Nonspecific DNAs Research Articles

A hairpin probe-mediated isothermal amplification method to detect target nucleic acid

We herein describe Hairpin probe-mediated Isothermal Amplification (HIAmp), a novel isothermal method to detect a target nucleic acid. This method employs a hairpin probe (HP) designed to be opened through binding to the target nucleic acid. Upon opening of the HP, the primer binds to the free stem of the opened HP followed by its extension by DNA polymerase, consequently displacing and recycling the target nucleic acid to open another HP and producing an intermediate product (IP) containing a nicking site. The IP then continuously produces a trigger probe (TP), which subsequently initiates the isothermal amplification cycles in two separate ways by binding to either the intact HP or the overhang region of the IP. Through the following well-designed interconnected pathways, a large amount of final double-stranded DNA products (FPs) is produced and a high fluorescent signal is generated from the duplex-specific fluorescent dye, SYBR Green I. By employing this isothermal strategy, target DNA was very sensitively detected down to 64 zmol with the capability to discriminate the target DNA against non-specific DNAs. This work would provide remarkable insight into the design of a new DNA network enabling isothermal amplification.

Electrochemical detection of plant virus using gold nanoparticle-modified electrodes

Tristeza is one of the destructive diseases of citrus causing by citrus tristeza virus (CTV). Historically, CTV has been associated with serious outbreaks of quick decline of citrus, therefore CTV monitoring is important aspect for avoiding such re-emerging epidemics, which would threat citrus production through the world. In this context, we have designed for the first time a label-free impedimetric biosensor for the detection of nucleic acid of CTV. The sensing platform based on a screen-printed carbon electrode (SPCE) was modified by electrodeposited gold nanoparticles (AuNPs), which allowed to efficiently immobilizing thiolated ssDNA probes as well to enhance the electrode conductivity. The growth of AuNPs was optimized and characterized using scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). We investigated the behavior of thiolated ssDNA probe layer and its hybridization with target DNA onto AuNP surfaces by EIS measurements in Fe(CN6)4-/Fe(CN6)3- red-ox system. The main sensor design aspects such as AuNPs size, probe DNA concentration and immobilization time together with DNA hybridization time were optimized so as to achieve the best performance. Impedance values of DNA hybridization increased with Citrus tristeza-related synthetic DNA concentration, showing a logarithmic relation in the range of 0.1–10 μM. The results also indicate that the biosensor was able to selectively detect CTV nucleic acids in the presence of other non-specific DNAs. Moreover, we have demonstrated the good performance of the system in a real plant sample matrix. In addition, the sensor reproducibility enhanced after the hybridization onto MCH/poly (AT) thiolated DNA probes which was confirmed by intra- and inter-day variability assays.

In-depth study of DNA binding of Cys2His2 finger domains in testis zinc-finger protein.

Previously, we identified that both fingers 1 and 2 in the three Cys2His2 zinc-finger domains (TZD) of testis zinc-finger protein specifically bind to its cognate DNA; however, finger 3 is non-sequence–specific. To gain insights into the interaction mechanism, here we further investigated the DNA-binding characteristics of TZD bound to non-specific DNAs and its finger segments bound to cognate DNA. TZD in non-specific DNA binding showed smaller chemical shift perturbations, as expected. However, the direction of shift perturbation, change of DNA imino-proton NMR signal, and dynamics on the 15N backbone atom significantly differed between specific and non-specific binding. Using these unique characteristics, we confirmed that the three single-finger segments (TZD1, TZD2 and TZD3) and the two-finger segment (TZD23) non-specifically bind to the cognate DNA. In comparison, the other two-finger segment (TZD12) binding to the cognate DNA features simultaneous non-specific and semi-specific binding, both slowly exchanged in terms of NMR timescale. The process of TZD binding to the cognate DNA is likely stepwise: initially TZD non-specifically binds to DNA, then fingers 1 and 2 insert cooperatively into the major groove of DNA by semi-specific binding, and finally finger 3 non-specifically binds to DNA, which promotes the specific binding on fingers 1 and 2 and stabilizes the formation of a specific TZD–DNA complex.

