Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications.

The synthesis, proof of structure, and the absorption and fluorescence properties of two new unsymmetrical cyanine dyes, thiazole orange dimer (TOTO; 1,1'-(4,4,7,7-tetramethyl-4,7- diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiaz ole)-2- methylidene]-quinolinium tetraiodide) and oxazole yellow dimer (YOYO; an analogue of TOTO with a benzo-1,3-oxazole in place of the benzo-1,3-thiazole) are reported. TOTO and YOYO are virtually non-fluorescent in solution, but form highly fluorescent complexes with double-stranded DNA (dsDNA), up to a maximum dye to DNA bp ratio of 1:4, with greater than 1000-fold fluorescence enhancement. The dsDNA-TOTO (lambda max 513 nm; lambda maxF 532 nm) and dsDNA-YOYO (lambda max 489 nm; lambda maxF 509 nm) complexes are completely stable to electrophoresis on agarose and acrylamide gels. Mixtures of restriction fragments pre-labeled with ethidium dimer (EthD; lambda maxF 616 nm) and those pre-labeled with either TOTO or YOYO were separated by electrophoresis. Laser excitation at 488 nm and simultaneous confocal fluorescence detection at 620-750 nm (dsDNA-EthD emission) and 500-565 nm (dsDNA-TOTO or dsDNA-YOYO emission) allowed sensitive detection, quantitation, and accurate sizing of restriction fragments ranging from 600 to 24,000 bp. The limit of detection of dsDNA-TOTO and YOYO complexes with a laser-excited confocal fluorescence gel scanner for a band 5-mm wide on a 1-mm thick agarose gel was 4 picograms, about 500-fold lower than attainable by conventional staining with ethidium bromide.     

Triplet fraction buildup effect of the DNA-YOYO complex studied with fluorescence correlation spectroscopy

DNA fragments of various lengths and YOYO-1 iodide (YOYO) were mixed at various ratios, and fluorescence was measured using fluorescence correlation spectroscopy. The number of substantially emitting YOYO molecules binding to the DNA and the binding intervals between the YOYO molecules were estimated for DNA-YOYO complexes of various lengths. In the present study, we found an interesting phenomenon: triplet buildup. Because fluorophores that fall into the triplet state do not emit fluorescence, a part of the dark period can be recovered by emitting photons from other excited YOYO molecules in the same DNA strings in the confocal elements. The remaining dark period can be considered to be the total miss-emission rate. Estimates of the total miss-emission rate are important for calculation of the length and amount of DNA.     

Intramolecular recombination R-triplex in solution: stabilization by bis-intercalator YOYO

Recognition of double-stranded DNA with a mixed nucleotide sequence by oligonucleotide is a long-term challenge. This aim can be achieved via formation of the recombination R-triplex, accommodating two identical DNA strands in parallel orientation, and antiparallel complementary strand. In the absence of proteins the R-triplex stability is low, however, so that intermolecular R-triplex is not formed by three DNA strands in a ligand-free system. Recently, recognition of DNA with mixed base sequence by single-stranded oligonucleotide in the presence of bis-intercalator YOYO was reported. Here, we describe thermodynamic characteristics of YOYO complexes with the model oligonucleotides 5'-GT-2AP-GACTGAG TTTT CTCAGTCTACGC GAA GCGTAGACTGAG-3' (R(2AP)CW) bearing a single reporting 2-aminopurine (2AP) in place of adenine and 5'-CTCAGTCTACGC GAA GCGTAGACTGAG-3' (CW). We found that each oligonucleotide is able to bind two YOYO molecules via intercalation mode in 0.5 M LiCl. Fluorescence intensity of YOYO intercalated in triplex R(2AP)CW and in CW hairpin increased 40-fold compared to the free YOYO. Remarkably, the melting temperature of the triplex (determined using temperature dependence of the 2AP fluorescence) increased from 19 degrees C to 33 degrees C upon binding two YOYO molecules. Further increase in the YOYO concentration resulted in binding of up to five YOYO molecules to R(2AP)CW triplex and up to six YOYO molecules to CW hairpin.     

