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Non-canonical DNA structures that retain programmability and structural predictability are increasingly being used in DNA nanotechnology applications, where they offer versatility beyond traditional Watson-Crick interactions. The d(CGA) triplet repeat motif is structurally dynamic and can transition between parallel-stranded homo-base paired duplex and anti-parallel unimolecular hairpin in a pH-dependent manner. Here, we evaluate the thermodynamic stability and nuclease sensitivity of oligonucleotides composed of the d(CGA) motif and several structurally related sequence variants. These results show that the structural transition resulting from decreasing the pH is accompanied by both a significant energetic stabilization and decreased nuclease sensitivity as unimolecular hairpin structures are converted to parallel-stranded homo-base paired duplexes. Furthermore, the stability of the parallel-stranded duplex form can be altered by changing the 5′-nucleobase of the d(CGA) triplet and the frequency and position of the altered triplets within long stretches of d(CGA) triplets. This work offers insight into the stability and versatility of the d(CGA) triplet repeat motif and provides constraints for using this pH-adaptive structural motif for creating DNA-based nanomaterials.STATEMENT OF SIGNIFICANCEThis article addresses the stability of the d(CGA) triplet motif and variants in solution. Our study reveals changes in thermodynamic stability and nuclease resistance in response to pH. The identity of the 5′-nucleobase within each triplet and the position and frequency of different triplets within stretches of d(CGA) triplets can tune parallel-stranded duplex stability. This tunability can be used for nanotechnological applications where the specificity of the 5′-nucleobase pairing interaction is used to order of long stretches of d(CGA) triplets. These results can inform the rational design of pH-sensitive structurally switchable DNA-based nanomaterials.

Synthetic nucleobases presenting non-Watson-Crick arrangements of hydrogen bond donor and acceptor groups can form additional nucleotide pairs that stabilize duplex DNA independent of the standard A:T and G:C pairs. The pair between 2-amino-3-nitropyridin-6-one 2'-deoxyriboside (presenting a {donor-donor-acceptor} hydrogen bonding pattern on the Watson-Crick face of the small component, trivially designated Z) and imidazo[1,2-a]-1,3,5-triazin-4(8H)one 2'-deoxyriboside (presenting an {acceptor-acceptor-donor} hydrogen bonding pattern on the large component, trivially designated P) is one of these extra pairs for which a substantial amount of molecular biology has been developed. Here, we report the results of UV absorbance melting measurements and determine the energetics of binding of DNA strands containing Z and P to give short duplexes containing Z:P pairs as well as various mismatches comprising Z and P. All measurements were done at 1 M NaCl in buffer (10 mM Na cacodylate, 0.5 mM EDTA, pH 7.0). Thermodynamic parameters (ΔH°, ΔS°, and ΔG°

DNAzymes with nucleic acid-cleaving catalytic activity are increasing in versatility through concerted efforts to discover new sequences with unique functions, and they are generating excitement in the sensing community as cheap, stable, amplifiable detection elements. This review provides a comprehensive list and detailed descriptions of the DNAzymes identified to date, classified by their associated small molecule or ion needed for catalysis; of note, this classification clarifies conserved regions of various DNAzymes that are not obvious in the literature. Furthermore, we detail the breadth of functionality of these DNA sequences as well as the range of reaction conditions under which they are useful. In addition, the utility of the DNAzymes in a variety of sensing and therapeutic applications is presented, detailing both their advantages and disadvantages.

The lac repressor protein (LacI) efficiently represses transcription of the lac operon in Escherichia coli by binding to two distant operator sites on the bacterial DNA and causing the intervening DNA to form a loop. We employed single-molecule tethered particle motion to observe LacI-mediated loop formation and breakdown in DNA constructs that incorporate optimized operator binding sites and intrinsic curvature favorable to loop formation. Previous bulk competition assays indirectly measured the loop lifetimes in these optimized DNA constructs as being on the order of days; however, we measured these same lifetimes to be on the order of minutes for both looped and unlooped states. In a range of single-molecule DNA competition experiments, we found that the resistance of the LacI-DNA complex to competitive binding is a function of both the operator strength and the interoperator sequence. To explain these findings, we present what we believe to be a new kinetic model of loop formation and DNA competition. In this proposed new model, we hypothesize a new unlooped state in which the unbound DNA-binding domain of the LacI protein interacts nonspecifically with nonoperator DNA adjacent to the operator site at which the second LacI DNA-binding domain is bound.

