Complementary Target Strand Research Articles

Design of a Sensitive and Selective Electrochemical Aptasensor for the Determination of the Complementary cDNA of miRNA-145 Based on the Intercalation and Electrochemical Reduction of Doxorubicin.

The aim of this research was the determination of a microRNA (miRNA) using a DNA electrochemical aptasensor. In this biosensor, the complementary complementary DNA (cDNA) of miRNA-145 (a sense RNA transcript) was the target strand and the cDNA of miRNA-145 was the probe strand. Both cDNAs can be the product of the reverse transcriptase-polymerase chain reaction of miRNA. The proposed aptasensor's function was based on the hybridization of target strands with probes immobilized on the surface of a working electrode and the subsequent intercalation of doxorubicin (DOX) molecules functioning as the electroactive indicators of any double strands that formed. Electrochemical transduction was performed by measuring the cathodic current resulting from the electrochemical reduction of the intercalated molecules at the electrode surface. In the experiment, because many DOX molecules accumulated on each target strand on the electrode surface, amplification was inherently easy, without a need for enzymatic or complicated amplification strategies. The proposed aptasensor also had the excellent ability to regenerate as a result of the melting of the DNA duplex. Moreover, the use of DNA probe strands obviated the challenges of working with an RNA probe, such as sensitivity to RNase enzyme. In addition to the linear relationship between the electrochemical signal and the concentration of the target strands that ranged from 2.0 to 80.0 nM with an LOD of 0.27 nM, the proposed biosensor was clearly capable of distinguishing between complementary (target strand) and noncomplementary sequences. The presented biosensor was successfully applied for the quantification of DNA strands corresponding to miRNA-145 in human serum samples.

Use of 3,3',5,5' tetramethylbenzidine as new electrochemical indicator of DNA hybridization and its application in genossensor

Electrochemical tools are important biosensor platforms for disease diagnosis, due to their speediness, easiness, low cost and portability. However, for DNA detection, the use of indicators and/or intercalators is necessary to improve electrochemical sensitivity.Currently, ethidium bromide (EthBr) is the cheapest and most used DNA intercalators, but presents carcinogenic and teratogenic properties. Other indicators may be important for DNA photonic detection, and besides being more expensive, they behave similarly to EthBr.This investigation shows for the first time the use of tetramethylbenzidine(TMB) as a new remarkable non-carcinogenic DNA indicator for genosensing purposes, which may be used for nucleic acid detection of microorganisms, based on complementarity of base-pairing between probe and target molecules.The results indicate that TMB can be used as a new electrochemical indicator readily applicable in genosensors, which is able to detect the hybridization of single stranded DNA probe with its complementary target strand. An additional advantage of TMB, beside its non-genotoxicity, is the electrochemical reduction property, which prevents interference of serum components and other oxidative samples in the electrochemical analysis.

Dynamic DNA Target Proofreading in a CRISPR-Cas System

CRISPR-Cas systems provide adaptive immunity against invading genetic elements such as phages and plasmids by degrading the invader DNA. In type I CRISPR-Cas systems the intrinsic RNA component of the Cascade effector complex recognizes a complementary DNA target strand (protospacer) through base pairing while displacing the non-target strand of the duplex. The resulting RNA-DNA hybrid is called R-loop. R-loop establishment recruits the helicase-nuclease Cas3, which subsequently degrades the target DNA. Here we use single-molecule DNA supercoiling experiments to investigate the recognition and verification of the target sequence by Cascade. To this end we carefully explore the effect of mutations across the target. We observe the occurrence of intermediate R-loops which lengths correspond exactly to the distance of the mutation from the start of the target sequence. These intermediate R-loops are unstable and their stability directly correlates with their length. When point mutations are eventually overcome, a full R-loop forms and becomes “locked” through conformational changes of the Cascade complex. These observations provide direct evidence for a directional R-loop formation and suggest a dynamic target scanning by Cascade, mediated through R-loop intermediates. Early encountered mutations challenge R-loop propagation and cause its collapse while distal mutations enable more stable and longer lived R-loops to overcome them. The target validation takes place exclusively at the end of the sequence when full R-loop formation induces conformational changes that initiate DNA degradation. Such a dynamic and directional R-loop propagation scheme offers several advantages for suitable homologous target recognition: (i) the complex spends little time on wrong targets that from the beginning on carry mismatches; (ii) however it supports that point mutations can be tolerated thus precluding invading DNA to escape degradation through protospacer mutations.

