Theoretical study of sequence-dependent nanopore unzipping of DNA

We theoretically investigate the unzipping of DNA electrically driven through a nanometer-size pore. Taking the DNA base sequence explicitly into account, the unpairing and translocation process is described by a biased random walk in a one-dimensional energy landscape determined by the sequential basepair opening. Distributions of translocation times are numerically calculated as a function of applied voltage and temperature. We show that varying these two parameters changes the dynamics from a predominantly diffusive behavior to a dynamics governed by jumps over local energy barriers. The work suggests experimentally studying sequence effects, by comparing the average value and standard deviation of the statistical distribution of translocation times.     

Free energy landscape and transition pathways from Watson–Crick to Hoogsteen base pairing in free duplex DNA

Houghton (HG) base pairing plays a central role in the DNA binding of proteins and small ligands. Probing detailed transition mechanism from Watson–Crick (WC) to HG base pair (bp) formation in duplex DNAs is of fundamental importance in terms of revealing intrinsic functions of double helical DNAs beyond their sequence determined functions. We investigated a free energy landscape of a free B-DNA with an adenosine–thymine (A–T) rich sequence to probe its conformational transition pathways from WC to HG base pairing. The free energy landscape was computed with a state-of-art two-dimensional umbrella molecular dynamics simulation at the all-atom level. The present simulation showed that in an isolated duplex DNA, the spontaneous transition from WC to HG bp takes place via multiple pathways. Notably, base flipping into the major and minor grooves was found to play an important role in forming these multiple transition pathways. This finding suggests that naked B-DNA under normal conditions has an inherent ability to form HG bps via spontaneous base opening events.     

Nanopore unzipping of individual DNA hairpin molecules

We have used the nanometer scale alpha-Hemolysin pore to study the unzipping kinetics of individual DNA hairpins under constant force or constant loading rate. Using a dynamic voltage control method, the entry rate of polynucleotides into the pore and the voltage pattern applied to induce hairpin unzipping are independently set. Thus, hundreds of unzipping events can be tested in a short period of time (few minutes), independently of the unzipping voltage amplitude. Because our method does not entail the physical coupling of the molecules under test to a force transducer, very high throughput can be achieved. We used our method to study DNA unzipping kinetics at small forces, which have not been accessed before. We find that in this regime the static unzipping times decrease exponentially with voltage with a characteristic slope that is independent of the duplex region sequence, and that the intercept depends strongly on the duplex region energy. We also present the first nanopore dynamic force measurements (time varying force). Our results are in agreement with the approximately logV dependence at high V (where V is the loading rate) observed by other methods. The extension of these measurements to lower loading rates reveals a much weaker dependence on V.     

Reconstruction and Identification of DNA Sequence Landscapes from Unzipping Experiments at Equilibrium

Two methods for reconstructing the free-energy landscape of a DNA molecule from the knowledge of the equilibrium unzipping force versus extension signal are introduced: a simple and fast procedure, based on a parametric representation of the experimental force signal, and a maximum-likelihood inference of coarse-grained free-energy parameters. In addition, we propose a force alignment procedure to correct for the drift in the experimental measure of the opening position, a major source of error. For unzipping data obtained by Huguet et al., the reconstructed basepair (bp) free energies agree with the running average of the true free energies on a 20–50 bp scale, depending on the region in the sequence. Features of the landscape at a smaller scale (5–10 bp) could be recovered in favorable regions at the beginning of the molecule. Based on the analysis of synthetic data corresponding to the 16S rDNA gene of bacteria, we show that our approach could be used to identify specific DNA sequences among thousands of homologous sequences in a database.     

Temperature and Electrolyte Optimization of the α-Hemolysin Latch Sensing Zone for Detection of Base Modification in Double-Stranded DNA

The latch region of the wild-type protein pore α-hemolysin (α-HL) constitutes a sensing zone for individual abasic sites (and furan analogs) in double-stranded DNA (dsDNA). The presence of an abasic site or furan within a DNA duplex, electrophoretically captured in the α-HL vestibule and positioned at the latch region, can be detected based on the current blockage prior to duplex unzipping. We investigated variations in blockage current as a function of temperature (12–35°C) and KCl concentration (0.15–1.0 M) to understand the origin of the current signature and to optimize conditions for identifying the base modification. In 1 M KCl solution, substitution of a furan for a cytosine base in the latch region results in an ∼8 kJ mol⁻¹ decrease in the activation energy for ion transport through the protein pore. This corresponds to a readily measured ∼2 pA increase in current at room temperature. Optimal resolution for detecting the presence of a furan in the latch region is achieved at lower KCl concentrations, where the noise in the measured blockage current is significantly lower. The noise associated with the blockage current also depends on the stability of the duplex (as measured from the melting temperature), where a greater noise in the measured blockage current is observed for less stable duplexes.     