Branched peptides containing histidines and lysines (HK) have been shown to be effective carriers for DNA and siRNA. We anticipate that elucidation of the binding mechanism of HK with siRNA will provide greater insight into the self-assembly and delivery of the HK:siRNA polyplex. Non-covalent bonds between histidine residues and nucleic acids may enhance the stability of siRNA polyplexes. We first compared the polyplex biophysical properties of a branched HK with those of branched asparagines-lysine peptide (NK). Consistent with siRNA silencing experiments, gel electrophoresis demonstrated that the HK siRNA polyplex maintained its integrity with prolonged incubation in serum, whereas siRNA in complex with NK was degraded in a time-dependent manner. Isothermal titration calorimetry of various peptides binding to siRNA at pH 7.3 showed that branched polylysine, interacted with siRNA was initially endothermic, whereas branched HK exhibited an exothermic reaction at initial binding. The exothermic interaction indicates formation of non-ionic bonds between histidines and siRNA; purely electrostatic interaction is entropy-driven and endothermic. To investigate the type of non-ionic bond, we studied the protonation state of imidazole rings of a selectively 15N labeled branched HK by heteronuclear single quantum coherence NMR. The peak of Nδ1-H tautomers of imidazole shifted downfield (in the direction of deprotonation) by 0.5 to 1.0 ppm with addition of siRNA, providing direct evidence that histidines formed hydrogen bonds with siRNA at physiological pH. These results establish that histidine-rich peptides form hydrogen bonds with siRNA, thereby enhancing the stability and biological activity of the polyplex in vitro and in vivo.

We characterized in this study the pharmacokinetics and antitumor efficacy of histidine-lysine (HK):siRNA nanoplexes modified with PEG and a cyclic RGD (cRGD) ligand targeting αvβ3 and αvβ5 integrins. With noninvasive imaging, systemically administered surface-modified HK:siRNA nanoplexes showed nearly 4-fold greater blood levels, 40% higher accumulation in tumor tissue, and 60% lower luciferase activity than unmodified HK:siRNA nanoplexes. We then determined whether the surface-modified HK:siRNA nanoplex carrier was more effective in reducing MDA-MB-435 tumor growth with an siRNA targeting Raf-1. Repeated systemic administration of the selected surface modified HK:siRNA nanoplexes targeting Raf-1 showed 35% greater inhibition of tumor growth than unmodified HK:siRNA nanoplexes and 60% greater inhibition of tumor growth than untreated mice. The improved blood pharmacokinetic results and tumor localization observed with the integrin-targeting surface modification of HK:siRNA nanoplexes correlated with greater tumor growth inhibition. This investigation reveals that through control of targeting ligand surface display in association with a steric PEG layer, modified HK: siRNA nanoplexes show promise to advance RNAi therapeutics in oncology and potentially other critical diseases.

The inflexibility of double-stranded DNA with respect to bending and twisting is well established in vitro. Understanding apparent DNA physical properties in vivo is a greater challenge. Here, we exploit repression looping with components of the Escherichia coli lac operon to monitor DNA flexibility in living cells. We create a minimal system for testing the shortest possible DNA repression loops that contain an E. coli promoter, and compare the results to prior experiments. Our data reveal that loop-independent repression occurs for certain tight operator/promoter spacings. When only loop-dependent repression is considered, fits to a thermodynamic model show that DNA twisting limits looping in vivo, although the apparent DNA twist flexibility is 2- to 4-fold higher than in vitro. In contrast, length-dependent resistance to DNA bending is not observed in these experiments, even for the shortest loops constraining

Sequence-dependent intrinsic curvature of DNA influences looping by regulatory proteins such as LacI and NtrC. Curvature can enhance stability and control shape, as observed in LacI loops formed with three designed sequences with operators bracketing an A-tract bend. We explore geometric, topological, and energetic effects of curvature with an analysis of a family of highly bent sequences, using the elastic rod model from previous work. A unifying straight-helical-straight representation uses two phasing parameters to describe sequences composed of two straight segments that flank a common helically supercoiled segment. We exercise the rod model over this two-dimensional space of phasing parameters to evaluate looping behaviors. This design space is found to comprise two subspaces that prefer parallel versus anti-parallel binding topologies. The energetic cost of looping varies from 4 to 12 kT. Molecules can be designed to yield distinct binding topologies as well as hyperstable or hypostable loops and potentially loops that can switch conformations. Loop switching could be a mechanism for control of gene expression. Model predictions for linking numbers and sizes of LacI-DNA loops can be tested using multiple experimental approaches, which coupled with theory could address whether proteins or DNA provide the observed flexibility of protein-DNA loops.