DNA-directed control of enzyme-inhibitor complex formation: a modular approach to reversibly switch enzyme activity.

DNA-templated reversible assembly of an enzyme-inhibitor complex is presented as a new and highly modular approach to control enzyme activity. TEM1-β-lactamase and its inhibitor protein BLIP were conjugated to different oligonucleotides, resulting in enzyme inhibition in the presence of template strand. Formation of a rigid dsDNA linker upon addition of a complementary target strand disrupts the enzyme-inhibitor complex and results in the restoration of enzyme activity, enabling detection of as little as 2 fmol DNA. The noncovalent assembly of the complex allows easy tuning of target and template strands without changing the oligonucleotide-functionalized enzyme and inhibitor domains. Using a panel of eight different template sequences, restoration of enzyme activity was only observed in the presence of the target viral DNA sequence. The use of stable, well-characterized protein domains and the intrinsic modularity of our system should allow easy integration with DNA/RNA-based logic circuits for applications in biomedicine and molecular diagnostics.

Single-molecule FRET supports the two-state model of Argonaute action

Argonaute can be found in all three domains of life and is the functional core of the eukaryotic RNA-silencing machinery. In order to shed light on the conformational changes that drive Argonaute action, we performed single-molecule FRET measurements employing a so far uncharacterized member of the Argonaute family, namely Argonaute from the archaeal organism Methanocaldococcus jannaschii (MjAgo). We show that MjAgo is a catalytically active Argonaute variant hydrolyzing exclusively DNA target strands out of a DNA/DNA hybrid. We studied the interplay between Argonaute and nucleic acids using fluorescent dyes covalently attached at different positions of the DNA guide as steric reporters. This allowed us to determine structurally confined parts of the protein scaffold and flexible regions of the DNA guide. Single-molecule FRET measurements demonstrate that the 3′end of the DNA guide is released from the PAZ domain upon target strand loading. This conformational change does not necessitate target strand cleavage but a fully complementary target strand. Thus, our data support the two state model for Argonaute action.

Silver Clusters as Both Chromophoric Reporters and DNA Ligands

Molecular silver clusters conjugated with DNA act as analyte sensors. Our studies evaluate a type of cluster-laden DNA strand whose structure and silver stoichiometry change with hybridization. The sensor strand integrates two functions: the 3' region binds target DNA strands through base recognition while the 5' sequence C(3)AC(3)AC(3)TC(3)A favors formation of a near-infrared absorbing and emitting cluster. This precursor form exclusively harbors an ∼11 silver atom cluster that absorbs at 400 nm and that condenses its single-stranded host. The 3' recognition site associates with a complementary target strand, thereby effecting a 330 nm red-shift in cluster absorption and a background-limited recovery of cluster emission at 790 nm. One factor underlying these changes is sensor unfolding and aggregation. Variations in salt and oligonucleotide concentrations control cluster development by influencing DNA association. Structural studies using fluorescence anisotropy, fluorescence correlation spectroscopy, and size exclusion chromatography show that the sensor-cluster conjugate opens and subsequently dimerizes with hybridization. A second factor contributing to the spectral and photophysical changes is cluster transformation. Empirical silver stoichiometries are preserved through hybridization, so hybridized, dimeric near-infrared conjugates host twice the amount of silver in relation to their violet absorbing predecessors. These DNA structure and net silver stoichiometry alterations provide insight into how DNA-silver conjugates recognize analytes.

