Quantitative analysis of DNA hybridization in a flowthrough microarray for molecular testing

Quantitative information about the nucleic acids hybridization reaction on microarrays is fundamental to designing optimized assays for molecular diagnostics. This study presents the kinetic, equilibrium, and thermodynamic analyses of DNA hybridization in a microarray system designed for fast molecular testing of pathogenic bacteria. Our microarray setup uses a porous, nylon membrane for probe immobilization and flowthrough incubation. The Langmuir model was used to determine the reaction rate constants of hybridization with antisense targets specific to Staphylococcus epidermidis and Staphylococcus aureus strains. The kinetic analysis revealed a sequence-dependent reaction rate, with association rate constants on the order of 10⁵ M⁻¹ s⁻¹ and dissociation rate constants of 10⁻⁴ s⁻¹. We found that by increasing the probe surface density from 10¹¹ to 10¹² molecules/cm², the hybridization rate and efficiency are suppressed while the melting temperature of the DNA duplex increases. The maximum fraction of hybridized capture probes at equilibrium did not exceed 50% for hybridization with antisense sequences and was below 6% for hybridization with long targets obtained from PCR. The van’t Hoff analysis of the temperature denaturation data showed that the DNA hybridization in our porous, flowthrough microarray is thermodynamically less favorable than the hybridization of the same sequences in solution.     

Eprobe Mediated Real-Time PCR Monitoring and Melting Curve Analysis

Real-time monitoring of PCR is one of the most important methods for DNA and RNA detection widely used in research and medical diagnostics. Here we describe a new approach for combined real-time PCR monitoring and melting curve analysis using a 3′ end-blocked Exciton-Controlled Hybridization-sensitive fluorescent Oligonucleotide (ECHO) called Eprobe. Eprobes contain two dye moieties attached to the same nucleotide and their fluorescent signal is strongly suppressed as single-stranded oligonucleotides by an excitonic interaction between the dyes. Upon hybridization to a complementary DNA strand, the dyes are separated and intercalate into the double-strand leading to strong fluorescence signals. Intercalation of dyes can further stabilize the DNA/DNA hybrid and increase the melting temperature compared to standard DNA oligonucleotides. Eprobes allow for specific real-time monitoring of amplification reactions by hybridizing to the amplicon in a sequence-dependent manner. Similarly, Eprobes allow for analysis of reaction products by melting curve analysis. The function of different Eprobes was studied using the L858R mutation in the human epidermal growth factor receptor (EGFR) gene, and multiplex detection was demonstrated for the human EGFR and KRAS genes using Eprobes with two different dyes. Combining amplification and melting curve analysis in a single-tube reaction provides powerful means for new mutation detection assays. Functioning as “sequence-specific dyes”, Eprobes hold great promises for future applications not only in PCR but also as hybridization probes in other applications.     

E2-mediated Small Ubiquitin-like Modifier (SUMO) Modification of Thymine DNA Glycosylase Is Efficient but Not Selective for the Enzyme-Product Complex

Thymine DNA glycosylase (TDG) initiates the repair of G·T mismatches that arise by deamination of 5-methylcytosine (mC), and it excises 5-formylcytosine and 5-carboxylcytosine, oxidized forms of mC. TDG functions in active DNA demethylation and is essential for embryonic development. TDG forms a tight enzyme-product complex with abasic DNA, which severely impedes enzymatic turnover. Modification of TDG by small ubiquitin-like modifier (SUMO) proteins weakens its binding to abasic DNA. It was proposed that sumoylation of product-bound TDG regulates product release, with SUMO conjugation and deconjugation needed for each catalytic cycle, but this model remains unsubstantiated. We examined the efficiency and specificity of TDG sumoylation using in vitro assays with purified E1 and E2 enzymes, finding that TDG is modified efficiently by SUMO-1 and SUMO-2. Remarkably, we observed similar modification rates for free TDG and TDG bound to abasic or undamaged DNA. To examine the conjugation step directly, we determined modification rates (k_obs) using preformed E2∼SUMO-1 thioester. The hyperbolic dependence of k_obs on TDG concentration gives k_max = 1.6 min⁻¹ and K_1/2 = 0.55 μm, suggesting that E2∼SUMO-1 has higher affinity for TDG than for the SUMO targets RanGAP1 and p53 (peptide). Whereas sumoylation substantially weakens TDG binding to DNA, TDG∼SUMO-1 still binds relatively tightly to AP-DNA (K_d ∼50 nm). Although E2∼SUMO-1 exhibits no specificity for product-bound TDG, the relatively high conjugation efficiency raises the possibility that E2-mediated sumoylation could stimulate product release in vivo. This and other implications for the biological role and mechanism of TDG sumoylation are discussed.     

