Double-stranded Form Research Articles

Deconstructing Structural Transitions of Biomolecules

Structural transitions appear everywhere: proteins fold, nanotubes collapse, DNA denatures, ice melts, and so on. In biology, these transitions play a role in processes such as transcription and also determine protein function. Yet, at the same time, they give examples of highly nonlinear processes that are challenging to model and understand. I will discuss one such transition - the denaturation of DNA, where its double stranded form unravels into two single strands. There are many models that can describe certain aspects of this transition equally well, such as the fraction of bound base pairs versus temperature. I will show, however, that two well-known models yield drastically different predictions for thermal transport. The latter can then be used to “peek inside” DNA and understand what is happening during the denaturation transition. Thus, on the one hand, thermal transport gives a method to probe structural transitions in biological molecules and other materials. On the other hand, molecular systems and materials with nonlinear structural transitions also give opportunities for developing novel thermal devices.

Extraordinary transgressive phenotypes of hybrid tomato are influenced by epigenetics and small silencing RNAs

Hybrid organisms may fail to develop, be sterile or they may be more vigorous than either of the parents. Examples of hybrid vigour or hybrid necrosis in the F1 are often not inherited stably in subsequent generations if they are associated with overdominance. There can also be transgressive phenotypes that are inherited stably in these later generations, but the underlying mechanisms are not well understood. Here we have investigated the possibility that stable transgressive phenotypes in the progeny of crosses between cultivated tomato (Solanum lycopersicum cv. M82) and a wild relative (Solanum pennellii, accession LA716) are associated with micro or small interfering(si) RNAs. We identified loci from which these small(s)RNAs were more abundant in hybrids than in either parent and we show that accumulation of such transgressive sRNAs correlated with suppression of the corresponding target genes. In one instance this effect was associated with hypermethylation of the corresponding genomic DNA. Our results illustrate a potential role of transgressive sRNAs in plant breeding and in natural evolution with wild plants.

Rare target enrichment for ultrasensitive PCR detection using cot–rehybridization and duplex-specific nuclease

Nucleic acid detection by polymerase chain reaction (PCR) is invaluable for the detection of dilute and rare sequences, including pathogens and infrequent species in complex clinical and environmental backgrounds. The presence of excess complex background nucleic acid can reduce sensitivity and specificity. This is because mispriming can cause failure of the amplification reaction. Here we describe a new approach to ultrasensitive PCR detection, using enrichment of rare target nucleic acid from abundant background by combining the classic technique of cot–rehybridization to convert the abundant background to double-stranded form, with the use of a newly described, highly processive duplex-specific crab nuclease. We show that trace sequences in a vast excess of background DNA can be undetectable by PCR, independent of the amount of the mixture added to the PCR, and that these sequences can be made detectable by background suppression using this method.

Merits of the double-stranded form of the actin filament revealed by structures of the filament ends

Actin forms a double-stranded filament, and the majority of actin filaments in the cell undergo the dynamic process of polymerization and depolymerization at both ends. Actin dynamics plays numerous important roles in eukaryotic cells. In order to understand actin dynamics, structural elucidation of the actin filament ends is particularly important because polymerization and depolymerization occurs only at the ends. We have developed original image analysis procedures to determine the structures of the actin filament ends from cryo-electron micrographs, and two structures have been determined. The structures revealed that the actin filament takes advantage of its double-stranded form to regulate its dynamics at both ends by a surprisingly simple mechanism.

Bioactive papaverine derivatives bind G-quadruplexes selectively

AbstractG-quadruplexes are a family of DNA secondary structures resulting from the folding of a guanine-rich sequence. Targeting quadruplexes by small molecules is an approach that is currently being studied with the aim of exploring their biological roles and developing new anti-cancer agents. There is evidence that the formation of G4 structures by telomeric DNA can be used to inhibit the enzyme activity of telomerase, and thereby to activate the pathway to senescence in tumour cells. It was previously shown that the papaverine oxidation products 6a,12a-diazadibenzo-[a,g]fluorenylium derivative (ligand I) and 2,3,9,10-tetramethoxy-12-oxo-12H-indolo[2,1-a]isoquinolinium chloride (ligand II) bind to G-quadruplex representing the human telomeric sequence. These ligands possess the ability to inhibit telomerase and polymerase action at the micromolar level. Here we report a DNA binding study on these two ligands and a new derivative 2-(2-carboxy-4,5-dimethoxyphenyl0-6,7-dimethoxyisoquiloliniuminner salt (ligand III) in order to evaluate their binding selectivity to samples of nucleic acids (ssDNA, dsDNA, triplexes, and quadruplexes). Simultaneous investigations on several DNA-ligand complexes carried out using an equilibrium dialysis approach revealed pronounced binding selectivity of ligand I and ligand II to tetraplex DNA structures over the doublestranded DNA forms.