Effect of Surface Binding on Heterogeneous DNA Melting Equilibria: A Monte Carlo Simulation Study

Deoxyribonucleic acid (DNA) microarrays are constructed with a surface-immobilized single-stranded probe sequence that hydrogen bonds with its complementary target strand from solution and is subsequently detected, making their hybridization equilibrium of central importance. Unexpectedly, the effect of surface immobilization is that if the sequences of probe and target are exchanged, the hybridization equilibrium shifts. Here, configurational-bias Monte Carlo simulations using a coarse-grained model for DNA were carried out for an undecamer double helix both in solution and bound to a surface to determine dissociation equilibria. Four possible surface binding orientations were independently investigated. Analysis shows that the effect of surface binding is to destabilize hydrogen-bonding interactions of bases proximal to the binding site and enhance those of distal bases due to the double helix lying flat on the surface. Results have implications for predicting surface-bound DNA hybridization equilibria.

High sensitivity DNA detection using gold nanoparticle functionalised polyaniline nanofibres

Polyaniline (PANI) nanofibres (PANI-NF) have been modified with chemically grown gold nanoparticles to give a nanocomposite material (PANI-NF–AuNP) and deposited on gold electrodes. Single stranded capture DNA was then bound to the gold nanoparticles and the underlying gold electrode and allowed to hybridise with a complementary target strand that is uniquely associated with the pathogen, Staphylococcus aureus ( S. aureus), that causes mastitis. Significantly, cyclic voltammetry demonstrates that deposition of the gold nanoparticles increases the area available for DNA immobilisation by a factor of approximately 4. EPR reveals that the addition of the Au nanoparticles efficiently decreases the interactions between adjacent PANI chains and/or motional broadening. Finally, a second horseradish peroxidase (HRP) labelled DNA strand hybridises with the target allowing the concentration of the target DNA to be detected by monitoring the reduction of a hydroquinone mediator in solution. The sensors have a wide dynamic range, excellent ability to discriminate DNA mismatches and a high sensitivity. Semi-log plots of the pathogen DNA concentration vs. faradaic current were linear from 150 × 10 −12 to 1 × 10 −6 mol L −1 and pM concentrations could be detected without the need for molecular, e.g., PCR or NASBA, amplification.

Immobilization of self-quenched DNA hairpin probe with a heterobifunctional reagent on a glass surface for sensitive detection of oligonucleotides

A new sensitive method for the detection of nucleic acids on a glass surface has been described. The self-quenched DNA hairpin probe is immobilized on a glass surface utilizing heterobifunctional reagent, N-(3-triethoxysilylpropyl)-4-(isothiocyanatomethyl)-cyclohexane-1-carboxamide (TPICC). In the closed state fluorescence intensity was quenched due to the presence of guanosine residues in close vicinity of fluorophore while on hybridization with perfectly matched complementary target strand fluorescence was restored. Efficiency and specificity of immobilization as well as thermal stability at variable temperature and pH conditions have been discussed in detail. The method employed has potential for the detection of single nucleotide variations and other diagnostic studies.

Triplex mediated delivery of a platinum complex to a specific DNA target site

Tethering an ethylene diamine linker to the 5 ′ terminus of an oligothymidine sequence provides a site for complexation with K 2PtCl 4. Due to the low reactivity of dT toward a platinum source, we chose dT 8 and dT 15 as our initial synthetic targets for platination. Post-synthetic reaction of the platinum reagent with the diamino oligothymidine generates the diamino dichloro platinum-DNA conjugate that can be used for DNA duplex targeting by oligodeoxyncleotide-mediated triplex formation. The dT 8 sequence is not sufficiently long to facilitate triplex formation and Pt-cross-linking, whereas with a dT 15 sequence cross-linking between the third strand and the duplex occurs exclusively with the duplex target strand directly involved in triplex formation. No examples of cross-linking to the complementary target strand, or of cross-linking to both target strands are observed. Most efficient cross-linking occurs when the dinucleotide d(GpG) is present in the target strand and no cross-linking occurs with the corresponding 7-deazaG dinucleotide target. Cross-linking is also observed when dC or dA residues are present in the target strand, or even with a single dG residue, but it is not observed in any cases to dT residues. Triplex formation provides the ability to target specific sequences of double-stranded DNA and the orientational control arising from triplex formation is sufficient to alter the binding preferences of platinum. Conjugates of the type described here offer the potential of delivering a platinum complex to a specific DNA site.

