Multiple DNA Strands Research Articles

Fast, Efficient, and Stable Conjugation of Multiple DNA Strands on Colloidal Quantum Dots.

A novel method for covalent conjugation of DNA to polymer coated quantum dots (QDs) is investigated in detail. This method is fast and efficient: up to 12 DNA strands can be covalently conjugated per QD in optimized reaction conditions. The QD-DNA conjugates can be purified using size exclusion chromatography and the QDs retain high quantum yield and excellent stability after DNA coupling. We explored single-stranded and double-stranded DNA coupling, as well as various lengths. We show that the DNA coupling is most efficient for short (15 mer) single-stranded DNA. The DNA coupling has been performed on QDs emitting at four different wavelengths, as well as on gold nanoparticles, suggesting that this technique can be generalized to a wide range of nanoparticles.

Single Fluorescence Channel-based Multiplex Detection of Avian Influenza Virus by Quantitative PCR with Intercalating Dye.

Since its invention in 1985 the polymerase chain reaction (PCR) has become a well-established method for amplification and detection of segments of double-stranded DNA. Incorporation of fluorogenic probe or DNA intercalating dyes (such as SYBR Green) into the PCR mixture allowed real-time reaction monitoring and extraction of quantitative information (qPCR). Probes with different excitation spectra enable multiplex qPCR of several DNA segments using multi-channel optical detection systems. Here we show multiplex qPCR using an economical EvaGreen-based system with single optical channel detection. Previously reported non quantitative multiplex real-time PCR techniques based on intercalating dyes were conducted once the PCR is completed by performing melting curve analysis (MCA). The technique presented in this paper is both qualitative and quantitative as it provides information about the presence of multiple DNA strands as well as the number of starting copies in the tested sample. Besides important internal control, multiplex qPCR also allows detecting concentrations of more than one DNA strand within the same sample. Detection of the avian influenza virus H7N9 by PCR is a well established method. Multiplex qPCR greatly enhances its specificity as it is capable of distinguishing both haemagglutinin (HA) and neuraminidase (NA) genes as well as their ratio.

Multicolor and erasable DNA photolithography.

The immobilization of DNA molecules onto a solid support is a crucial step in biochip research and related applications. In this work, we report a DNA photolithography method based on photocleavage of 2-nitrobenzyl linker-modified DNA strands. These strands were subjected to ultraviolet light irradiation to generate multiple short DNA strands in a programmable manner. Coupling the toehold-mediated DNA strand-displacement reaction with DNA photolithography enabled the fabrication of a DNA chip surface with multifunctional DNA patterns having complex geometrical structures at the microscale level. The erasable DNA photolithography strategy was developed to allow different paintings on the same chip. Furthermore, the asymmetrical modification of colloidal particles was carried out by using this photolithography strategy. This strategy has broad applications in biosensors, nanodevices, and DNA-nanostructure fabrication.

DNA Interactions in Crowded Nanopores

The motion of DNA in crowded environments is a common theme in physics and biology. Examples include gel electrophoresis and the self-interaction of DNA within cells and viral capsids. Here we study the interaction of multiple DNA molecules within a nanopore by tethering the DNA to a bead held in a laser optical trap to produce a "molecular tug-of-war". We measure this tether force as a function of the number of DNA molecules in the pore and show that the force per molecule decreases with the number of molecules. A simple scaling argument based on a mean field theory of the hydrodynamic interactions between multiple DNA strands explains our observations. At high salt concentrations, when the Debye length approaches the size of the counterions, the force per molecule becomes essentially independent of the number of molecules. We attribute this to a sharp decrease in electroosmotic flow which makes the hydrodynamic interactions ineffective.

Spectroscopic study of cyanine dyes interacting with the biopolymer, DNA

Dyes intercalated into DNA strands or bound to grooves show fluorescence intensity changes and aggregate formation depending on the conditions. In order to establish some empirical rules concerning dye intercalation, spectroscopic studies for the effects of DNA on several series of cyanine dyes with different aromatic rings, conjugated chain length and alkyl substituents were made. Absorption spectra, fluorescence intensity and circular dichroism spectra showed strong dependence on the species of dyes. Combination of preceding studies and these present results indicates that cyanine dyes tend to intercalate into DNA strand if their polymethine bridge was composed of only one carbon. For molecules with the longer chains irregular aggregates were formed by small amounts of DNA, which transformed into complexes composed of multiple dye and DNA strands. These results would serve as a useful guideline for designing of optical functional materials and devices utilizing DNA complex.