Enhanced loop DNA folding induced by thymine-CH3 group contact and perpendicular guanine-thymine interaction

A remarkable stabilizing effect induced by T-CH₃ group and perpendicular guanine–thymine interactions in the DNA loop conformation has been demonstrated for the d(TTTG) loop structure using UV melting, high resolution NMR, distance geometry, and molecular dynamics studies. Contrary to the previously published d(TTCG) sequence that exhibits no specific inter-nucleotide interaction, we have found that d(TTTG), which differs only by one nucleotide with the d(TTCG) sequence (C7 T7), forms a rather stable and well-defined loop structure. Two characteristic structural features account for the stabilization of an otherwise flexible loop structure; the second loop T (T6) residue folds into the minor groove and engages in perpendicular interaction with the G8-NH₂, while the third loop T (T7) residue stacks well upon the closing T5G8 wobble base pair and exhibits good contacts with many of the loop T5 and T6 sugar protons, which may form a hydrophobic core in the loop region. The importance of the bulky T7-CH₃ was also proved by the UV melting study; while d(TTCG) hairpin exhibits a lower melting point (74.5°C ) than d(TTTG) hairpin (80.5°C ), d(TT^5–methylCG) hairpin resumes the same higher melting point (80°C ). Similarly, the fact that the melting temperature (74°C ) of d(TTTI) is lower than that of d(TTTG) indicates the critical role played by the G8-NH₂ group. Our structural studies of the d(TTTG) loop indicate that DNA and RNA use a different strategy to establish stable tertiary folds. Comparison with several other pyrimidine-rich loop hairpins suggests that different minor-groove folding modes exist for the folding thymidine residue.     

Comparative study of sequence-dependent hybridization kinetics in solution and on microspheres

Hybridization kinetics of DNA sequences with known secondary structures and random sequences designed with similar melting temperatures were studied in solution and when one strand was bound to 5 μm silica microspheres. The rates of hybridization followed second-order kinetics and were measured spectrophotometrically in solution and fluorometrically in the solid phase. In solution, the rate constants for the model sequences varied by almost two orders of magnitude, with a decrease in the rate constant with increasing amounts of secondary structure in the target sequence. The random sequences also showed over an order of magnitude difference in the rate constant. In contrast, the hybridization experiments in the solid phase with the same model sequences showed almost no change in the rate constant. Solid phase rate constants were approximately three orders of magnitude lower compared with the solution phase constants for sequences with little or no single-stranded structure. Sequences with a known secondary structure yielded solution phase rate constants as low as 3 × 10³ M⁻¹ s⁻¹ with solid phase rate constants for the same sequences measured at 2.5 × 10² M⁻¹ s⁻¹. The results from these experiments indicate that (i) solid phase hybridization occurs three orders of magnitude slower than solution phase, (ii) trends observed in structure-dependent kinetics of solution phase hybridization may not be applicable to solid phase hybridization and (iii) model probes with known secondary structure decrease reaction rates; however, even random sequences with no known internal single-stranded structure can yield a broad range of reaction rates.     

Calibration of the change in thermal stability of DNA duplexes and degree of base pair mismatch

Summary One method of determining the degree of base pair divergence between two sources of DNA (different strains, species, etc.) is to determine the decrease in thermal stability of hybrid duplex DNA due to mismatching of base pairs. Attempts to calibrate the change in median melting temperature (T_m) to base pair mismatch have led to conflicting results. We have studied the T_m between DNAs of known sequence over a range of from 0.55% to 7.2% base pair mismatch. The relationship of T_m and percent base pair mismatch is remarkably linear over this range with a correlation coefficient >0.98. A T_m of 1°C corresponds to 1.7% base pair mismatch. This conversion is higher than that usually assumed and, therefore, rates of DNA evolution estimated by DNA-DNA hybridization studies are likely faster than previously thought.     