Proton assignment of nuclear magnetic resonance (NMR) spectra of homopyrimidine/homopurine tract oligonucleotides becomes extremely challenging with increasing helical length due to severe cross-peak overlap. As an alternative to the more standard practice of (15)N and (13)C labeling of oligonucleotides, here, we describe a method for assignment of highly redundant DNA sequences that uses single-site substitution of the thymine isostere 2,4-difluoro-5-methylbenzene (dF). The impact of this approach in facilitating the assignment of intractable spectra and analyzing oligonucleotide structure and dynamics is demonstrated using A-tract and TATA box DNA and two polypurine tract-containing RNA:DNA hybrids derived from HIV-1 and the Saccharomyces cerevisiae long-terminal repeat-containing retrotransposon Ty3. Only resonances proximal to the site of dF substitution exhibit sizable chemical shift changes, providing spectral dispersion while still allowing chemical shift mapping of resonances from unaffected residues distal to the site of modification directly back to the unmodified sequence. It is further illustrated that dF incorporation can subtly alter the conformation and dynamics of homopyrimidine/homopurine tract oligonucleotides, and how these NMR observations can be correlated, in the cases of the TATA box DNA, with modulation in the TATA box-binding protein interaction using an orthogonal gel assay.

The intrinsic stiffness of DNA limits its ability to be bent and twisted over short lengths, but such deformations are required for gene regulation. One classic paradigm is DNA looping in the regulation of the Escherichia coli lac operon. Lac repressor protein binds simultaneously to two operator sequences flanking the lac promoter. Analysis of the length dependence of looping-dependent repression of the lac operon provides insight into DNA deformation energetics within cells. The apparent flexibility of DNA is greater in vivo than in vitro, possibly because of host proteins that bind DNA and induce sites of flexure. Here we test DNA looping in bacterial strains lacking the nucleoid proteins HU, IHF or H-NS. We confirm that deletion of HU inhibits looping and that quantitative modeling suggests residual looping in the induced operon. Deletion of IHF has little effect. Remarkably, DNA looping is strongly enhanced in the absence of H-NS, and an explanatory model is proposed. Chloroquine titration, psoralen crosslinking and supercoiling-sensitive reporter assays show that the effects of nucleoid proteins on looping are not correlated with their effects on either total or unrestrained supercoiling. These results suggest that host nucleoid proteins can directly facilitate or inhibit DNA looping in bacteria.

We wish to understand the role of electrostatics in DNA stiffness and bending. The DNA charge collapse model suggests that mutual electrostatic repulsions between neighboring phosphates significantly contribute to DNA stiffness. According to this model, placement of fixed charges near the negatively charged DNA surface should induce bending through asymmetric reduction or enhancement of these inter-phosphate repulsive forces. We have reported previously that charged variants of the elongated basic-leucine zipper (bZIP) domain of Gcn4p bend DNA in a manner consistent with this charge collapse model. To extend this result to a more globular protein, we present an investigation of the dimeric basic-helix-loop-helix (bHLH) domain of Pho4p. The 62 amino acid bHLH domain has been modified to position charged amino acid residues near one face of the DNA double helix. As observed for bZIP charge variants, DNA bending toward appended cations (away from the protein:DNA interface) is observed. However, unlike bZIP proteins, DNA is not bent away from bHLH anionic charges. This finding can be explained by the structure of the more globular bHLH domain which, in contrast to bZIP proteins, makes extensive DNA contacts along the binding face.