Determination of DNA guanine sites forming photo-adducts with Ru(II)-labeled oligonucleotides; DNA polymerase inhibition by the resulting photo-crosslinking.

The influence of the distance between the anchoring site of the tethered [Ru(TAP)(2)dip](2+) complex (TAP=1,4,5,8-tetraazaphenanthrene; dip=4,7-diphenyl-1,10-phenanthroline) on a probe sequence and the guanines of the complementary target strand was studied by (1) the luminescence quenching of the complex (by electron transfer) and (2) the oligodeoxyribonucleotide adduct (ODN adduct) formation which results in photo-crosslinking of the two strands. Moving the guanine moieties away from the complex induces an important decrease of the efficiency of both processes, but clearly affects the ODN adduct formation more specifically than the quenching process. From these results, we determined the positions of the guanine bases in the duplex ODN that are able to form a photo-adduct with the tethered complex. We also examined the possible competition between a long-range hole migration in the duplex ODN and the formation of a photo-adduct by using a sequence labeled with the complex at the 5'-phosphate end. Such a hole migration appears to be inefficient as compared to the ODN adduct formation. Finally, we studied the influence of the photo-crosslinking on the function of two different DNA polymerases. A 17-mer Ru(II)-labeled ODN was hybridized to its complementary sequence located on the 5'-side of a 40-mer matrix. After illumination, the elongation of a 13-mer DNA primer hybridized to the 3'-extremity of the same matrix was stopped at a position corresponding to the formation of the ODN adduct.

Electric-field-pulse-assisted covalent immobilization of DNA in the nanosecond time scale

A single square voltage pulse is used to achieve the selective covalent bonding of DNA probes to a functionalized thin-film surface (SiO2) on a plastic substrate. Significant covalent bonding occurs with only 100 ns of pulse duration, representing a decrease in immobilization time of more than ten orders of magnitude from the 2 h needed without electric field. A systematic study of the effect of the duration and rise/fall times of the voltage pulse on the immobilization of DNA probe molecules is presented. Successful hybridization of the DNA probes immobilized using this technique with a complementary target strand was achieved.

Cross-linking of a DNA conjugate tethering a cis-bifunctional platinated complex to a target DNA duplex.

Tethering an ethylene diamine linker to the 5' terminus of an oligothymidine sequence provides a ligand for complexation with K2PtCl4. Post-synthetic reaction of the platinum reagent with the diamino oligothymidine generates the diamino dichloro platinum-DNA conjugate that can be used for DNA duplex targeting by oligodeoxyncleotide-mediated triplex formation. Cross-linking between the third strand and the duplex occurs exclusively with the duplex target strand directly involved in triplex formation. No examples of cross-linking to the complementary target strand or cases of cross-linking to both target strands are observed. Most efficient cross-linking occurs when the dinucleotide d(GpG) is present in the target strand and no cross-linking occurs with the corresponding 7-deazaG dinucleotide target. Cross-linking is also observed when dC or dA residues are present in the target strand, or even with a single dG residue, but it is not observed in any cases to dT residues. Triplex formation provides the ability to target specific sequences of double-stranded DNA; conjugates of the type described here offer the potential of delivering a platinum complex to a specific DNA site.

The design of antisense RNA.

Antisense nucleic acids comprise short-chain synthetic oligonucleotides, often oligodeoxyribonucleotides (ODN) of less than approximately 30 nucleotides and substantially longer sequences formed by ribonucleic acids (RNA). Both groups differ with respect to several properties, including their generation, the mode of delivery, and their structure-function relationship. Long-chain antisense RNA transcribed in vitro or endogenously from recombinant genes fold into three-dimensional structures. The pairing reaction with their complementary target strand occurs via largely unknown annealing mechanisms and, depending on the phylogenetic cellular background, in different cellular compartments. The annealing pathway is influenced by a variety of biologic and biochemical parameters that are as yet poorly understood. However, the basal biochemical mechanisms underlying the relationship between RNA structure and efficient annealing could allow one to derive more general rules for the design of in vivo effective antisense RNA in a way that is not dependent on specific cell types. Here, some of the criteria are discussed that are currently thought to have major impact on the design of long-chain antisense RNA.