8‐Vinyl‐2′‐deoxyguanosine as a Fluorescent 2′‐Deoxyguanosine Mimic for Investigating DNA Hybridization and Topology

A one light strand: 2′-Deoxyguanosine was made fluorescent by attaching a vinyl group at the C8 position (see structure). The resulting fluorophore is highly sensitive towards DNA double-strand formation and alterations in the secondary structure of its parent oligonucleotide, such as formation of different quadruplex structures. These differences can be readily detected in the emission spectrum (see picture).

Species-specific Typing of DNA Based on Palindrome Frequency Patterns

DNA in its natural, double-stranded form may contain palindromes, sequences which read the same from either side because they are identical to their reverse complement on the sister strand. Short palindromes are underrepresented in all kinds of genomes. The frequency distribution of short palindromes exhibits more than twice the inter-species variance of non-palindromic sequences, which renders palindromes optimally suited for the typing of DNA. Here, we show that based on palindrome frequency, DNA sequences can be discriminated to the level of species of origin. By plotting the ratios of actual occurrence to expectancy, we generate palindrome frequency patterns that allow to cluster different sequences of the same genome and to assign plasmids, and in some cases even viruses to their respective host genomes. This finding will be of use in the growing field of metagenomics.

Citrus tristeza virus infection induces the accumulation of viral small RNAs (21–24-nt) mapping preferentially at the 3′-terminal region of the genomic RNA and affects the host small RNA profile

To get an insight into the host RNA silencing defense induced by Citrus tristeza virus (CTV) and into the counter defensive reaction mediated by its three silencing suppressors (p25, p20 and p23), we have examined by deep sequencing (Solexa-Illumina) the small RNAs (sRNAs) in three virus-host combinations. Our data show that CTV sRNAs: (i) represent more than 50% of the total sRNAs in Mexican lime and sweet orange (where CTV reaches relatively high titers), but only 3.5% in sour orange (where the CTV titer is significantly lower), (ii) are predominantly of 21-22-nt, with a biased distribution of their 5' nucleotide and with those of (+) polarity accumulating in a moderate excess, and (iii) derive from essentially all the CTV genome (ca. 20kb), as revealed by its complete reconstruction from viral sRNA contigs, but adopt an asymmetric distribution with a prominent hotspot covering approximately the 3'-terminal 2,500 nt. These results suggest that the citrus homologues of Dicer-like (DCL) 4 and 2 most likely mediate the genesis of the 21 and 22 nt CTV sRNAs, respectively, and show that both ribonucleases act not only on the genomic RNA but also on the 3' co-terminal subgenomic RNAs and, particularly, on their double-stranded forms. The plant sRNA profile, very similar and dominated by the 24-nt sRNAs in the three mock-inoculated controls, was minimally affected by CTV infection in sour orange, but exhibited a significant reduction of the 24-nt sRNAs in Mexican lime and sweet orange. We have also identified novel citrus miRNAs and determined how CTV influences their accumulation.

Two Distinct Overstretched DNA States

Double-stranded DNA can undergo an “overstretching” transition within a narrow force range when the external force reaches around 60 pN. The basic question of the overstretched DNA is whether the strands are separated or not after transition. Despite numbers of studies, this question still remains controversial. In this research, we directly show that DNA overstretching transition actually involves two distinct types of double-helix reorganization: slow hysteretic “unpeeling” of one strand off the other; and a fast, non-hysteretic transition to an elongated double stranded form. The competing between these two overstretched forms is sensitive to factors that affect DNA base pair stability: DNA sequence, salt concentration, and temperature. The balance between the two forms shifts near physiological solution conditions. This result clearly demonstrates that an overstretched double stranded state does exit. It also shows that DNA double helix physical properties, unpeeling or overextension, can be selected via small changes in molecule environment.