The Escherichia coli lactose repressor protein (LacI) provides a classic model for understanding protein-induced DNA looping. LacI has a C-terminal four-helix bundle tetramerization domain that may act as a flexible hinge. In previous work, several DNA constructs, each containing two lac operators bracketing a sequence-induced bend, were designed to stabilize different possible looping geometries. The resulting hyperstable LacI-DNA loops exist as both a compact “closed” form with a V-shaped repressor and also a more “open” form with an extended hinge. The “9C14” construct was of particular interest because footprinting, electrophoretic mobility shift, and ring closure experiments suggested that it forms both geometries. Previous fluorescence resonance energy transfer (FRET) measurements gave an efficiency of energy transfer (ET) of 70%, confirming the existence of a closed form. These measurements could not determine whether open form or intermediate geometries are populated or the timescale of interconversion. We have now applied single-molecule FRET to Cy3, Cy5 double-labeled LacI-DNA loops diffusing freely in solution. By using multiple excitation wavelengths and by carefully examining the behavior of the zero-ET peak during titration with LacI, we show that the LacI-9C14 loop exists exclusively in a single closed form exhibiting essentially 100% ET.

Transcriptional activation by the E.coli NtrC protein can occur via DNA looping between a DNA-bound activator and the target sigma(54) RNA polymerase. NtrC forms an octamer on DNA that is capable of binding two DNA molecules. Its ATPase activity is required for open complex formation. Geometric requirements for activation were assessed using a library of DNA bending sequences created by random ligation of A-tract oligonucleotides, as well as several designed sequences. Thirty random or designed sequences with a variety of DNA lengths and bending geometries were cloned in plasmids, and the library was used to replace the spacer between the NtrC binding sites and the core glnAp2 promoter. The activity of each promoter construct under nitrogen limitation was determined in vivo, in a lambda phage lacZ reporter system integrated as a single-copy lysogen to avoid titrating NtrC or polymerase. A wide variety of bending geometries was found to support a similar level of transcriptional activation ( approximately 3-4-fold). Computer modeling of the DNA trajectories suggests that the most inactive promoters have short spacer DNA and the NtrC sites on the opposite side of the helix as the wild-type sites; otherwise, the loop can form effectively. Flexibility and multivalency of the NtrC-Esigma(54) interaction apparently provides substantial independence from DNA stiffness constraints, and in general activation requires less efficient looping than repression. However, none of the random templates were as active as wild-type promoter. Subsidiary activator binding sites in the wild-type were found to be required for full activity, but, surprisingly, these sites could not be functionally replaced by strong binding sites. This suggests that one or more protomers in the NtrC octamer must form and then release contacts with DNA in order to complete the ATPase cycle and act as an AAA(+) activator of the Esigma(54). This dynamic DNA wrapping around the NtrC octamer is proposed to be necessary for efficient activation, and the wrapping may also reduce adventitious activation of other promoters.

The design of modified nucleic acid probes, primers, and therapeutics is improved by considering their thermodynamics. Locked nucleic acid (LNA) is one of the most useful modified backbones, with incorporation of a single LNA providing a substantial increase in duplex stability. In this work, the hybridization DeltaH(o), DeltaS(o), and melting temperature (T(M)) were measured from absorbance melting curves for 100 duplex oligonucleotides with single internal LNA nucleotides on one strand, and the results provided DeltaDeltaH(o), DeltaDeltaS(o), DeltaDelta, and DeltaT(M) relative to reference DNA oligonucleotides. LNA pyrimidines contribute more stability than purines, especially A(L), but there is substantial context dependence for each LNA base. Both the 5' and 3' neighbors must be considered in predicting the effect of an LNA incorporation, with purine neighbors providing more stability. Enthalpy-entropy compensation in DeltaDeltaH(o) and DeltaDeltaS(o) is observed across the set of sequences, suggesting that LNA can stabilize the duplex by either preorganization or improved stacking, but not both simultaneously. Singular value decomposition analysis provides predictive sequence-dependent rules for hybridization of singly LNA-substituted DNA oligonucleotides to their all-DNA complements. The results are provided as sets of DeltaDeltaH(o), DeltaDeltaS(o), and DeltaDelta parameters for all 32 of the possible nearest neighbors for LNA+DNA:DNA hybridization (5' MX(L) and 5' X(L)N, where M, N, and X = A, C, G, or T and X(L) represents LNA). The parameters are applicable within the standard thermodynamic prediction algorithms. They provide T(M) estimates accurate to within 2 degrees C for LNA-containing oligonucleotides, which is significantly better accuracy than previously available.