Identification of binding mechanisms in single molecule-DNA complexes

Changes in the elastic properties of single deoxyribonucleic acid (DNA) molecules in the presence of different DNA-binding agents are identified using atomic force microscope single molecule force spectroscopy. We investigated the binding of poly(dG-dC) dsDNA with the minor groove binder distamycin A, two supposed major groove binders, an alpha-helical and a 3(10)-helical peptide, the intercalants daunomycin, ethidium bromide and YO, and the bis-intercalant YOYO. Characteristic mechanical fingerprints in the overstretching behavior of the studied single DNA-ligand complexes were observed allowing the distinction between different binding modes. Docking of ligands to the minor or major groove of DNA has the effect that the intramolecular B-S transition remains visible as a distinct plateau in the force-extension trace. By contrast, intercalation of small molecules into the double helix is characterized by the vanishing of the B-S plateau. These findings lead to the conclusion that atomic force microscope force spectroscopy can be regarded as a single molecule biosensor and is a potent tool for the characterization of binding motives of small ligands to DNA.     

Single- and double-strand photocleavage of DNA by YO, YOYO and TOTO.

Photocleavage of dsDNA by the fluorescent DNA stains oxazole yellow (YO), its dimer YOYO) and the dimer TOTO of thiazole orange (TO) has been investigated as a function of binding ratio. On visible illumination, both YO and YOYO cause single-strand cleavage, with an efficiency that varies with the dye/DNA binding ratio in a manner which can be rationalized in terms of free dye being an inefficient photocleavage reagent and externally bound dye being more efficient than intercalated dye. Moreover, the photocleavage mechanism changes with binding mode. Photocleavage by externally bound dye is, at least partly, oxygen dependent with scavenger studies implicating singlet oxygen as the activated oxygen intermediate. Photocleavage by intercalated dye is essentially oxygen-independent but can be inhibited by moderate concentrations of beta- mercaptoethanol--direct attack on the phosphoribose backbone is a possible mechanism. TOTO causes single-strand cleavage approximately five times less efficiently than YOYO. No direct double-strand breaks (dsb) are detected with YO or YOYO, but in both cases single-strand breaks (ssb) are observed to accumulate to eventually produce double-strand cleavage. With intercalated YO the accumulation occurs in a manner consistent with random generation of strand lesions, while with bisintercalated YOYO the yield of double-strand cleavage (per ssb) is 5-fold higher. A contributing factor is the slow dissociation of the bis-intercalated dimer, which allows for repeated strand-attack at the same binding site, but the observation that the dsb/ssb yield is considerably lower for externally bound than for bis-intercalated YOYO at low dye/DNA ratios indicates that the binding geometry and/or the cleavage mechanism are also important for the high dsb-efficiency. In fact, double-strand cleavage yields with bis-intercalated YOYO are higher than those predicted by simple models, implying a greater than statistical probability for a second cleavage event to occur adjacent to the first (i.e. to be induced by the same YOYO molecule). With TOTO the efficiency of the ssb-accumulation is comparable to that observed with YOYO.     

Enzymatic conversion of long DNA to small DNA fragments for the construction of short hairpin RNA expression libraries

Several techniques to enzymatically construct a short hairpin RNA (shRNA) expression library have been reported as tools for comprehensive genetic analyses by RNA interference. Our technique constructs an shRNA expression library from 25- to 35-bp DNA fragments by fragmenting given double-stranded DNA (dsDNA). We compared the following two procedures to efficiently prepare such small DNA fragments: one is the cleavage of dsDNA with deoxyribonuclease I (DNase I) in the presence of Mn²⁺ followed by blunting with T4 DNA polymerase, and the other is the introduction of nicks with DNase I in the presence of Mg²⁺ followed by blunting with the Klenow fragment. Consequently, the latter yielded the DNA fragments more efficiently. However, these DNA fragments were contaminated with fused DNA fragments that had originated from two regions of original dsDNA. Therefore, we used single-strand-specific exonucleases and succeeded in suppressing the production of such fused DNA fragments. Our technique allows the efficient conversion of given dsDNA to small DNA fragments.     