Unzipping DNA from the condensed globule state-effects of unraveling

We have studied theoretically the unzipping of a double-stranded DNA from a condensed globule state by an external force. At constant force, we found that the double-stranded DNA unzips an at critical force Fc and the number of unzipped monomers M goes as M approximately (Fc - F)-3, for both the homogeneous and heterogeneous double-stranded DNA sequence. This is different from the case of unzipping from an extended coil state in which the number of unzipped monomers M goes as M approximately (Fc - F)-chi, where the exponent chi is either 1 or 2 depending on whether the double-stranded DNA sequence is homogeneous or heterogeneous, respectively. In the case of unzipping at constant extension, we found that for a double-stranded DNA with a very large number N of base pairs, the force remains almost constant as a function of the extension, before the unraveling transition, at which the force drops abruptly to zero. Right at the unraveling transition, the number of base pairs remaining in the condensed globule state is still very large and goes as N(3/4), in agreement with theoretical predictions of the unraveling transition of polymers stretched by an external force.     

Rfold: an exact algorithm for computing local base pairing probabilities

MOTIVATION: Base pairing probability matrices have been frequently used for the analyses of structural RNA sequences. Recently, there has been a growing need for computing these probabilities for long DNA sequences by constraining the maximal span of base pairs to a limited value. However, none of the existing programs can exactly compute the base pairing probabilities associated with the energy model of secondary structures under such a constraint. RESULTS: We present an algorithm that exactly computes the base pairing probabilities associated with the energy model under the constraint on the maximal span W of base pairs. The complexity of our algorithm is given by O(NW2) in time and O(N+W2) in memory, where N is the sequence length. We show that our algorithm has a higher sensitivity to the true base pairs as compared to that of RNAplfold. We also present an algorithm that predicts a mutually consistent set of local secondary structures by maximizing the expected accuracy function. The comparison of the local secondary structure predictions with those of RNALfold indicates that our algorithm is more accurate. Our algorithms are implemented in the software named 'Rfold.' AVAILABILITY: The C++ source code of the Rfold software and the test dataset used in this study are available at http://www.ncrna.org/software/Rfold/.     

Structural consequences of a 3′ → 3′ DNA interstrand cross-link by a trinuclear platinum complex: unique formation of two such cross-links in a 10-mer duplex

Combined multidimensional nuclear magnetic resonance spectroscopy and electrospray mass spectrometry was used to analyze the platinated DNA adduct of the phase II anticancer drug [{trans-PtCl(NH₃)₂}₂-μ-{trans-Pt(NH₃)₂(NH₂(CH₂)₆NH₂)₂}](NO₃)₄ (BBR3464) with [5′-d(ACG*TATACG*T)-3′]₂. Two 1,2-interstrand cross-links were formed by concomitant binding of two trinuclear moieties to the oligonucleotide. The four DNA-bound platinum atoms coordinated in the major groove at N7 positions of guanines in the 3′ → 3′ direction and the central platinum unit is expected to lie in the DNA minor groove. This is the first report of such a DNA lesion. The melting temperature of the adduct is 76 °C and is 42 °C higher than that of the unplatinated DNA. The sugar residues of the platinated bases are in the N-type conformation and the G9 nucleoside is in the syn orientation, while the G3 nucleoside appears to retain the anti configuration. The secondary structure of DNA was significantly changed upon cross-linking of the two BBR3464 molecules. Base destacking occurs between A1/C2 and C2/G3 and weakened stacking is seen for the C8/G9 and G9/T10 bases. The lack of Watson–Crick base pairing is also seen for A1–T10 and C2–G9 base pairs, whereas Watson–Crick base pairs in the central sequence of the DNA (T4 → A7) are well maintained. While DNA repair proteins may “see” different platinated adducts as bulky “lesions”, the subtle differences involved in base pairing and stacking, as summarized here, may extend to their role as a substrate for repair enzymes. Thus, differences in protein recognition and repair efficiency among the various interstrand cross-links are likely and a subject worthy of detailed exploration. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Dynamics of the DNA duplex formation studied by single molecule force measurements