Lac repressor (LacI) forms DNA loops which are critical for efficient operator binding and transcriptional repression. Designed DNA loops formed on three constructs with lac operators bracketing phased A-tract bends were characterized by mobility shift, footprinting, and DNA cyclization and topology. Operator dyad axes point either in or out relative to the sequence-induced curvature. Possible conformations suggested from X-ray structures of LacI and LacI·DNA include “wrapping away” (WA), “simple loop” (SL), and “wrapping toward” (WT) models. The WA loop should be preferentially stabilized by the outward operators, the SL and WT loops by the inward operators. Competition experiments demonstrated increased loop stability for all the bent constructs, with the SL/WT construct supporting hyperstable loops (t1/2 of days). This offers a general approach to stabilizing multi-protein DNA complexes on short DNA. DNA cyclization of loops gave minicircle products with altered topologies. WA constructs afforded relaxed and positive topoisomers, and cyclization kinetics indicated slow interconversion of precursors to the two topoisomers. The SL/WT construct gave a relaxed topoisomer, with a small amount of negative supercoil. These results suggest that while it is possible to force the WA loop to form (as in a model proposed from the LacI·DNA structure), the most stable loop geometry is different, probably a U-shape around an extended LacI tetramer. The topological results show how a protein-induced positive supercoil can be reconciled with LacI’s preference for binding negatively supercoiled DNA. We suggest that looping proteins can affect the assembly of subsequent proteins by controling loop topology.

We have studied DNA minicircles containing the ATF/CREB binding site for GCN4 by using a combination of cyclization kinetics experiments and Monte Carlo simulations. Cyclization rates were determined with and without GCN4 for DNA constructs containing the ATF/CREB site separated from a phased A-tract multimer bend by a variable length phasing adaptor. The cyclization results show that GCN4 binding does not significantly change the conformation of the ATF/CREB site, which is intrinsically slightly bent toward the major groove. Monte Carlo simulations quantitate the ATF/CREB site structure as an 8 degrees bend toward the major groove in a coordinate frame near the center of the site. The ATF/CREB site is underwound by 53 degrees relative to the related AP-1 site DNA. The effect of GCN4 binding can be modeled either as a decrease in the local flexibility, corresponding to an estimated 60% increase in the persistence length for the 10-bp binding site, or possibly as a small decrease (1 degrees) in intrinsic bend angle. Our results agree with recent electrophoretic and crystallographic studies and demonstrate that cyclization and simulation can characterize subtle changes in DNA structure and flexibility.

Gene delivery is a promising way to treat hereditary diseases and cancer; however, there is little understanding of DNA:carrier complex mechanical properties, which may be critical for the protection and release of nucleic acids. We applied optical tweezers to directly measure single-molecule mechanical properties of DNA condensed using 19-mer poly-L-lysine (PLL) or branched histidine–lysine (HK) peptides. Force–extension profiles indicate that both carriers condense DNA actively, showing force plateaus during stretching and relaxation cycles. As the environment such as carrier concentration, pH, and the presence of zinc ions changes, DNA:HK complexes showed dynamically regulated mechanical properties at multiple force levels. The fundamental knowledge from this study can be applied to design a mechanically tailored complex which may enhance transfection efficiency by controlling the stability of the complex temporally and spatially.

Artificial DNA looping peptides were engineered to study the roles of protein and DNA flexibility in controlling the geometry and stability of protein-mediated DNA loops. These LZD (leucine zipper dual-binding) peptides were derived by fusing a second, C-terminal, DNA-binding region onto the GCN4 bZip peptide. Two variants with different coiled-coil lengths were designed to control the relative orientations of DNA bound at each end. Electrophoretic mobility shift assays verified formation of a sandwich complex containing two DNAs and one peptide. Ring closure experiments demonstrated that looping requires a DNA-binding site separation of 310 bp, much longer than the length needed for natural loops. Systematic variation of binding site separation over a series of 10 constructs that cyclize to form 862-bp minicircles yielded positive and negative topoisomers because of two possible writhed geometries. Periodic variation in topoisomer abundance could be modeled using canonical DNA persistence length and torsional modulus values. The results confirm that the LZD peptides are stiffer than natural DNA looping proteins, and they suggest that formation of short DNA loops requires protein flexibility, not unusual DNA bendability. Small, stable, tunable looping peptides may be useful as synthetic transcriptional regulators or components of protein–DNA nanostructures.