Development of an in situ assay for simultaneous detection of the genomic and replicative form of PCV2 using padlock probes and rolling circle amplification

BackgroundIn this study we utilized padlock probes and rolling circle amplification as a mean to detect and study the replication of porcine circovirus type 2 (PCV2) in cultured cells and in infected tissue. Porcine circovirus type 2 is a single-stranded circular DNA virus associated with several severe diseases, porcine circovirus diseases (PCVD) in pigs, such as postweaning multisystemic wasting syndrome. The exact reason and mechanisms behind the trigger of PCV2 replication that is associated with these diseases is not well-known. The virus replicates with rolling circle replication and thus also exists as a double-stranded replicative form.ResultsBy applying padlock probes and rolling circle amplification we could not only visualise the viral genome but also discriminate between the genomic and the replicative strand in situ. The genomic strand existed in higher numbers than the replicative strand. The virus accumulated in certain nuclei but also spread into the cytoplasm of cells in the surrounding tissue. In cultured cells the average number of signals increased with time after infection.ConclusionsWe have developed a method for detection of both strands of PCV2 in situ that can be useful for studies of replication and in situ detection of PCV2 as well as of DNA viruses in general.

Quantitative high-resolution sensing of DNA hybridization using magnetic tweezers with evanescent illumination

We applied the combined approach of evanescent nanometry and force spectroscopy using magnetic tweezers to quantify the degree of hybridization of a single synthetic single-stranded DNA oligomer to a resolution approaching a single-base. In this setup, the 200 nucleotide long DNA was covalently attached to the surface of an optically transparent solid support at one end and to the surface of a superparamagnetic fluorescent microsphere (force probe) at the other end. The force was applied to the probes using an electromagnet. The end-to-end molecular distance (i.e. out-of-image-plane position of the force probe) was determined from the intensity of the probe fluorescence image observed with total-internal reflectance microscopy. An equation of state for single stranded DNA molecules under tension (extensible freely jointed chain) was used to derive the penetration depth of the evanescent field and to calibrate the magnetic properties of the force probes. The parameters of the magnetic response of the force probes obtained from the equation of state remained constant when changing the penetration depth, indicating a robust calibration procedure. The results of such a calibration were also confirmed using independently measured probe-surface distances for probes mounted onto cantilevers of an atomic force microscope. Upon hybridization of the complementary 50 nucleotide-long oligomer to the surface-bound 200-mer, the changes in the force-distance curves were consistent with the quantitative conversion of 25% of the original single-stranded DNA to its double-stranded form, which was modeled as an elastic rod. The method presented here for quantifying the hybridization state of the single DNA molecules has potential for determining the degree of hybridization of individual molecules in a single molecule array with high accuracy.

Interaction of Escherichia coli RNA Polymerase σ70 Subunit with Promoter Elements in the Context of Free σ70, RNA Polymerase Holoenzyme, and the β′-σ70 Complex

Promoter recognition by RNA polymerase is a key point in gene expression and a target of regulation. Bacterial RNA polymerase binds promoters in the form of the holoenzyme, with the σ specificity subunit being primarily responsible for promoter recognition. Free σ, however, does not recognize promoter DNA, and it has been proposed that the intrinsic DNA binding ability is masked in free σ but becomes unmasked in the holoenzyme. Here, we use a newly developed fluorescent assay to quantitatively study the interactions of free σ(70) from Escherichia coli, the β'-σ complex, and the σ(70) RNA polymerase (RNAP) holoenzyme with non-template strand of the open promoter complex transcription bubble in the context of model non-template oligonucleotides and fork junction templates. We show that σ(70), free or in the context of the holoenzyme, recognizes the -10 promoter element with the same efficiency and specificity. The result implies that there is no need to invoke a conformational change in σ for recognition of the -10 element in the single-stranded form. In the holoenzyme, weak but specific interactions of σ are increased by contacts with DNA downstream of the -10 element. We further show that region 1 of σ(70) is required for stronger interaction with non-template oligonucleotides in the holoenzyme but not in free σ. Finally, we show that binding of the β' RNAP subunit is sufficient to allow specific recognition of the TG motif of the extended -10 promoter element by σ(70). The new fluorescent assay, which we call a protein beacon assay, will be instrumental in quantitative dissection of fine details of RNAP interactions with promoters.