Dynamics of Translocation and Substrate Binding in Individual Complexes Formed with Active Site Mutants of Φ29 DNA Polymerase

The Φ29 DNA polymerase (DNAP) is a processive B-family replicative DNAP. Fluctuations between the pre-translocation and post-translocation states can be quantified from ionic current traces, when individual Φ29 DNAP-DNA complexes are held atop a nanopore in an electric field. Based upon crystal structures of the Φ29 DNAP-DNA binary complex and the Φ29 DNAP-DNA-dNTP ternary complex, residues Tyr-226 and Tyr-390 in the polymerase active site were implicated in the structural basis of translocation. Here, we have examined the dynamics of translocation and substrate binding in complexes formed with the Y226F and Y390F mutants. The Y226F mutation diminished the forward and reverse rates of translocation, increased the affinity for dNTP in the post-translocation state by decreasing the dNTP dissociation rate, and increased the affinity for pyrophosphate in the pre-translocation state. The Y390F mutation significantly decreased the affinity for dNTP in the post-translocation state by decreasing the association rate ∼2-fold and increasing the dissociation rate ∼10-fold, implicating this as a mechanism by which this mutation impedes DNA synthesis. The Y390F dissociation rate increase is suppressed when complexes are examined in the presence of Mn²⁺ rather than Mg²⁺. The same effects of the Y226F or Y390F mutations were observed in the background of the D12A/D66A mutations, located in the exonuclease active site, ∼30 Å from the polymerase active site. Although translocation rates were unaffected in the D12A/D66A mutant, these exonuclease site mutations caused a decrease in the dNTP dissociation rate, suggesting that they perturb Φ29 DNAP interdomain architecture.     

A Phenomenological Model for Predicting Melting Temperatures of DNA Sequences

We report here a novel method for predicting melting temperatures of DNA sequences based on a molecular-level hypothesis on the phenomena underlying the thermal denaturation of DNA. The model presented here attempts to quantify the energetic components stabilizing the structure of DNA such as base pairing, stacking, and ionic environment which are partially disrupted during the process of thermal denaturation. The model gives a Pearson product-moment correlation coefficient (r) of ∼0.98 between experimental and predicted melting temperatures for over 300 sequences of varying lengths ranging from 15-mers to genomic level and at different salt concentrations. The approach is implemented as a web tool (www.scfbio-iitd.res.in/chemgenome/Tm_predictor.jsp) for the prediction of melting temperatures of DNA sequences.     

DNA assembler,an in vivo genetic method for rapid construction of biochemical pathways

The assembly of large recombinant DNA encoding a whole biochemical pathway or genome represents a significant challenge. Here, we report a new method, DNA assembler, which allows the assembly of an entire biochemical pathway in a single step via in vivo homologous recombination in Saccharomyces cerevisiae. We show that DNA assembler can rapidly assemble a functional d-xylose utilization pathway (∼9 kb DNA consisting of three genes), a functional zeaxanthin biosynthesis pathway (∼11 kb DNA consisting of five genes) and a functional combined d-xylose utilization and zeaxanthin biosynthesis pathway (∼19 kb consisting of eight genes) with high efficiencies (70–100%) either on a plasmid or on a yeast chromosome. As this new method only requires simple DNA preparation and one-step yeast transformation, it represents a powerful tool in the construction of biochemical pathways for synthetic biology, metabolic engineering and functional genomics studies.     

A ruthenium dimer complex with a flexible linker slowly threads between DNA bases in two distinct steps

Several multi-component DNA intercalating small molecules have been designed around ruthenium-based intercalating monomers to optimize DNA binding properties for therapeutic use. Here we probe the DNA binding ligand [μ-C4(cpdppz)₂(phen)₄Ru₂]⁴⁺, which consists of two Ru(phen)₂dppz²⁺ moieties joined by a flexible linker. To quantify ligand binding, double-stranded DNA is stretched with optical tweezers and exposed to ligand under constant applied force. In contrast to other bis-intercalators, we find that ligand association is described by a two-step process, which consists of fast bimolecular intercalation of the first dppz moiety followed by ∼10-fold slower intercalation of the second dppz moiety. The second step is rate-limited by the requirement for a DNA-ligand conformational change that allows the flexible linker to pass through the DNA duplex. Based on our measured force-dependent binding rates and ligand-induced DNA elongation measurements, we are able to map out the energy landscape and structural dynamics for both ligand binding steps. In addition, we find that at zero force the overall binding process involves fast association (∼10 s), slow dissociation (∼300 s), and very high affinity (K_d ∼10 nM). The methodology developed in this work will be useful for studying the mechanism of DNA binding by other multi-step intercalating ligands and proteins.     