DNA is partly denatured in vitro by applying a force that mechanically separates the two strands of the double helix. Sudden reduction of the imposed displacement triggers spontaneous reannealing of the molecule. The corresponding force signals are measured by optical trapping interferometry for backward steps of various amplitudes and base sequence intervals. The measured signals frequently show plateaus of varying duration at discrete values that are dependent on the base sequence. Additional measurements are performed with proteins bound to the double helix. When the opening fork encounters such a protein during mechanical unzipping, force increases until the protein is ejected. This ejection induces fast release of tension and fast unzipping. Comparing our different measurements, we find that both DNA unzipping and the relaxation of tension in DNA are faster than the formation of the double helix.     

Single-molecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase

Helicases are molecular motors that separate DNA strands for efficient replication of genomes. We probed the kinetics of individual ring-shaped T7 helicase molecules as they unwound double-stranded DNA (dsDNA) or translocated on single-stranded DNA (ssDNA). A distinctive DNA sequence dependence was observed in the unwinding rate that correlated with the local DNA unzipping energy landscape. The unwinding rate increased approximately 10-fold (approaching the ssDNA translocation rate) when a destabilizing force on the DNA fork junction was increased from 5 to 11 pN. These observations reveal a fundamental difference between the mechanisms of ring-shaped and nonring-shaped helicases. The observed force-velocity and sequence dependence are not consistent with a simple passive unwinding model. However, an active unwinding model fully supports the data even though the helicase on its own does not unwind at its optimal rate. This work offers insights into possible ways helicase activity is enhanced by associated proteins.     

Hierarchical Cascades of Instability Govern the Mechanics of Coiled Coils: Helix Unfolding Precedes Coil Unzipping

Coiled coils are a fundamental emergent motif in proteins found in structural biomaterials, consisting of α-helical secondary structures wrapped in a supercoil. A fundamental question regarding the thermal and mechanical stability of coiled coils in extreme environments is the sequence of events leading to the disassembly of individual oligomers from the universal coiled-coil motifs. To shed light on this phenomenon, here we report atomistic simulations of a trimeric coiled coil in an explicit water solvent and investigate the mechanisms underlying helix unfolding and coil unzipping in the assembly. We employ advanced sampling techniques involving steered molecular dynamics and metadynamics simulations to obtain the free-energy landscapes of single-strand unfolding and unzipping in a three-stranded assembly. Our comparative analysis of the free-energy landscapes of instability pathways shows that coil unzipping is a sequential process involving multiple intermediates. At each intermediate state, one heptad repeat of the coiled coil first unfolds and then unzips due to the loss of contacts with the hydrophobic core. This observation suggests that helix unfolding facilitates the initiation of coiled-coil disassembly, which is confirmed by our 2D metadynamics simulations showing that unzipping of one strand requires less energy in the unfolded state compared with the folded state. Our results explain recent experimental findings and lay the groundwork for studying the hierarchical molecular mechanisms that underpin the thermomechanical stability/instability of coiled coils and similar protein assemblies.     

The post-SCF quantum chemistry characteristics of inter- and intra-strand stacking interactions in d(CpG) and d(GpC) steps found in B-DNA, A-DNA and Z-DNA crystals

The energies of intra- and inter-strand stacking interactions in model d(GpC) and d(CpG) two-base-pair steps were estimated by MP2/aug-cc-pVDZ single point calculations corrected for basis superposition errors. The stacked two-nucleobase pairs were constructed using experimental values of base pair and base step parameters taken from Nucleic Acid Database (). Three distinct polymorphic forms were analysed, namely A-, B- and Z-DNA. The applied methodology enables statistical analysis of structural and energetic diversities. The structural relationships between polymorphic forms are quite complex and depend on the sequence of pairs. The variability of parameters such as shift and tilt is almost the same irrespective of the polymorphic form and sequence of steps analysed. In contrast, shift and twist distributions easily discriminate all three polymorphic forms of DNA. Interestingly, despite significant structural diversities, the energies of the most frequent energy ranges are comparable irrespective of the polymorphic form and base sequence. There was observed compensation of inter- and intra-strand interactions, especially for d(GpC) and d(CpG) steps found in A- and B-DNA. Thus, among many other roles, these pairs act as a kind of energetic buffer, balancing the double helix. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Reduction of gene repair by selenomethionine with the use of single-stranded oligonucleotides

Background  

The repair of single base mutations in mammalian genes can be directed by single-stranded oligonucleotides in a process known as targeted gene repair. The mechanism of this reaction is currently being elucidated but likely involves a pairing step in which the oligonucleotide align in homologous register with its target sequence and a correction step in which the mutant base is replaced by endogenous repair pathways. This process is regulated by the activity of various factors and proteins that either elevate or depress the frequency at which gene repair takes place.     