New roles of DNA and SopB in polymerization of SopA of Escherichia coli F plasmid.

Active equi-paritioning of the F plasmid is achieved by its sopABC gene. SopA binds to the sopAB promoter region and SopB binds to sopC. SopA also polymerizes in the presence of ATP and Mg(II), which is stimulated by SopB. Non-specific DNA is known to inhibit SopA polymerization and disassemble SopA polymer. This study followed kinetics of polymerization and de-polymerization of SopA by turbidity measurement and found new effects by DNA and SopB. Plasmid DNA, at low concentrations, shortened the lag (nucleation) phase of SopA polymerization and also caused an initial 'burst' of turbidity. Results with two non-specific 20-bp DNAs indicated sequence/length dependence of these effects. sopAB operator DNA only showed inhibition of SopA polymerization. Results of turbidity decrease of pre-formed SopA polymer in the presence of ethylenediaminetetraacetic acid showed that SopB also accelerates disassembly of the SopA polymer. The steady-state level of turbidity in the presence of SopB and plasmid DNA indicated synergy between SopB and DNA in the disassembly. SopB protein showed no effect on SopA polymerization, when SopB was specifically bound to DNA. This result and others with truncation mutants of SopB suggested that a proper configuration of the domains of SopB is important for SopA-SopB interactions.

Structural basis of X chromosome DNA recognition by the MSL2 CXC domain during Drosophila dosage compensation.

The male-specific lethal dosage compensation complex (MSL-DCC) selectively assembles on the X chromosome in Drosophila males and activates gene transcription by twofold through histone acetylation. An MSL recognition element (MRE) sequence motif nucleates the initial MSL association, but how it is recognized remains unknown. Here, we identified the CXC domain of MSL2 specifically recognizing the MRE motif and determined its crystal structure bound to specific and nonspecific DNAs. The CXC domain primarily contacts one strand of DNA duplex and employs a single arginine to directly read out dinucleotide sequences from the minor groove. The arginine is flexible when bound to nonspecific sequences. The core region of the MRE motif harbors two binding sites on opposite strands that can cooperatively recruit a CXC dimer. Specific DNA-binding mutants of MSL2 are impaired in MRE binding and X chromosome localization in vivo. Our results reveal multiple dynamic DNA-binding modes of the CXC domain that target the MSL-DCC to X chromosomes.

Microfluidic method for rapid turbidimetric detection of the DNA of Mycobacterium tuberculosis using loop-mediated isothermal amplification in capillary tubes

We describe a microfluidic method for rapid isothermal turbidimetric detection of the DNA of Mycobacterium tuberculosis. Loop-mediated isothermal amplification is accomplished in capillary tubes for amplifying DNA in less than 15 min, and sensitivity and specificity were compared to conventional loop-mediated isothermal amplification (LAMP). The method can detect as little as 1 pg mL−1 DNA in a sample. Results obtained with clinical specimens indicated 90 % sensitivity and 95 % specificity for microfluidic LAMP in comparison to culture methods. No interference occurred due to the presence of nonspecific DNAs. The findings demonstrate the power of the new microfluidic LAMP test for rapid molecular detection of microorganisms even when using bare eyes.

A robust chromatin immunoprecipitation protocol for studying transcription factor-DNA interactions and histone modifications in wood-forming tissue.