Yeast Pif1 Helicase Exhibits a One-base-pair Stepping Mechanism for Unwinding Duplex DNA

Kinetic analysis of the DNA unwinding and translocation activities of helicases is necessary for characterization of the biochemical mechanism(s) for this class of enzymes. Saccharomyces cerevisiae Pif1 helicase was characterized using presteady state kinetics to determine rates of DNA unwinding, displacement of streptavidin from biotinylated DNA, translocation on single-stranded DNA (ssDNA), and ATP hydrolysis activities. Unwinding of substrates containing varying duplex lengths was fit globally to a model for stepwise unwinding and resulted in an unwinding rate of ∼75 bp/s and a kinetic step size of 1 base pair. Pif1 is capable of displacing streptavidin from biotinylated oligonucleotides with a linear increase in the rates as the length of the oligonucleotides increased. The rate of translocation on ssDNA was determined by measuring dissociation from varying lengths of ssDNA and is essentially the same as the rate of unwinding of dsDNA, making Pif1 an active helicase. The ATPase activity of Pif1 on ssDNA was determined using fluorescently labeled phosphate-binding protein to measure the rate of phosphate release. The quantity of phosphate released corresponds to a chemical efficiency of 0.84 ATP/nucleotides translocated. Hence, when all of the kinetic data are considered, Pif1 appears to move along DNA in single nucleotide or base pair steps, powered by hydrolysis of 1 molecule of ATP.     

The depletion of DNA methyltransferase-1 and the epigenetic effects of 5-aza-2′deoxycytidine (decitabine) are differentially regulated by cell cycle progression

5-Aza-2′-deoxycytidine (decitabine) is a drug targeting the epigenetic abnormalities of tumors. The basis for its limited efficacy in solid tumors is unresolved, but may relate to their indolent growth, their p53 genotype or both. We report that the primary molecular mechanism of decitabine—depletion of DNA methyltransferase-1 following its “suicide” inactivation—is not absolutely associated with cell cycle progression in HCT 116 colon cancer cells, but is associated with their p53 genotype. Control experiments affirmed that the secondary molecular effects of decitabine on global and promoter-specific CpG methylation and MAGE-A1 mRNA expression were S-phase dependent, as expected. Secondary changes in CpG methylation occurred only in growing cells ∼24–48 h after decitabine treatment; these epigenetic changes coincided with p53 accumulation, an index of DNA damage. Conversely, primary depletion of DNA methyltransferase-1 began immediately after a single exposure to 300 nM decitabine and it progressed to completion within ∼8 h, even in confluent cells arrested in G₁ and G₂/M. Our results suggest that DNA repair and remodeling activity in arrested, confluent cells may be sufficient to support the primary molecular action of decitabine, while its secondary, epigenetic effects require cell cycle progression through S-phase.Key words: 5-aza-2′-deoxycytidine, decitabine, DNA methyltransferase-1, suicide inactivation, p53, S-phase, cell cycle     

Sequence-dependent gating of an ion channel by DNA hairpin molecules

DNA hairpins produce ionic current signatures when captured by the alpha-hemolysin nano-scale pore under conditions of single molecule electrophoresis. Gating patterns produced by individual DNA hairpins when captured can be used to distinguish differences of a single base pair or even a single nucleotide [Vercoutere,W.A. et al. (2003) Nucleic Acids Res., 31, 1311–1318]. Here we investigate the mechanism(s) that may account for the ionic current gating signatures. The ionic current resistance profile of conductance states produced by DNA hairpin molecules with 3–12 bp stems showed a plateau in resistance between 10 and 12 bp, suggesting that hairpins with 10–12 bp stems span the pore vestibule. DNA hairpins with 9–12 bp stems produced gating signatures with the same relative conductance states. Systematic comparison of the conductance state dwell times and apparent activation energies for a series of 9–10 bp DNA hairpins suggest that the 3′ and 5′ ends interact at or near the limiting aperture within the vestibule of the alpha-hemolysin pore. The model presented may be useful in predicting and interpreting DNA detection using nanopore detectors. In addition, this well-defined molecular system may prove useful for investigating models of ligand-gated channels in biological membranes.     