Intramolecular Recombination R-Triplex in Solution: Stabilization by bis-intercalator YOYO

Abstract

Recognition of double-stranded DNA with a mixed nucleotide sequence by oligonucleotide is a long-term challenge. This aim can be achieved via formation of the recombination R-triplex, accommodating two identical DNA strands in parallel orientation, and antiparallel complementary strand. In the absence of proteins the R-triplex stability is low, however, so that intermolecular R-triplex is not formed by three DNA strands in a ligand-free system. Recently, recognition of DNA with mixed base sequence by single-stranded oligonucleotide in the presence of bis-intercalator YOYO was reported. Here, we describe thermodynamic characteristics of YOYO complexes with the model oligonucleotides 5′-GT- 2AP-GACTGAG TTTT CTCAGTCTACGC GAA GCGTAGACTGAG-3′ (R^2APCW) bearing a single reporting 2-aminopurine (2AP) in place of adenine and 5′- CTCAGTCTACGC GAA GCGTAGACTGAG-3′ (CW). We found that each oligonucleotide is able to bind two YOYO molecules via intercalation mode in 0.5 M LiCl. Fluorescence intensity of YOYO intercalated in triplex R^2APCW and in CW hairpin increased 40-fold compared to the free YOYO. Remarkably, the melting temperature of the triplex (determined using temperature dependence of the 2AP fluorescence) increased from 19° C to 33° C upon binding two YOYO molecules. Further increase in the YOYO concentration resulted in binding of up to five YOYO molecules to R^2APCW triplex and up to six YOYO molecules to CW hairpin.     

Ligand binding of a ribosome-displayed protein detected in solution at the single molecule level by fluorescence correlation spectroscopy

Interaction of a single-chain antibody fragment (scFv) with its cognate antigen while still attached to the ribosome was studied by fluorescence correlation spectroscopy (FCS). In experiments with purified scFv, FCS was capable of resolving the difference in diffusion time between free and antibody-bound labelled antigen. Ribosome-displayed antibody fragments generated by in vitro translation, in which neither the protein nor the mRNA leaves the ribosome owing to the absence of a stop codon and stabilizing buffer conditions, could be shown to specifically bind the antigen. The antibody-antigen interaction was specific, as shown by inhibition or displacement with unlabelled antigen and by control experiments with a non-cognate antibody fragment.     

Rapid Synthesis of a Long Double-Stranded Oligonucleotide from a Single-Stranded Nucleotide Using Magnetic Beads and an Oligo Library

Chemical synthesis of oligonucleotides is a widely used tool in the field of biochemistry. Several methods for gene synthesis have been introduced in the growing area of genomics. In this paper, a novel method of constructing dsDNA is proposed. Short (28-mer) oligo fragments from a library were assembled through successive annealing and ligation processes, followed by PCR. First, two oligo fragments annealed to form a dsDNA molecule. The double-stranded oligo was immobilized onto magnetic beads (solid support) via streptavidin-biotin binding. Next, single-stranded oligo fragments were added successively through ligation to form the complete DNA molecule. The synthesized DNA was amplified through PCR and gel electrophoresis was used to characterize the product. Sanger sequencing showed that more than 97% of the nucleotides matched the expected sequence. Extending the length of the DNA molecule by adding single-stranded oligonucleotides from a basis set (library) via ligation enables a more convenient and rapid mechanism for the design and synthesis of oligonucleotides on the go. Coupled with an automated dispensing system and libraries of short oligo fragments, this novel DNA synthesis method would offer an efficient and cost-effective method for producing dsDNA.     

Comparison by restriction fragment differential display RT-PCR of gene expression pattern in bovine oocytes matured in the presence or absence of fetal calf serum