Woody cells and tissues are recalcitrant to standard chromatin immunoprecipitation (ChIP) procedures. However, we recently successfully implemented ChIP in wood-forming tissue of the model woody plant Populus trichocarpa. Here we provide the detailed ChIP protocol optimized for wood-forming tissue that we used in those studies. By using stem-differentiating xylem (SDX; a wood-forming tissue), we identified all steps that were ineffective in standard ChIP protocols and systematically modified them to develop and optimize a robust ChIP protocol. The protocol includes tissue collection, cross-linking, nuclear isolation, chromatin extraction, DNA fragmentation, immunoprecipitation, DNA purification and sequence analysis. The protocol takes 2.5 d to complete and allows a robust 8-10-fold enrichment of transcription factor (TF)-bound genomic fragments (~150 ng/g of SDX) over nonspecific DNAs. The enriched DNAs are of high quality and can be used for subsequent PCR and DNA-seq analyses. We used this protocol to identify genome-wide specific TF-DNA interactions during wood formation and histone modifications associated with regulation of wood formation. Our protocol, which may be suitable for many tissue types, is so far the only working ChIP system for wood-forming tissue.

RESPAC: Method to Determine Partial Charges in Coarse-Grained Protein Model and Its Application to DNA-Binding Proteins

While coarse-grained (CG) molecular simulations for large biomolecular complexes have become popular, their electrostatic treatment is often rather simplistic. Here, for Cα-based CG models of globular proteins, we developed a method to obtain an optimal partial charge set and applied it to 17 proteins that bind to DNA. The method follows the restrained electrostatic potential (RESP) fitting method widely used for determination of atomic partial charges in all-atom (AA) molecular mechanics. The proposed method, called the RESPAC method, finds optimal partial charges on surface Cα CG beads so that these charges best approximate the electrostatic potential of the AA model under a restraint term. Comparison of the AA and CG electrostatic potentials showed that the RESPAC charges outperformed simplistic integer-valued charges. Then, the RESPAC method was applied to lac repressor binding to a nonspecific DNA sequence. We found that the CG simulations correlated well with AA molecular dynamics simulations. We also performed CG simulations of 16 other transcription factors. The differences in binding interfaces between nonspecific and specific DNAs were, on average, reduced by using the RESPAC charges. Yet, for several proteins, the nonspecific DNA binding interface was quite different from that of the specific binding interface, which is in accord with a previous report.

Transcription Factor-dependent DNA Bending Governs Promoter Recognition by the Mitochondrial RNA Polymerase

Promoter recognition is the first and the most important step during gene expression. Our studies of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) transcription machinery provide mechanistic understandings on the basic problem of how the mt RNA polymerase (RNAP) with the help of the initiation factor discriminates between promoter and non-promoter sequences. We have used fluorescence-based approaches to quantify DNA binding, bending, and opening steps by the core mtRNAP subunit (Rpo41) and the transcription factor (Mtf1). Our results show that promoter recognition is not achieved by tight and selective binding to the promoter sequence. Instead, promoter recognition is achieved by an induced-fit mechanism of transcription factor-dependent differential conformational changes in the promoter and non-promoter DNAs. While Rpo41 induces a slight bend upon binding both the DNAs, addition of the Mtf1 results in severe bending of the promoter and unbending of the non-promoter DNA. Only the sharply bent DNA results in the catalytically active open complex. Such an induced-fit mechanism serves three purposes: 1) assures catalysis at promoter sites, 2) prevents RNA synthesis at non-promoter sites, and 3) provides a conformational state at the non-promoter sites that would aid in facile translocation to scan for specific sites.

A Reactive Peptidic Linker for Self-Assembling Hybrid Quantum Dot−DNA Bioconjugates