Manipulation of single polymerase-DNA complexes: A mechanical view of DNA unwinding during replication

Comment on: Morin JA, et al. Proc Natl Acad Sci USA 2012; 109:8115-20.DNA replication requires overcoming the energetic barrier associated with the base pair melting of its double helix and a fine-tuned coordination between the processes of DNA unwinding and DNA replication. One intriguing question that remains poorly understood is the exact mechanism of the coupling of these two reactions. In some organisms, these activities are coupled within the same protein, like in the case of the phage Phi29 DNA polymerase. This polymerase works as a hybrid polymerase-helicase, because it presents an amino acid insertion that together with other protein domains forms a narrow tunnel around the template strand. This topological restriction is similar to the one imposed by hexameric helicases at the fork junction and promotes the separation of the fork ahead.¹ The Phi29 DNA polymerase, therefore, constitutes a simple, good model system to understand the basic mechanistic principles of the coupling between DNA replication and unwinding activities: the polymerase may behave as a “passive” unwinding motor, if translocation of the protein traps transient unwinding fluctuations of the fork, or as an “active” motor, if the polymerase actively destabilizes the duplex DNA at the junction. Therefore, factors that affect the stability of the fork junction, as DNA sequence or mechanical destabilization of the fork, will have a stronger effect on the unwinding kinetics of a “passive” motor than on an “active” one.To determine the DNA unwinding mechanism of the Phi29 DNA polymerase, we used optical tweezers to measure at single molecule level the effect of DNA sequence and destabilizing forces on the fork on the rates of strand displacement (replication and unwinding are tightly coupled, Δx₁, Fig. 1A) and primer extension (replication of the displaced complementary strand without unwinding, Δx₂, Fig. 1A) of two polymerases: the wild-type Phi29 DNA polymerase and a strand displacement deficient variant, which bears a couple of mutations that may affect the stability of the tunnel required for unwinding.² We quantified the free energy of interaction between the polymerase and the DNA fork, ΔG_int, and the range of this interaction, M, through a theoretical analysis of the dependence of the replication, unwinding and pause kinetics on the DNA sequence and force.^3,4Open in a separate windowFigure 1. (A) Schematic representation of the experimental design (not to scale). A single DNA hairpin was attached to functionalized beads inside a fluidics chamber. One strand of the hairpin is attached through a dsDNA handle to a bead held in the optical trap (top), while the complementary strand is attached to a bead on top of a mobile micropipette (bottom). At a constant force, after flowing the nucleotides into the reaction chamber, the strand displacement and primer extension rates of the polymerase are detected as a change in distance between the beads, Δx₁ and Δx₂, respectively. (B) Representative replication activity of a single mutant polymerase molecule. Long pauses are observed only during the strand displacement reaction. (C) Mechanistic distinction between passive and active unwinding. The cartoon illustrates the degree of activeness in DNA unwinding of different replicative helicases⁶ and the Phi29 DNA polymerase.Our results show that while the primer extension rates of both polymerases are force- and sequence-independent their average unwinding rates are sensitive to these two variables, although with different intensity. As expected, the dsDNA fork presents a much stronger physical barrier to the mutant polymerase unwinding. Qualitative reasoning might suggest that the observed differences imply different “activeness” of the unwinding mechanism of each polymerase. However, the inclusion of the pause kinetics of each polymerase in our model revealed that they use the same active mechanism; they both destabilize the two nearest base pairs of the fork (M = 2) with an interaction energy ΔG_int = 2 k_BT per base pair. These results suggest that mutations affecting the stability of the tunnel required for unwinding do not decrease the “activeness” of the motor but instead increase the probability of the unwinding mechanism to fail upon encountering a closed fork junction, inducing the entrance of the mutant polymerase into a long-lived inactive pause state (Fig. 1B). These results bring out the importance to consider pause kinetics to accurately quantify the actual unwinding mechanism of the Phi29 DNA polymerase or any other nucleic acid unwinding motor in which pauses are relevant during its operation. The presence of pauses obscures the actual pause-free rates of the motor and can lead to misleading results when they are not properly accounted.Our data are consistent with a model in which the closed template tunnel that wraps around the template strand allows the Phi29 DNA polymerase to maintain a sharp bending of this strand (essential for template reading in all replicative polymerases) and a bending of the complementary strand, due to its steric exclusion, at a closed fork junction. Bending of the two strands would generate mechanical stress at the junction promoting its active destabilization. A less stable tunnel, as in the mutant polymerase, will not be able to keep the mechanical stress at a closed fork junction, in this case the fork pressure would induce loosening of the correct protein-DNA interactions favoring the entrance to a polymerization inactive state.Similar mechanisms for mechanical destabilization of the fork junction can be envisioned for other DNA replication systems in which a DNA polymerase and a helicase work in coordination. In these systems, the leading strand can be sharply bent by the steric exclusion induced by the helicase and by the functional binding of the polymerase generating effective mechanical stress at the fork junction to account for efficient unwinding during replication. These implications are further supported by recent single molecule studies using magnetic tweezers that describe a collaborative coupling of this nature between the activities of the bacteriophage T4 DNA polymerase and DNA helicase.⁵     