A novel restriction fragment differential display (RFDD) RT-PCR has been used to compare patterns of mRNA expression in bovine oocytes matured in vitro in the presence (10%) or absence of fetal calf serum (FCS). Total RNA extracted from matured and denuded oocytes was processed using display Profile kit (Display System Biotech). RFDD RT-PCR products were separated on 6% polyacrylamide gel and analyzed using a Storm 860 scanner. Selected bands representing potentially differentially expressed fragments were excised from the gel and re-amplified. Re-amplified fragments with size matched to the original fragment were cloned into the TA vector and sequenced. Initially, 10 and 15 differentially expressed fragments were isolated from oocytes matured in the presence and absence of FCS, respectively. Eight out of 10 and 10 out of 15 fragments were re-amplified successfully as evidenced by size similarity to the original fragments. Finally, the size of six inserts sequenced from each group matched the size of corresponding original as well as re-amplified fragments. Sequence comparison search revealed similarity of some isolated fragments to 18s ribosomal RNA, bovine apolipoprotein A-I, bovine mitochondrion DNA, human CGI-79 mRNA, human Ab1-interactor protein, and bovine satellite DNA. The other sequenced fragments may represent novel genes. We showed that RFDD RT-PCR can be effectively applied to contrast gene expression pattern in bovine oocytes and that presence or absence of FCS during maturation interval affects gene expression pattern in matured bovine oocytes.     

Sequence-specific fluorescent labeling of double-stranded DNA observed at the single molecule level

Fluorescent labeling of a short sequence of double-stranded DNA (dsDNA) was achieved by ligating a labeled dsDNA fragment to a stem–loop triplex forming oligonucleotide (TFO). After the TFO has wound around the target sequence by ligand-induced triple helix formation, its extremities hybridize to each other, leaving a dangling single-stranded sequence, which is then ligated to a fluorescent dsDNA fragment using T4 DNA ligase. A non-repeated 15 bp sequence present on lambda DNA was labeled and visualized by fluorescence microscopy after DNA combing. The label was found to be attached at a specific position located at 4.2 ± 0.5 kb from one end of the molecule, in agreement with the location of the target sequence for triple helix formation (4.4 kb from one end). In addition, an alternative combing process was noticed in which a DNA molecule becomes attached to the combing slide from the label rather than from one of its ends. The method described herein provides a new tool for the detection of very short sequences of dsDNA and offers various perspectives in the micromanipulation of single DNA molecules.     

Direct observation of the reversible unwinding of a single DNA molecule caused by the intercalation of ethidium bromide

Ethidium bromide (EtBr) is the conventional intercalator for visualizing DNA. Previous studies suggested that EtBr lengthens and unwinds double-stranded DNA (dsDNA). However, no one has observed the unwinding of a single dsDNA molecule during intercalation. We developed a simple method to observe the twisting motions of a single dsDNA molecule under an optical microscope. A short dsDNA was attached to a glass surface of a flow chamber at one end and to a doublet bead as a rotation marker at the other end. After the addition and removal of EtBr, the bead revolved in opposite directions that corresponded to the unwinding and rewinding of a dsDNA, respectively. The amount of intercalating EtBr was estimated from the revolutions of the bead. EtBr occupied 57% of base pairs on a single dsDNA at 1 mM of EtBr, indicating that EtBr molecules could bind at contiguous sites to each other. The isotherm of intercalation showed that negative cooperativity existed between adjoining EtBr molecules. The association constant of EtBr and dsDNA (1.9 (±0.1) × 10⁵ M⁻¹) was consistent with that of previous results. Our system is useful to investigate the twisting of a single dsDNA interacting with various chemicals and biomolecules.     

DNA interference: a simple and efficient gene-silencing system for high-throughput functional analysis in the fern adiantum

RNA interference (RNAi) has become a powerful tool for determining gene function and is used in a wide variety of organisms. Since it is necessary to generate double-stranded RNA (dsRNA) as an inducer for RNAi, preparation of RNAi-inducing constructs is somewhat cumbersome and time consuming, especially for the thousands of genes used in a genome-wide analysis. To overcome these problems, we have developed a more convenient gene-silencing method in the fern Adiantum using double-stranded DNA (dsDNA) as a model system for functional analysis in plants. Delivery of dsDNA fragments homologous to an endogenous gene into gametophytic cells can induce sequence-specific gene silencing. As it only requires dsDNA fragments homologous to a target gene, PCR-amplified fragments are enough to trigger gene silencing. Maximum gene silencing efficiencies of >90% have been achieved for transformed plants. In addition, simultaneous transfer of dsDNA fragments corresponding to multiple genes still has a silencing effect for individual genes. We term this approach 'DNA interference'.     