Self-assembly of proteins, peptides, DNA, and other biomolecules to semiconductor quantum dots (QD) is an attractive bioconjugation route that can circumvent many of the problems associated with covalent chemistry and subsequent purification. Polyhistidine sequences have been shown to facilitate self-assembly of proteins and peptides to ZnS-overcoated CdSe QDs via complexation to unoccupied coordination metal sites on the nanocrystal surface. We describe the synthesis and characterization of a thiol-reactive hexahistidine peptidic linker that can be chemically attached to thiolated-DNA oligomers and mediate their self-assembly to CdSe-ZnS core-shell QDs. The self-assembly of hexahistidine-appended DNA to QDs is probed with gel electrophoresis and fluorescence resonance energy transfer techniques, and the results confirm high-affinity conjugate formation with control over the average molar ratio of DNA assembled per QD. To demonstrate the potential of this reactive peptide linker strategy, a prototype QD-DNA-dye molecular beacon is self-assembled and tested against both specific and nonspecific target DNAs. This conjugation route is potentially versatile, as altering the reactivity of the peptide linker may allow targeting of different functional groups such as amines and facilitate self-assembly of other nanoparticle-biomolecule structures.

Voltammetric Behavior of Osmium-Labeled DNA at Mercury Meniscus-Modified Solid Amalgam Electrodes. Detecting DNA Hybridization

The complex of osmium tetroxide with 2,2′-bipyridine has been utilized as a probe of DNA structure and an electroactive marker of DNA in DNA hybridization sensors. It produces several voltammetric signals, the most negative of them has been observed only at mercury electrodes. This signal is of catalytic nature affording a high sensitivity of DNA determination. The catalytic current due to evolution of hydrogen in voltammetry of DNA modified by complex of osmium tetroxide with 2,2′-bipyridine (DNA-Os,bipy) was studied. Solid amalgam electrodes (modified with mercury menisci) of silver (m-AgSAE), copper (m-CuSAE), gold, and of combined bismuth and silver, were used as possible substitutes for mercury electrodes. Besides the hanging mercury drop electrode (HMDE), the catalytic current was observed only on m-AgSAE and m-CuSAE. Electrodes of gold and bismuth amalgams did not give the catalytic current. The detection limit of DNA-Os,bipy on HMDE was 0.1 ng mL−1 (RSD=2.3 %, N=11), and on m-AgSAE 0.2 ng mL−1 (RSD=3.1%, N=11). The m-AgSAE was successfully applied as a detection electrode in double-surface DNA hybridization experiments offering highly specific discrimination between complementary (target) and nonspecific DNAs, as well as determination of the length of a repetitive DNA sequence. The m-AgSAE has proved a convenient alternative to the HMDE or carbon electrodes used for similar purposes in previous work.

DNA Hybridization on Membrane‐Modified Carbon Electrodes

The DNA‐modified membrane electrode was prepared by casting a mixture of nitrocellulose (NC) with target DNA (tDNA) in organic solvent on glassy carbon electrode (GCE). Unlabeled polymerase chain reaction (PCR)‐amplified human genomic sequence (628 bp) or synthetic oligodeoxynucleotides (ODNs) were used as tDNAs, creating a recognition layer. Biotinylated ODNs were used as hybridization probes to recognize specific nucleotide sequences. The hybridization events were detected via an enzyme‐linked electrochemical assay involving binding of streptavidin‐coupled alkaline phosphatase (SALP) to the biotin labels of the probe bound to tDNA. After the probe hybridization and SALP binding, the electrode was immersed into an electroinactive enzyme substrate (1‐naphthyl phosphate). The alkaline phosphatase converted the inactive substrate into electroactive 1‐naphthol that penetrated through the NC membrane to the GCE surface and was subsequently detected using an anodic voltammetric signal. The optimized method offered a good discrimination between complementary and nonspecific DNAs and yielded well‐defined responses for both single‐copy and repetitive tDNA sequences. In contrast to previously published methods using electrodes with mechanically attached membranes, the previously mentioned electrode is easily amenable to parallel DNA analysis. On the occasion of the eightieth birthday of Professor Petr Zuman.

TATA-box DNA binding activity and subunit composition for RNA polymerase III transcription factor IIIB from Xenopus laevis.