Empirical comparison of cross-platform normalization methods for gene expression data

Background

The prediction of secondary structure, i.e. the set of canonical base pairs between nucleotides, is a first step in developing an understanding of the function of an RNA sequence. The most accurate computational methods predict conserved structures for a set of homologous RNA sequences. These methods usually suffer from high computational complexity. In this paper, TurboFold, a novel and efficient method for secondary structure prediction for multiple RNA sequences, is presented.

Results

TurboFold takes, as input, a set of homologous RNA sequences and outputs estimates of the base pairing probabilities for each sequence. The base pairing probabilities for a sequence are estimated by combining intrinsic information, derived from the sequence itself via the nearest neighbor thermodynamic model, with extrinsic information, derived from the other sequences in the input set. For a given sequence, the extrinsic information is computed by using pairwise-sequence-alignment-based probabilities for co-incidence with each of the other sequences, along with estimated base pairing probabilities, from the previous iteration, for the other sequences. The extrinsic information is introduced as free energy modifications for base pairing in a partition function computation based on the nearest neighbor thermodynamic model. This process yields updated estimates of base pairing probability. The updated base pairing probabilities in turn are used to recompute extrinsic information, resulting in the overall iterative estimation procedure that defines TurboFold. TurboFold is benchmarked on a number of ncRNA datasets and compared against alternative secondary structure prediction methods. The iterative procedure in TurboFold is shown to improve estimates of base pairing probability with each iteration, though only small gains are obtained beyond three iterations. Secondary structures composed of base pairs with estimated probabilities higher than a significance threshold are shown to be more accurate for TurboFold than for alternative methods that estimate base pairing probabilities. TurboFold-MEA, which uses base pairing probabilities from TurboFold in a maximum expected accuracy algorithm for secondary structure prediction, has accuracy comparable to the best performing secondary structure prediction methods. The computational and memory requirements for TurboFold are modest and, in terms of sequence length and number of sequences, scale much more favorably than joint alignment and folding algorithms.

Conclusions

TurboFold is an iterative probabilistic method for predicting secondary structures for multiple RNA sequences that efficiently and accurately combines the information from the comparative analysis between sequences with the thermodynamic folding model. Unlike most other multi-sequence structure prediction methods, TurboFold does not enforce strict commonality of structures and is therefore useful for predicting structures for homologous sequences that have diverged significantly. TurboFold can be downloaded as part of the RNAstructure package at http://rna.urmc.rochester.edu.     

Unzipping DNA with optical tweezers: high sequence sensitivity and force flips

Force measurements are performed on single DNA molecules with an optical trapping interferometer that combines subpiconewton force resolution and millisecond time resolution. A molecular construction is prepared for mechanically unzipping several thousand-basepair DNA sequences in an in vitro configuration. The force signals corresponding to opening and closing the double helix at low velocity are studied experimentally and are compared to calculations assuming thermal equilibrium. We address the effect of the stiffness on the basepair sensitivity and consider fluctuations in the force signal. With respect to earlier work performed with soft microneedles, we obtain a very significant increase in basepair sensitivity: presently, sequence features appearing at a scale of 10 basepairs are observed. When measured with the optical trap the unzipping force exhibits characteristic flips between different values at specific positions that are determined by the base sequence. This behavior is attributed to bistabilities in the position of the opening fork; the force flips directly reflect transitions between different states involved in the time-averaging of the molecular system.     