T4 DNA ligase is more than an effective trap of cyclized dsDNA

T4 DNA ligase is used in standard cyclization assays to trap double-stranded DNA (dsDNA) in low-probability, cyclic or highly bent conformations. The cyclization probability, deduced from the relative yield of cyclized product, can be used in conjunction with statistical mechanical models to extract the bending stiffness of dsDNA. By inserting the base analog 2-aminopurine (2-AP) at designated positions in 89bp and 94bp dsDNA fragments, we find that T4 DNA ligase can have a previously unknown effect. Specifically, we observe that addition of T4 ligase to dsDNA in proportions comparable to what is used in the cyclization assay leads to a significant increase in fluorescence from 2-AP. This effect is believed to originate from stabilization of local base-pair opening by formation of transient DNA-ligase complexes. Non-specific binding of T4 ligase to dsDNA is also confirmed using fluorescence correlation spectroscopy (FCS) experiments, which reveal a systematic reduction of dsDNA diffusivity in the presence of ligase. ATP competes with regular DNA for non-covalent binding to the T4 ligase and is found to significantly reduce DNA-ligase complexation. For short dsDNA fragments, however, the population of DNA-ligase complexes at typical ATP concentrations used in DNA cyclization studies is determined to be large enough to dominate the cyclization reaction.     

Separation of large DNA restriction fragments on a size-exclusion column by a nonideal mechanism

Double-strand DNA (dsDNA) restriction fragments were chromatographed on the DuPont Bioseries GF-250 column. Two anomolous chromatographic properties were observed. (1) A triphasic dependence of retention on dsDNA chain length was observed. Small DNA fragments (less than 500 base pairs) displayed typical size exclusion, intermediate size DNA (800-5000 base pairs) eluted in the void volume, and larger DNA fragments were increasingly retained. (2) The void volume for nucleic acids was less than that for large polypeptides. The retention of moderately large DNA fragments increased linearly as the square root of the chain length over the range 5.5 to 50 kilobase pairs (ca. 3-30 X 10(6) Mr). A number of eluant manipulations were carried out in order to examine the mechanism by which the larger DNA fragments were being retained and separated. Evidence was not obtained to support either ion exchange or reverse phase as the retention mechanism. The usefulness of such a column for molecular biological manipulations is illustrated by the rapid isolation of homogeneous viral DNA fragments resected from their cloning vectors with restriction endonucleases.     

Single molecule linear analysis of DNA in nano-channel labeled with sequence specific fluorescent probes

An array of nano-channels was fabricated from silicon based semiconductor materials to stretch long, native dsDNA. Here we present a labeling scheme in which it is possible to identify the location of specific sequences along the stretched DNA molecules. The scheme proceeds by first using the strand displacement activity of the Vent (exo-) polymerase to generate single strand flaps on nicked dsDNA. These single strand flaps are hybridized with sequence specific fluorophore-labeled probes. Subsequent imaging of the DNA molecules inside a nano-channel array device allows for quantitative identification of the location of probes. The highly efficient DNA hybridization on the ss-DNA flaps is an excellent method to identify the sequence motifs of dsDNA as it gives us unique ability to control the length of the probe sequence and thus the frequency of hybridization sites on the DNA. We have also shown that this technique can be extended to a multi color labeling scheme by using different dye labeled probes or by combining with a DNA- polymerase-mediated incorporation of fluorophore-labeled nucleotides on nicking sites. Thus this labeling chemistry in conjunction with the nano-channel platform can be a powerful tool to solve complex structural variations in DNA which is of importance for both research and clinical diagnostics of genetic diseases.     