Distinguishable Populations Report on the Interactions of Single DNA Molecules with Solid-State Nanopores

Solid-state nanopores have received increasing interest over recent years because of their potential for genomic screening and sequencing. In particular, small nanopores (2–5 nm in diameter) allow the detection of local structure along biological molecules, such as proteins bound to DNA or possibly the secondary structure of RNA molecules. In a typical experiment, individual molecules are translocated through a single nanopore, thereby causing a small deviation in the ionic conductance. A correct interpretation of these conductance changes is essential for our understanding of the process of translocation, and for further sophistication of this technique. Here, we present translocation measurements of double-stranded DNA through nanopores down to the diameter of the DNA itself (1.8–7 nm at the narrowest constriction). In contrast to previous findings on such small nanopores, we find that single molecules interacting with these pores can cause three distinct levels of conductance blockades. We attribute the smallest conductance blockades to molecules that briefly skim the nanopore entrance without translocating, the intermediate level of conductance blockade to regular head-to-tail translocations, and the largest conductance blockades to obstruction of the nanopore entrance by one or multiple (duplex) DNA strands. Our measurements are an important step toward understanding the conductance blockade of biomolecules in such small nanopores, which will be essential for future applications involving solid-state nanopores.

Contact Printing Beyond Surface Roughness: Liquid Supramolecular Nanostamping

Liquid supramolecular nanostamping (LiSuNS) is an efficient stamping technique that replicates various DNA features with no need of solid surface contact. LiSuNS is based on the specific interaction of complementary DNA strands. An original pattern composed of multiple DNA strands on a rough surface can be copied onto a liquid prepolymer that is subsequently hardened, leading to a solid surface that displays the mirror image of the master used.

Demethylation of βA-Globin Gene in Avian Primary Erythroid Cells Is Developmentally Regulated, Strand Asymmetrical and an Active Process.

Differential expression of globin genes has provided an interesting model system for the better understanding of commonly inherited diseases such as thalassemia. In the avian β-type globin cluster (5′- ρ-βA- βA - ε - 3′), silencing of the embryonic ρ-globin gene occurs concomitantly with the activation of adult βA-globin gene during the embryonic development. The pattern of cytosine methylation plays an important role in gene regulation. We have previously shown that de novo methylation of the embryonic ρ-globin gene during development is strand-asymmetrical and spreads from the proximal transcribed region in the early definitive chicken erythroid cells. In the present study, we examined the methylation pattern of βA- globin gene during development in primary chicken erythroid cells. Using bisulfite genomic sequencing we observed a progressive demethylation of βA- globin gene during embryonic development. Maximum methylation is seen in the different regions of the βA- globin gene on day 5, which reduces on day 8 by about 50 %, and on day 11 the degree of methylation is minimal. Further, strand and regional asymmetry was seen with respect to loss of methylation. The template strand gets demethylated first and demethylation of the coding strand lags behind. The promoter and exon 1 get completely demethylated by day 11 whereas residual methylation is retained on exons 2 and 3. To investigate the mechanism of loss of methylation, we examined the methylation pattern of multiple DNA strands derived from primitive and early definitive embryonic erythroid cells. The primitive embryonic erythroid cells demonstrated methylation of the βA- globin promoter and exon 1 on most of the CpG dinucleotides. Discontinuous loss of methylation in early definitive erythroid cells suggested an active mechanism of demethylation since the DNA replication mediated passive demethylation is likely to be continuous. To investigate if an active process contributes to the demethylation of βA- globin gene, we used a modified methylation sensitive single nucleotide primer extension assay (MS-SNuPE) assay for quantitation of demethylase activity. Demethylase assay was carried out using synthetic unmethylated, hemimethylated, and completely methylated oligonucleotides derived from the βA-globin promoter region and nuclear extracts from embryonic chicken erythroid cells. The demethylase activity in day 5, 8 and 11 nuclear extracts was 6%, 76%, and 24% respectively. In contrast to the previously reported 5-methylcytosine glycosylase, which shows a significant substrate specificity for the hemimethylated DNA, we observed the demethylase activity in the embryonic chicken erythroid cells on both fully methylated and hemimethylated βA-globin promoter sequences. Since human and chicken methyl binding domain 4 (MBD4) proteins exhibit 5-methylcytosine glycosylase activity, we determined the in vivo binding of MBD4 to βA- globin promoter in chicken erythroid cells using Chromatin ImmunoPrecipitation (ChIP) followed by real time polymerase chain reaction. MBD4 binding to βA- globin promoter was seen in day 8 but not day 5 chicken erythroid cells implicating a possible role of chicken MBD4 in mediating the active demethylation of the avian βA- globin gene. Our findings demonstrate that demethylation of βA globin gene in developing primary chicken erythroid cells shows strand and regional asymmetry and occurs at least in part by an active process.