The RNA polymerase III transcription initiation factor TFIIIB contains the TATA-box-binding protein (TBP) and polymerase III-specific TBP-associated factors (TAFs). Previous studies have shown that DNA oligonucleotides containing the consensus TATA-box sequence inhibit polymerase III transcription, implying that the DNA binding domain of TBP is exposed in TFIIIB. We have investigated the TATA-box DNA binding activity of Xenopus TFIIIB, using transcription inhibition assays and a gel mobility shift assay. Gel shift competition assays with mutant and nonspecific DNAs demonstrate the specificity of the TFIIIB-TATA box DNA complex. The apparent dissociation constant for this protein-DNA interaction is approximately 0.4 nM, similar to the affinity of yeast TBP for the same sequence. TFIIIB transcriptional activity and TATA-box binding activity cofractionate during a series of four ion-exchange chromatographic steps, and reconstituted transcription reactions demonstrate that the TATA-box DNA-protein complex contains TFIIIB TAF activity. Polypeptides with apparent molecular masses of 75 and 92 kDa are associated with TBP in this complex. These polypeptides were renatured after elution from sodium dodecyl sulfate-gels and tested individually and in combination for TFIIIB TAF activity. Recombinant TBP along with protein fractions containing the 75- and 92-kDa polypeptides were sufficient to reconstitute TFIIIB transcriptional activity and DNA binding activity, suggesting that Xenopus TFIIIB is composed of TBP along with these polypeptides.

DNA Recognition by the EcoK Methyltransferase : The Influence of DNA Methylation and the Cofactor S-adenosyl- l-methionine

The methyltransferase of the EcoK type I restriction/modification system is trimeric, M 2S 1, where the S subunit determines the sequence specificity of the enzyme. The methyltransferase has a strong preference for hemimethylated substrate DNA and, therefore, we have investigated the effect of the methylation state of DNA on binding by the enzyme, together with the effects on binding of the cofactor S -adenosyl- l-methionine. Our results indicate that the methyltransferase has two non-interacting S-adenosyl-L-methionine binding sites, each with a dissociation constant of 3·60(± 0·42) μM determined by equilibrium dialysis, or 2·21(±0·29) μM determined by the displacement of a fluorescent probe. Ultraviolet light-induced crosslinking showed that S -adenosyl- l-methionine binds strongly only to the modification (M) subunits. Changes in the sedimentation velocity of the methyltransferase imply a protein conformational change due to S -adenosyl- l-methionine binding. Gel retardation results show that the binding of S-adenosyl- l-methionine to the methyltransferase enhances binding to both specific and non-specific DNAs, but the enhancement is greater for the specific DNA. Differences in binding affinities contribute to the recognition of the specific nucleotide sequence AAC(N) 6GTGC by the methyltransferase in preference to a non-specific sequence. In contrast, although the complexes of unmodified and hemimethylated DNAs with the methyltransferase have different mobilities in non-denaturing gels, there appears to be no contribution of binding affinity to the distinction between these two substrates. Therefore, the preference for a hemimethylated substrate must be due to a difference in catalysis.

Multiple functional enhancer motifs of rat ribosomal gene

Previous studies from this laboratory have characterized a 174 bp enhancer element which is located 2 kb upstream of the initiation site. Half of the enhancer action is controlled by a 37 bp element at the 3' end of the 174 bp region. We now report that a 43 bp adjacent domain which is located upstream of the 37 bp element constitutes an additional motif of the rDNA enhancer. When the plasmid consisting of the 43 bp DNA upstream of the rDNA core promoter was transcribed in a fractionated rat tumor cell extract (fraction DE-B), transcription of rDNA was augmented 4 fold. Electrphoretic mobility shift and DNAase I footprinting analyses showed that the purified 37 bp enhancer (E1)-binding protein, (E1BF) not only interacted with the enhancer motif E1 but also interacted with the neighbouring 43 bp enhancer domain E2. The specificity of the binding was demonstrated by competition with unlabelled 37 bp and 43 bp fragment and lack of competition with nonspecific DNAs in the mobility shift assay. These studies have shown that a single pol I transcription factor can bind to multiple enhancer domains with no significant sequence homologies and such multiple interactions may result in maximal transcription of ribosomal gene from the core promoter.

