNA strand, enabling its handover to Cas3 for cleavage. The combination of both negative and positive checkpoints ensures stringent yet efficient target degradation in type I CRISPR-Cas systems.

Sequence specific recognition of HIV-1 dsDNA in the large amount of normal dsDNA based upon nicking enzyme signal amplification and triplex DNA

A sensitive fluorescent strategy for sequence specific recognition of HIV dsDNA was established based upon Nicking Enzyme Signal Amplification (NESA) and triplex formation. dsDNA sequence from the site 7960 to site 7991 of the HIV1 dsDNA gene was designed as target dsDNA, which was composed of two complementary strands Oligonucleotide 1 with the sequence of 3′-CTT CCT TAT CTT CTT CTT CCA CCT CTC TCT CT-5′ (Oligo-1) and Oligonucleotide 2 with the sequence of 5′-GAA GGA ATA GAA GAA GAA GGT GGA GAG AGA GA-3′ (Oligo-2). As a proof of concept, Oligonucleotide 5′-6-FAM-GAG GTG GAG CTG CGC GAC TCC TCC TCT CTC TCT CTC CAC CTC-BHQ-1–3′(Oligo-4) acted as molecular beacon(MB) probe, Oligonucleotide 5′-CTT CCT TAT CTT CTT CTT CCA AAA GGA GTC GCG-3′ (Oligo-7) acted as assistant probe. In the presence of target dsDNA, Oligo-4 and Oligo-7 hybridized with target dsDNA through triplex formation and formed Y-shaped structure, NESA occurred with further addition of Nt.BbvCI, accompanied with the release of fluorescent DNA fragment circularly, resulted in the increase of fluorescence intensity. Under the optimum conditions, the fluorescence intensity was linear with the concentration of target dsDNA over the range from 100pM to 200nM, the linear regression equation was I = 1.266 C + 84.3 (C: nmol/L, R2 = 0.991), with a detection limit of 65pM. Moreover, the effect of coexisted other dsDNA was investigated as well, and satisfactory results were obtained.

Pipette tip biosensors for bacterial double-stranded DNA using bioluminescence induced by zinc finger luciferase

The authors describe a pipette type of biosensor for detecting target genes and using a zinc finger protein fused to luciferase (ZF luciferase). The ZF protein binds to a specific DNA sequence, and the target double-stranded (ds) DNA can be detected by monitoring the enzymatic activity of ZF luciferase. A small avidin-immobilized reaction plate is placed on a plastic pipette tip (referred to as Biologi tip). The dsDNA detection procedures are carried out by using a programmable dispensing robot equipped with a photodetector. These procedures include (a) the aspiration of an analyte to capture the biotinylated target dsDNA (a product of a polymerase chain reaction) on the small reaction plate inside the pipette tip, (b) the introduction of ZF luciferase and luciferin into the pipette tip, and (c) migration of the pipette tip to the detection port to measure bioluminescence on the small reaction plate. The emission originating from luciferase activity is observed on the reaction plate containing immobilized biotin-tagged target dsDNA, whereas plates containing non-target or biotinylated single-stranded DNA only do not yield a signal. The intensity of emission increases proportionally to the concentration of dsDNA, and the detection limit of the target dsDNA is as low as 62 pM. An actual genomic DNA sample from Escherichia coli O157 was successfully detected by this automatic analyzer using the Biologi tip equipped with a reaction plate. This indicates that this system has a large potential for practical applications, including in particular point-of-care analyses in hygiene control, food safety testing, and clinical diagnosis.

Electrochemical Quantitative Analysis of Nucleic Acids Using β‐Cyclodextrin Modified Gold Electrode

AbstractWe present an electrochemical biosensor for the analysis of nucleic acids upon hybridization on the β‐cyclodextrin (β‐CD)‐modified gold electrode. The strategy is based on the following: The 5’‐ferrocene‐labeled single stranded capture probe DNA (5’‐fc‐ss‐DNA) was incorporated into the cavity of thiolated β‐CD which was immobilized on the surface of gold electrode. After hybridization of complementary target DNA, hybridized double stranded DNA (ds‐DNA) was released from the cavity of β‐CD. The difference of electrochemical properties on the modified gold electrode was characterized by cyclic voltametry and surface plasmon resonance. We successfully applied this method to the investigation of the sensor responses due to hybridization on various concentrations of applied target DNA. As a result, the label‐free electrochemical DNA sensor can detect the target DNA with a detection limit of 1.08 nM. Finally, our method does not require either hybridization indicators or other signalling molecules such as DNA intercalaters which most of electrochemical hybridization detection systems require.

Recognition of mixed-sequence DNA using double-stranded probes with interstrand zipper arrangements of O2'-triphenylene- and coronene-functionalized RNA monomers.

Development of hybridization-based probes that enable recognition of specific mixed-sequence double-stranded DNA (dsDNA) regions is of considerable interest due to their potential applications in molecular biology, biotechnology, and medicine. We have recently demonstrated that nucleic acid duplexes with +1 interstrand zipper arrangements of intercalator-functionalized nucleotides such as 2'-O-(pyren-1-yl)methyl RNA monomers are inherently activated for recognition of mixed-sequence dsDNA targets, including chromosomal DNA. In the present work, we follow up on our previous structure-activity relationship studies and explore if the dsDNA-recognition efficiency of these so-called Invader probes can be improved by using larger intercalators than pyrene. Oligodeoxyribonucleotides modified with 2'-O-(triphenylen-2-yl)methyl-uridine monomer X and 2'-O-(coronen-1-yl)methyl-uridine monomer Z form extraordinarily stabilized duplexes with complementary DNA (ΔTm's per modification of up to 13 °C and 20 °C, respectively). Invader probes based on X- and Z-monomers are shown to recognize model dsDNA targets with exceptional binding specificity, but are less efficient than reference probes modified with 2'-O-(pyren-1-yl)methyl-uridine monomer Y. The insight from this study will inform further optimization of Invader probes.

A Novel Electrochemical Sensor Based on [Ru(NH3)6]Cl3 as a Redox Indicator for the Detection of G-G Mismatched DNA.

In this paper, a novel electrochemical sensor was developed for the rapid detection of G-G mismatched DNA based on hexaammineruthenium(III) chloride ([Ru(NH3)6]Cl3) as a redox indicator. The sensor platform was constructed by immobilizing small molecules (NC-linker) on the gold electrode via amide bonds. The as-prepared NC-linker as the nucleic acids recognition molecule can interact with the G base of DNA. After the sensor was incubated with G-G mismatched DNA, the double-stranded DNA (dsDNA) acted as carriers of the signal tags-[Ru(NH3)6]Cl3, which resulted in a remarkable electrochemical signal. More binding of [Ru(NH3)6]Cl3 led to increases of the electrochemical signal. Other mismatched DNA produced only a low response, as well as complementary DNA. Thus G-G mismatched DNA can be easily discriminated from other mismatched and complementary DNA based on the sensor. Furthermore, the method was simple, rapid and repeatable for the detection of G-G mismatched DNA. The selective detection of target dsDNA was achieved by a relative current ratio of the target and control DNA. These results demonstrated that this strategy could provide great promise for the rapid and specific detection of other sequence-specific DNA.