Solution AFM Studies of Human Swi-Snf and Its Interactions with MMTV DNA and Chromatin

ATP-dependent nucleosome remodeling complexes are crucial for relieving nucleosome repression during transcription, DNA replication, recombination, and repair. Remodeling complexes can carry out a variety of reactions on chromatin substrates but precisely how they do so remains a topic of active inquiry. Here, a novel recognition atomic force microscopy (AFM) approach is used to characterize human Swi-Snf (hSwi-Snf) nucleosome remodeling complexes in solution. This information is then used to locate hSwi-Snf complexes bound to mouse mammary tumor virus promoter nucleosomal arrays, a natural target of hSwi-Snf action, in solution topographic AFM images of surface-tethered arrays. By comparing the same individual chromatin arrays before and after hSwi-Snf activation, remodeling events on these arrays can be monitored in relation to the complexes bound to them. Remodeling is observed to be: inherently heterogeneous; nonprocessive; able to occur near and far from bound complexes; often associated with nucleosome height decreases. These height decreases frequently occur near sites of DNA release from chromatin. hSwi-Snf is usually incorporated into nucleosomal arrays, with multiple DNA strands entering into it from various directions,+or−ATP; these DNA paths can change after hSwi-Snf activation. hSwi-Snf appears to interact with naked mouse mammary tumor virus DNA somewhat differently than with chromatin and ATP activation of surface-bound DNA/hSwi-Snf produces no changes detectable by AFM.

DNA Sequence Bias During Tn 5 Transposition

Transposition is one of the primary mechanisms causing genome instability. This phenomenon is mechanistically related to other DNA rearrangements such as V(D)J recombination and retroviral DNA integration. In the Tn 5 system, only one protein, the transposase (Tnp), is required for all of the catalytic steps involved in transposon movement. The complexity involved in moving multiple DNA strands within one active site suggests that, in addition to the specific contacts maintained between Tnp and its recognition sequence, Tnp also interacts with the flanking DNA sequence. Here, we demonstrate that Tnp interacts with the donor DNA region. Tnp protects the donor DNA from DNase I digestion, suggesting that Tnp is in contact with, or otherwise distorts, the donor DNA during synapsis. In addition, changes in the donor DNA sequence within this region alter the affinity of Tnp for DNA by eightfold during synapsis. In vitro selection for more stable synaptic complexes reveals an A/T sequence bias for this region. We further show that certain donor DNA sequences, which favor synapsis, also appear to serve as hot spots for strand transfer. The TTATA donor sequence represents the best site. Most surprising is the fact that this sequence is found within the Tnp recognition sequence. Preference for insertion into a site within the Tnp recognition sequence would effectively inactivate one copy of the element and form clusters of the Tn 5 transposon. In addition, the fact that several donor DNA sequences, which favor synapsis, appear to serve as hot spots for transposon insertion suggest that similar criteria may exist for Tnp–donor DNA and Tnp–target DNA interactions.

Direct DNA binding by Brca1.

The tumor suppressor Brca1 plays an important role in protecting mammalian cells against genomic instability, but little is known about its modes of action. In this work we demonstrate that recombinant human Brca1 protein binds strongly to DNA, an activity conferred by a domain in the center of the Brca1 polypeptide. As a result of this binding, Brca1 inhibits the nucleolytic activities of the Mre11/Rad50/Nbs1 complex, an enzyme implicated in numerous aspects of double-strand break repair. Brca1 displays a preference for branched DNA structures and forms protein-DNA complexes cooperatively between multiple DNA strands, but without DNA sequence specificity. This fundamental property of Brca1 may be an important part of its role in DNA repair and transcription.

Complete transposition requires four active monomers in the mu transposase tetramer.

A tetramer of Mu transposase (MuA) cleaves and joins multiple DNA strands to promote transposition. Derivatives of MuA altered at two acidic residues that are conserved among transposases and retroviral integrases form tetramers but are defective in both cleavage and joining. These mutant proteins were used to analyze the contribution of individual monomers to the activity of the tetramer. The performance of different protein combinations demonstrates that not all monomers need to be catalytically competent for the complex to promote an individual cleavage or joining reaction. Furthermore, the results indicate that each pair of essential residues is probably donated to the active complex by a single monomer. Although stable, tetramers composed of a mixture of mutant and wild-type MuA generate products cleaved at only one end and with only one end joined to the target DNA. The abundance of these abortive products and the ratios of the two proteins in complexes stalled at different steps indicate that the complete reaction requires the activity of all four monomers. Thus, each subunit of MuA appears to use the conserved acidic amino acids to promote one DNA cleavage or one DNA joining reaction.