Rational Design of High-Number dsDNA Fragments Based on Thermodynamics for the Construction of Full-Length Genes in a Single Reaction

Gene synthesis is frequently used in modern molecular biology research either to create novel genes or to obtain natural genes when the synthesis approach is more flexible and reliable than cloning. DNA chemical synthesis has limits on both its length and yield, thus full-length genes have to be hierarchically constructed from synthesized DNA fragments. Gibson Assembly and its derivatives are the simplest methods to assemble multiple double-stranded DNA fragments. Currently, up to 12 dsDNA fragments can be assembled at once with Gibson Assembly according to its vendor. In practice, the number of dsDNA fragments that can be assembled in a single reaction are much lower. We have developed a rational design method for gene construction that allows high-number dsDNA fragments to be assembled into full-length genes in a single reaction. Using this new design method and a modified version of the Gibson Assembly protocol, we have assembled 3 different genes from up to 45 dsDNA fragments at once. Our design method uses the thermodynamic analysis software Picky that identifies all unique junctions in a gene where consecutive DNA fragments are specifically made to connect to each other. Our novel method is generally applicable to most gene sequences, and can improve both the efficiency and cost of gene assembly.     

Kinetics of DNA binding with chloroquine phosphate using capacitive sensing method

The capacitive sensing method has been applied to study the binding of DNA with chloroquine phosphate. DNA was immobilized on a gold electrode surface, self-assembled with thioglycolic acid. The results of a quartz crystal impedance (QCI) study indicate that the reaction of double-strand DNA (dsDNA) with chloroquine includes a fast electrostatic attraction and a slow intercalation of chloroquine into double-strand helix. The real-time experimental data obtained by capacitive sensing also revealed two distinctive kinetics stages during binding of dsDNA with chloroquine, while only one stage exists during reaction of single-strand DNA (ssDNA) with chloroquine. The kinetic parameters were obtained by fitting the real-time experimental data using a two stage reaction model. The rate constants of electrostatic attraction for dsDNA and ssDNA are estimated as 0.014 and 0.018 s(-1), respectively. The rate constant of the second stage of dsDNA is 0.0011 s(-1).     

DNA Replication Catalyzed by Herpes Simplex Virus Type 1 Proteins Reveals Trombone Loops at the Fork

Using purified replication factors encoded by herpes simplex virus type 1 and a 70-base minicircle template, we obtained robust DNA synthesis with leading strand products of >20,000 nucleotides and lagging strand fragments from 600 to 9,000 nucleotides as seen by alkaline gel electrophoresis. ICP8 was crucial for the synthesis on both strands. Visualization of the deproteinized products using electron microscopy revealed long, linear dsDNAs, and in 87%, one end, presumably the end with the 70-base circle, was single-stranded. The remaining 13% had multiple single-stranded segments separated by dsDNA segments 500 to 1,000 nucleotides in length located at one end. These features are diagnostic of the trombone mechanism of replication. Indeed, when the products were examined with the replication proteins bound, a dsDNA loop was frequently associated with the replication complex located at one end of the replicated DNA. Furthermore, the frequency of loops correlated with the fraction of DNA undergoing Okazaki fragment synthesis.     

Nanostructured dna-protein aggregates consisting of covalent oligonucleotide-streptavidin conjugates

Covalent conjugates consisting of streptavidin and a 24-mer single-stranded DNA oligonucleotide have been oligomerized by cross-linking with a 5',5'-bis-biotinylated 169-base-pair double-stranded DNA (dsDNA) fragment. The oligomeric conjugates formed have been analyzed by nondenaturing gel electrophoresis and scanning-force microscopy (SFM). The comparison of analogous oligomers, prepared from native STV and the bis-biotinylated dsDNA fragment, revealed that the covalent STV-oligonucleotide hybrid conjugates self-assemble to generate oligomeric aggregates of significant smaller size, containing on average only about 2.5 times less dsDNA fragments per aggregate. Likely, this is a consequence of electrostatic or steric repulsion between the dsDNA and the single-stranded oligomer covalently attached to the hybrid, as indicated from control experiments. Nevertheless, the single-stranded oligonucleotide moiety within the oligomeric conjugates can be used as a selective molecular handle for further functionalization and manipulation. For instance, it was used for specific DNA-directed immobilization at a surface, previously functionalized with complementary capture oligonucleotides. Moreover, we demonstrate that macromolecules, such as STV and antibody molecules, which are tagged with the complementary oligonucleotide, specifically bind to the supramolecular DNA-STV oligomeric conjugates. This leads to a novel class of functional DNA-protein conjugates, suitable, for instance, as reagents in immuno-PCR or as building blocks in molecular nanotechnology.