Coupling translocation with nucleic acid unwinding by NS3 helicase

We present a semiquantitative model for translocation and unwinding activities of monomeric nonstructural protein 3 (NS3) helicase. The model is based on structural, biochemical, and single-molecule measurements. The model predicts that the NS3 helicase actively unwinds duplex by reducing more than 50% the free energy that stabilizes base pairing/stacking. The unwinding activity slows the movement of the helicase in a sequence-dependent manner, lowering the average unwinding efficiency to less than 1 bp per ATP cycle. When bound with ATP, the NS3 helicase can display significant translocational diffusion. This increases displacement fluctuations of the helicase, decreases the average unwinding efficiency, and enhances the sequence dependence. Also, interactions between the helicase and the duplex stabilize the helicase at the junction, facilitating the helicase's unwinding activity while preventing it from dissociating. In the presence of translocational diffusion during active unwinding, the dissociation rate of the helicase also exhibits sequence dependence. Based on unwinding velocity fluctuations measured from single-molecule experiments, we estimate the diffusion rate to be on the order of 10 s^− 1 . The generic features of coupling single-stranded nucleic acid translocation with duplex unwinding presented in this work may apply generally to a class of helicases.     

In Vivo Selection of Rous Sarcoma Virus Mutants with Randomized Sequences in the Packaging Signal

Retrovirus genomes contain a sequence at the 5′ end which directs their packaging into virions. In Rous sarcoma virus, previous studies have identified important segments of the packaging signal, Ψ, and support elements of a secondary-structure prediction. To further characterize this sequence, we used an in vivo selection strategy to test large collections of mutants. We generated pools of full-length viral DNA molecules with short stretches of random sequence in Ψ and transfected each pool into avian cells. Resulting infectious virus was allowed to spread by multiple passages, so that sequences could compete and the best could be selected. This method provides information on the kinds of sequences allowed, as well as those that are most fit. Several predicted stem-loop structures in Ψ were tested. A stem at the base of element O3 was highly favored; only sequences which maintained base pairing were selected. Two other stems, at the base and in the middle of element L3, were not conserved: neither base pairing nor sequence was maintained. A single mutation, G213U, was seen upstream of the randomized region in all selected L3 stem mutants; we interpret this to mean that it compensates for the defects in L3. Randomized mutations adjacent to G213 maintained the wild-type base composition but not its sequence. The kissing-loop sequence at end of L3, postulated to function in genome dimerization, was not required for infectivity but was selected for over time. Finally, a deletion of L3 was constructed and found to be poorly infectious.     

Two-phase partitioning bioreactors in environmental biotechnology

Operation of microbial electrolysis cells (MECs) without an ion exchange membrane could help to lower the construction costs while lowering the ohmic cell resistance and improving MEC conversion rates by minimizing the pH gradient between anode and cathode. In this research, we demonstrate that membraneless MECs with plain graphite can be operated for methane production without pH adjustment and that the ohmic cell resistance could be lowered with approximately 50% by removing the cation exchange membrane. As a result, the current production increased from 66 ± 2 to 156 ± 1 A m⁻³ MEC by removing the membrane with an applied voltage of −0.8 V. Methane was the main energetic product despite continuous operation under carbonate-limited and slightly acidified conditions (pH 6.1–6.2). Our results suggest that continuous production of hydrogen in membraneless MECs will be challenging since methane production might not be avoided easily. The electrical energy invested was not always completely recovered under the form of an energy-rich biogas; however, our results indicate that membraneless MECs might be a viable polishing step for the treatment of the effluent of anaerobic digesters as methane was produced under low organic loading conditions and at room temperature. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Base pairing at the 5' splice site with U1 small nuclear RNA promotes splicing of the upstream intron but may be dispensable for slicing of the downstream intron.

We previously reported that exon skipping in vivo due to point mutations in the 5' splice site (5'ss) signal of an internal mammalian exon can be prevented by coexpression of U1 small nuclear RNAs, termed shift-U1s, with complementarity to sequence upstream or downstream of the mutated site. We now show by S1 nuclease protection experiments that a typical shift-U1 restores splicing of the upstream intron, but not necessarily of the down stream intron. This indicates that the normal 5'ss sequence acts as an enhancer for splicing of the upstream intron, that it owes this activity to base pairing with U1, and that the enhancer activity is reproduced by base pairing of U1 with other sequences in the area. Shift-U1s are dispensable when the 3'ss sequence of the upstream intron is improved, which suggests that base pairing of U1 with sequences at or near the downstream end of the exon normally functions by compensating for a weakness in the upstream 3'ss. Accordingly, U1 appears to be involved in communication across the exon, but our data indicate at the same time that extensive base pairing between U1 and the 5'ss sequence is not necessary for accurate splicing of the downstream intron. These findings are discussed in relation to the coordinate selection exon termini proposed by the exon definition model.