Comparison of Methods for Accurate Quantification of DNA Mass Concentration with Traceability to the International System of Units

Accurate estimation of total DNA concentration (mass concentration, e.g., ng/muL) that is traceable to the International System of Units (SI) is a crucial starting point for improving reproducible measurements in many applications involving nucleic acid testing and requires a DNA reference material which has been certified for its total DNA concentration. In this study, the concentrations of six different lambda DNA preparations were determined using different measurement platforms: UV Absorbance at 260 nm (A(260)) with and without prior sodium hydroxide (NaOH) treatment of the DNA, PicoGreen assay, and digital polymerase chain reaction (dPCR). DNA concentration estimates by A(260) with and without prior NaOH treatment were significantly different for five of the six samples tested. There were no significant differences in concentration estimates based on A(260) with prior NaOH treatment, PicoGreen analysis, and dPCR for two of the three samples tested using dPCR. Since the measurand in dPCR is amount (copy number) concentration (copies/muL), the results suggest that accurate estimation of DNA mass concentration based on copy number concentration is achievable provided the DNA is fully characterized and in the double-stranded form or amplification is designed to be initiated from only one of the two complementary strands.

Effect of Molecular Crowding and Ionic Strength on the Isothermal Hybridization of Oligonucleotides

The isothermal hybridization of complementary oligonucleotides, 15-mer, 25-mer, 35-mer, and a molecular beacon, was investigated under varying conditions of molecular crowding and ionic strength, using hypochromicity to follow strand pairing and polyethylene glycol as a crowding agent. Thermodynamic analysis of the results revealed the addition of counterions to the oligonucleotide backbones, DeltaPsi, to be dependent on the strand GC content and the molecular crowding. A decrease in DeltaPsi was observed, with both increasing GC% and solution PEG content. In contrast, the number of bound water molecules depended on the activity of Na(+), where two regimes were observed. At a(Na(+)) < 0.05 and increasing molecular crowding, water molecules were released into the DNA solutions, and oligonucleotide pairing was favored with both increasing hydrophobic forces, whereas at a(Na(+)) >or= 0.05, water molecules were bound to the strands, and the extent of double strand formation decreased with increasing PEG wt %.

The first crystal structure of a gramicidin complex with sodium: high-resolution study of a nonstoichiometric gramicidin D–NaI complex

The crystal structure of the nonstoichiometric complex of gramicidin D with NaI has been studied using synchrotron radiation at 100 K. The limiting resolution was 1.25 A and the R factor was 16% for 19 883 observed reflections. The general architecture of the antiparallel two-stranded gramicidin dimers in the studied crystal was a right-handed antiparallel double-stranded form that closely resembles the structures of other right-handed species published to date. However, there were several surprising observations. In addition to the significantly different composition of linear gramicidins identified in the crystal structure, including the absence of the gramicidin C form, only two cationic sites were found in each of the two independent dimers (channels), which were partially occupied by sodium, compared with the seven sites found in the RbCl complex of gramicidin. The sum of the partial occupancies of Na(+) was only 1.26 per two dimers and was confirmed by the similar content of iodine ions (1.21 ions distributed over seven sites), which was easily visible from their anomalous signal. Another surprising observation was the significant asymmetry of the distributions and occupancies of cations in the gramicidin dimers, which was in contrast to those observed in the high-resolution structures of the complexes of heavier alkali metals with gramicidin D, especially that of rubidium.

Two distinct overstretched DNA states

The DNA double helix undergoes an ‘overstretching’ transition in a narrow force range near 65 pN. Despite numerous studies the basic question of whether the strands are separated or not remains controversial. Here we show that overstretching in fact involves two distinct types of double-helix reorganization: slow hysteretic ‘unpeeling’ of one strand off the other; and a fast, non-hysteretic transition to an elongated double-stranded form. We demonstrate that the relative fraction of these two overstretched forms is sensitive to factors that affect DNA base pair stability, including DNA sequence, salt concentration and temperature. The balance between the two forms shifts near physiological solution conditions. This result, in addition to establishing the existence of an overstretched double-stranded state, also shows that double helix physical properties are tuned so that either unpeeling or overextension can be selected via small changes in molecule environment.