Mechanism of Regulation of Native Cardiac Muscle Thin Filaments by Rigor Cardiac Myosin-S1 and Calcium

We have studied the mechanism of activation of native cardiac thin filaments by calcium and rigor myosin. The acceleration of the rate of 2′-deoxy-3′-O-(N-methylanthraniloyl)ADP (mdADP) dissociation from cardiac myosin-S1-mdADP-P_i and cardiac myosin-S1-mdADP by native cardiac muscle thin filaments was measured using double mixing stopped-flow fluorescence. Relative to inhibited thin filaments (no bound calcium or rigor S1), fully activated thin filaments (with both calcium and rigor-S1 bound) increase the rate of product dissociation from the physiologically important pre-power stroke myosin-mdADP-P_i by a factor of ∼75. This can be compared with only an ∼6-fold increase in the rate of nucleotide diphosphate dissociation from nonphysiological myosin-mdADP by the fully activated thin filaments relative to the fully inhibited thin filaments. These results show that physiological levels of regulation are not only dependent on the state of the thin filament but also on the conformation of the myosin. Less than 2-fold regulation is due to a change in affinity of myosin-ADP-P_i for thin filaments such as would be expected by a simple “steric blocking” of the myosin-binding site of the thin filament by tropomyosin. Although maximal activation requires both calcium and rigor myosin-S1 bound to the cardiac filament, association with a single ligand produces ∼70% maximal activation. This can be contrasted with skeletal thin filaments in which calcium alone only activated the rate of product dissociation ∼20% of maximum, and rigor myosin produces ∼30% maximal activation.     

Conserved secondary structures in Aspergillus

Background

Recent evidence suggests that the number and variety of functional RNAs (ncRNAs as well as cis-acting RNA elements within mRNAs ) is much higher than previously thought; thus, the ability to computationally predict and analyze RNAs has taken on new importance. We have computationally studied the secondary structures in an alignment of six Aspergillus genomes. Little is known about the RNAs present in this set of fungi, and this diverse set of genomes has an optimal level of sequence conservation for observing the correlated evolution of base-pairs seen in RNAs.

Methodology/Principal Findings

We report the results of a whole-genome search for evolutionarily conserved secondary structures, as well as the results of clustering these predicted secondary structures by structural similarity. We find a total of 7450 predicted secondary structures, including a new predicted ∼60 bp long hairpin motif found primarily inside introns. We find no evidence for microRNAs. Different types of genomic regions are over-represented in different classes of predicted secondary structures. Exons contain the longest motifs (primarily long, branched hairpins), 5′ UTRs primarily contain groupings of short hairpins located near the start codon, and 3′ UTRs contain very little secondary structure compared to other regions. There is a large concentration of short hairpins just inside the boundaries of exons. The density of predicted intronic RNAs increases with the length of introns, and the density of predicted secondary structures within mRNA coding regions increases with the number of introns in a gene.