Optoelectric potato sorting machine with scanner and reflected light spectrum analyser : Zamotaev AI DE-3490661; 13 February 1992

Polyaniline (PANI) nanofibres (PANI-NF) have been modified with chemically grown gold nanoparticles to give a nanocomposite material (PANI-NF–AuNP) and deposited on gold electrodes. Single stranded capture DNA was then bound to the gold nanoparticles and the underlying gold electrode and allowed to hybridise with a complementary target strand that is uniquely associated with the pathogen, Staphylococcus aureus (S. aureus), that causes mastitis. Significantly, cyclic voltammetry demonstrates that deposition of the gold nanoparticles increases the area available for DNA immobilisation by a factor of approximately 4. EPR reveals that the addition of the Au nanoparticles efficiently decreases the interactions between adjacent PANI chains and/or motional broadening. Finally, a second horseradish peroxidase (HRP) labelled DNA strand hybridises with the target allowing the concentration of the target DNA to be detected by monitoring the reduction of a hydroquinone mediator in solution. The sensors have a wide dynamic range, excellent ability to discriminate DNA mismatches and a high sensitivity. Semi-log plots of the pathogen DNA concentration vs. faradaic current were linear from 150 × 10−12 to 1 × 10−6 mol L−1 and pM concentrations could be detected without the need for molecular, e.g., PCR or NASBA, amplification.

Site-specific DNA cleavage by antisense oligonucleotides covalently linked to phenazine di-N-oxide.

Site-specific degradation of DNA was achieved by the use of DNA oligonucleotides covalently tethered to phenazine 5,10-di-N-oxide. When annealed to a complementary DNA target strand, the antisense oligonucleotide effected alkylation of guanosine residues in proximity to the phenazine di-N-oxide prosthetic group. Admixture of dithiothreitol to the formed duplex resulted in reductive activation of the phenazine di-N-oxide moiety with concomitant generation of diffusible oxygen radicals; the latter effected strand scission of the target DNA oligonucleotide. Several parameters of DNA degradation were studied, including the effect on DNA degradation of chain length in the tether connecting the oligonucleotides and prosthetic group, the relative efficiencies of DNA cleavage when the prosthetic group was in the middle or at the end of the antisense oligonucleotide, and the effect of O2 on DNA degradation. Also studied was the actual chemistry of DNA oligonucleotide degradation and the ability of individual diastereomers of the modified oligonucleotides to mediate degradation of the target DNA.

Alkali reversal of psoralen cross-link for the targeted delivery of psoralen monoadduct lesion.

Psoralen intercalates into double-stranded DNA and photoreacts mainly with thymines to form monoadducts and interstrand cross-links. We used an oligonucleotide model to demonstrate a novel mechanism: the reversal of psoralen cross-links by base-catalyzed rearrangement at 90 degrees C (BCR). The BCR reaction is more efficient than the photoreversal reaction. We show that the BCR occurs predominantly on the furan side of a psoralen cross-link. The cleavage does not result in the breaking of the DNA backbone, and the thymine base freed from the cross-link by the cleavage reaction appears to be unmodified. Similarly, BCR of the furan-side monoadduct of psoralen removed the psoralen molecule and regenerated the unaltered native oligonucleotide. The pyrone-side psoralen monoadduct is relatively resistant to BCR. One can use BCR to perform efficient oligonucleotide-directed, site-specific delivery of a psoralen monoadduct. As a demonstration of this approach, we have hybridized a 19 base long oligonucleotide vehicle containing a furan-side psoralen monoadduct to a 56 base long complementary oligonucleotide target strand and formed a specific cross-link at the target site with 365-nm UV. Subsequent BCR released the oligonucleotide vehicle and deposited the psoralen at the target site.