Purification and Characterization of a Novel Factor Which Stimulates Rat Ribosomal Gene Transcription In Vitro by Interacting with Enhancer and Core Promoter Elements

Previous studies in our laboratory have characterized a 174-base-pair (bp) enhancer sequence in the rat ribosomal DNA spacer region that exhibits all of the characteristics of a polymerase (Pol) II enhancer. Further studies showed that at least half of the enhancer activity resides in a 37-bp motif (E1) within the 174-bp spacer sequence that is located between positions -2.183 and -2.219 kilobase pairs upstream of the initiation site. To identify the factor(s) that binds specifically to the 37-bp enhancer domain, we fractionated whole-cell extract from rat adenocarcinoma ascites cells by chromatography on a series of columns, including an oligodeoxynucleotide affinity column. The final preparation contained two polypeptides of molecular weights 79,400 and 89,100 and was completely devoid of RNA Pol I activity. Electrophoretic mobility shift analysis showed that the polypeptides in the purified preparation (designated E1BF) interacted with both the enhancer element and the core promoter. To determine whether each polypeptide can separately bind to the core promoter and the enhancer, the individual components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, renatured, and subjected to gel retardation analysis. This experiment demonstrated that both polypeptides interacted with the two cis-acting sequences. The specificity of the binding was demonstrated by competition with unlabeled 37-bp and core promoter fragments and lack of competition with nonspecific DNAs in the mobility shift assay. The 37-bp enhancer as well as the downstream sequence of the core promoter were protected by E1BF in the DNase I footprinting assay. Addition of E1BF to limiting amounts of fraction DE-B, which contains all factors essential for Pol I-directed transcription, resulted in three- to fourfold stimulation of ribosomal DNA transcription. Comparison of molecular weights and footprinting profiles did not reveal any relationship between E1BF and other Pol I trans-acting factors.

Purification and characterization of a novel factor which stimulates rat ribosomal gene transcription in vitro by interacting with enhancer and core promoter elements.

Previous studies in our laboratory have characterized a 174-base-pair (bp) enhancer sequence in the rat ribosomal DNA spacer region that exhibits all of the characteristics of a polymerase (Pol) II enhancer. Further studies showed that at least half of the enhancer activity resides in a 37-bp motif (E1) within the 174-bp spacer sequence that is located between positions -2.183 and -2.219 kilobase pairs upstream of the initiation site. To identify the factor(s) that binds specifically to the 37-bp enhancer domain, we fractionated whole-cell extract from rat adenocarcinoma ascites cells by chromatography on a series of columns, including an oligodeoxynucleotide affinity column. The final preparation contained two polypeptides of molecular weights 79,400 and 89,100 and was completely devoid of RNA Pol I activity. Electrophoretic mobility shift analysis showed that the polypeptides in the purified preparation (designated E1BF) interacted with both the enhancer element and the core promoter. To determine whether each polypeptide can separately bind to the core promoter and the enhancer, the individual components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, renatured, and subjected to gel retardation analysis. This experiment demonstrated that both polypeptides interacted with the two cis-acting sequences. The specificity of the binding was demonstrated by competition with unlabeled 37-bp and core promoter fragments and lack of competition with nonspecific DNAs in the mobility shift assay. The 37-bp enhancer as well as the downstream sequence of the core promoter were protected by E1BF in the DNase I footprinting assay. Addition of E1BF to limiting amounts of fraction DE-B, which contains all factors essential for Pol I-directed transcription, resulted in three- to fourfold stimulation of ribosomal DNA transcription. Comparison of molecular weights and footprinting profiles did not reveal any relationship between E1BF and other Pol I trans-acting factors.