Fluorescence Aptameric Sensor for Strand Displacement Amplification Detection of Cocaine

A new fluorescence method based on aptamer-target interactions has been developed for cocaine detection with target-induced strand displacement. Here we describe new probes, the hairpin-probe and the single strand-probe (ss-probe), that possess two recognition sequences of cocaine aptamer. In the presence of cocaine, both probes would associate with the target to form a tripartite complex. The conformational change in the hairpin-probe causes the opening of a hairpin structure and the hybridization to primer. With polymerase and the dNTPs, the replication of the single-stranded domain of hairpin-probe triggers the process of primer extension. When the hairpin-probe is converted into a fully double-stranded form, the ss-probe and cocaine are displaced to bind another hairpin-probe and initiate new amplification cycles. Fluorescence signal generation would be observed upon SYBR Green I intercalating into the new DNA double helix. The new protocol design permits detection of as low as 2 nM cocaine in a closed tube, offering a convenient approach for a homogeneous assay. Compared with previously reported cocaine aptameric sensors, our new method is highly sensitive, selective, and economical.

Evidence for the Failure of Adeno-associated Virus Serotype 5 to Package a Viral Genome ≥8.2 kb

Limited packaging capacity hinders adeno-associated virus (AAV) gene therapy. A recent study seems to have provided a solution to this problem. Allocca et al. reported that AAV-5 could package an 8.9 kb vector genome. Here we tested whether this approach can be used to deliver a large genome for Duchenne muscular dystrophy (DMD) gene therapy. We first evaluated AAV-5 packaging of an 8.2 kb genome. This vector carries two independent reporter gene cassettes, one for alkaline phosphatase (AP) and another for LacZ. Viral yield was log-fold lower than that of a regular AAV-5. Nevertheless, both AP and LacZ genes were detected in purified virus. Injection to dystrophic muscle resulted in both AP and LacZ expression. On electron microscopy, virion structure appeared normal. Surprisingly, we did not find the full-length single-stranded viral genome by alkaline gel electrophoresis. Neither did we see the full-length double-stranded replication forms in adenovirus coinfected cells. We suspect that AP and LacZ expression may have come from partially packaged 5' or 3'-half of the genome. Additional studies revealed failure of AAV-5 to package and express an 8.7 kb minidystrophin gene cassette. In summary, our results do not support the extraordinary packaging capacity of AAV-5.

Inconvenient facts about pathological amyloid fibrils.

Mass per-length measurements of Alzheimer's amyloid fibrils, now reported in a recent issue of PNAS (1), contradict the conviction that such fibrils should be constructed with an integral number of molecules in their ≈4.7-A axial repeat period, which is the signature periodicity of the amyloid cross-β structure. Solid-state NMR spectra of various pathological amyloids show that their cross-β spines are built from layers of parallel in-register β-strands (2), which requires an integral number of identically bonded molecules per layer. Schmidt et al. (1) count close to 2½ Aβ molecules per layer in a single protofilament form of the 42-residue Alzheimer's Aβ peptide and in protofilaments of two distinct double-stranded forms of the more common 40-residue Aβ peptide; their image reconstructions from electron cryomicrographs suggest similar morphology for the different protofilaments. Where is the fractional molecule?

Separate Production of Single-Stranded DNA is not Necessary: Circuit Denaturation of Double-Stranded DNA Followed by Hybridization of Single Strands on Oligonucleotide Microchips

An approach to circuit renaturation-hybridization of dsDNA on oligonucleotide microchips is described. A close circuit cycling device has been developed, and the feasibility of the proposed technique was demonstrated on two platforms. First, a commercial microchip for detection of rifampicin resistance in Mycobacterium tuberculosis was used. Hybridization of a 126 nt long single-stranded DNA (ssDNA) fragment of the rpoB gene according to manufacturer's protocol has been compared to hybridization of the same double-stranded DNA (dsDNA) fragment using the developed approach. Hybridization signals obtained by both methods were comparable in intensity and correlated closely. Second, a 22 nt long hairpin-forming oligonucleotide was designed and hybridized with a custom microchip containing probes complementary to both strands of the oligonucleotide. Conventional hybridization of this oligonucleotide did not yield any significant signals. Cleavage of the hairpin loop resulted in the formation of a 9 bp long intermolecular duplex. Hybridization of the duplex using the suggested technique yielded strong signals. The proposed approach allows analyzing target DNA in double-stranded form bypassing the preparation of single-stranded targets. Moreover, both complementary chains could be analyzed simultaneously, providing a reliable internal control. Being combined with fragmentation this method opens new possibilities in analyzing ssDNA with complex secondary structure.