Conclusions/Sigificance

There are many conserved, high-confidence RNAs of unknown function in these Aspergillus genomes, as well as interesting spatial distributions of predicted secondary structures. This study increases our knowledge of secondary structure in these aspergillus organisms.     

Thermodynamic parameters for loop formation in RNA and DNA hairpin tetraloops.

We determined the melting temperatures (Tm) and thermodynamic parameters of 15 RNA and 19 DNA hairpins at 1 M NaCl, 0.01 M sodium phosphate, 0.1 mM EDTA, at pH 7. All these hairpins have loops of four bases, the most common loop size in 16S and 23S ribosomal RNAs. The RNA hairpins varied in loop sequence, loop-closing base pair (A.U, C.G, or G.C), base sequence of the stem, and stem size (four or five base pairs). The DNA hairpins varied in loop sequence, loop-closing base pair (C.G, or G.C), and base sequence of the four base-pair stem. Thermodynamic properties of a hairpin may be represented by nearest-neighbor interactions of the stem plus contributions from the loop. Thus, we obtained thermodynamic parameters for the formation of RNA and DNA tetraloops. For the tetraloops we studied, a free energy of loop formation (at 37 degrees C) of about +3 kcal/mol is most common for either RNA or DNA. There are extra stable loops with delta G degrees 37 near +1 kcal/mol, but the sequences are not necessarily the same for RNA and DNA. The closing base pair is also important; changing from C.G to G.C lowered the stability of several tetraloops in both RNA and DNA. These values will be useful in predicting RNA and DNA secondary structures.     

Local and global effects of strong DNA bending induced during molecular dynamics simulations

DNA bending plays an important role in many biological processes, but its molecular and energetic details as a function of base sequence remain to be fully understood. Using a recently developed restraint, we have studied the controlled bending of four different B-DNA oligomers using molecular dynamics simulations. Umbrella sampling with the AMBER program and the recent parmbsc0 force field yield free energy curves for bending. Bending 15-base pair oligomers by 90° requires roughly 5kcalmol⁻¹, while reaching 150° requires of the order of 12kcalmol⁻¹. Moderate bending occurs mainly through coupled base pair step rolls. Strong bending generally leads to local kinks. The kinks we observe all involve two consecutive base pair steps, with disruption of the central base pair (termed Type II kinks in earlier work). A detailed analysis of each oligomer shows that the free energy of bending only varies quadratically with the bending angle for moderate bending. Beyond this point, in agreement with recent experiments, the variation becomes linear. An harmonic analysis of each base step yields force constants that not only vary with sequence, but also with the degree of bending. Both these observations suggest that DNA is mechanically more complex than simple elastic rod models would imply.     

Solution structure determination of the two DNA-binding domains in the Schizosaccharomyces pombe Abp1 protein by a combination of dipolar coupling and diffusion anisotropy restraints

We have solved the solution structure of the N-terminal region of the fission yeast centromere protein, Abp1, bound to a 21-base pair DNA fragment bearing its recognition site (Mw = 30 kDa). Although the two DNA-binding domains in the Abp1 protein were defined well by a conventional NOE-based NMR methodology, the overall structure of the Abp1 protein was poorly defined, due to the lack of interdomain distance restraints. Therefore, we additionally used residual dipolar couplings measured in a weakly aligned state, and rotational diffusion anisotropies. Neither the NH residual dipolar couplings nor the backbone ¹⁵N T ₁/T ₂ data were sufficient to determine the overall structure of the Abp1 protein, due to spectral overlap. We used a combination of these two orientational restraints (residual dipolar coupling and rotational diffusion anisotropy), which significantly improved the convergence of the overall structures. The range of the observed T ₁/T ₂ ratios was wider (20–50 for the secondary structure regions of Abp1) than the previously reported data for several globular proteins, indicating that the overall shape of the Abp1DNA complex is ellipsoid. This extended form would facilitate the recognition of the two separate sites in the relatively long DNA sequence by the DNA-binding domains of Apb1.