The E. coli Lac repressor (LacI) tetramer binds simultaneously to a promoter-proximal DNA binding site (operator) and an auxiliary operator, resulting in a DNA loop, which increases repression efficiency. Induction of the lac operon by allolactose reduces the affinity of LacI for DNA, but induction does not completely prevent looping in vivo. Our previous work on the conformations of LacI loops used a hyperstable model DNA construct, 9C14, that contains a sequence directed bend flanked by operators. Single-molecule fluorescence resonance energy transfer (SM-FRET) on a dual fluorophore-labeled LacI-9C14 loop showed that it adopts a single, stable, high-FRET V-shaped LacI conformation. Ligand-induced changes in loop geometry can affect loop stability, and the current work assesses loop population distributions for LacI-9C14 complexes containing the synthetic inducer IPTG. SM-FRET confirms that the high-FRET LacI-9C14 loop is only partially destabilized by saturating IPTG. LacI titration experiments and FRET fluctuation analysis suggest that the addition of IPTG induces loop conformational dynamics and re-equilibration between loop population distributions that include a mixture of looped states that do not exhibit high-efficiency FRET. The results show that repression by looping even at saturating IPTG should be considered in models for regulation of the operon. We propose that persistent DNA loops near the operator function biologically to accelerate rerepression upon exhaustion of inducer.

DNA looping mediated by the Lac repressor is an archetypal test case for modeling protein and DNA flexibility. Understanding looping is fundamental to quantitative descriptions of gene expression. Systematic analysis of LacI•DNA looping was carried out using a landscape of DNA constructs with lac operators bracketing an A-tract bend, produced by varying helical phasings between operators and the bend. Fluorophores positioned on either side of both operators allowed direct Forster resonance energy transfer (FRET) detection of parallel (P1) and antiparallel (A1, A2) DNA looping topologies anchored by V-shaped LacI. Combining fluorophore position variant landscapes allows calculation of the P1, A1 and A2 populations from FRET efficiencies and also reveals extended low-FRET loops proposed to form via LacI opening. The addition of isopropyl-β-d-thio-galactoside (IPTG) destabilizes but does not eliminate the loops, and IPTG does not redistribute loops among high-FRET topologies. In some cases, subsequent addition of excess LacI does not reduce FRET further, suggesting that IPTG stabilizes extended or other low-FRET loops. The data align well with rod mechanics models for the energetics of DNA looping topologies. At the peaks of the predicted energy landscape for V-shaped loops, the proposed extended loops are more stable and are observed instead, showing that future models must consider protein flexibility.

Intrinsic DNA bending or curvature is a phenomenon that has been shown to play an important role in a variety of DNA transactions. Large curvature occurs when short homopolymeric (dA.dT)4-6 runs (A-tracts) are repeated in phase with the helical screw. We have used electrophoretic mobility modulation to examine how bending depends on the nature of the 5 bp DNA sequence between the A tracts in molecules of the form (A5-6N5)n. We show that A-tract-induced DNA curvature can indeed be affected by other sequence elements, although by only about +/- 10%. The small observed curvature modulation implies that the overall helix axis deflection contributed by 5-bp B-DNA segments between A-tracts varies little from one sequence to another. This result validates, to first order, the assumption that DNA curvature results from inserting A-tracts at integral turn phasing into generic B-DNA. Therefore, if, as has been proposed, A-tracts have zero roll between the base-pairs and all curvature results from positive roll in the B-DNA segments, then this must be a general property of approximately 5 bp B-DNA sequences, not just special cases. This interpretation would require that the canonical structure of B-DNA be revised to include systematic roll between the base-pairs of about 6 degrees. Alternatively, the data are also consistent with zero average roll in the B-DNA sequences, and wedge angles dominated by negative roll in the A-tracts, or with an appropriate mixture of the two models. It is not possible to resolve this ambiguity using comparative electrophoresis or existing structural data. We show that published wedge angle parameters successfully predict the measured direction and, with appropriate rescaling, the magnitude of curvature due to a non-A-tract sequence containing the protein-free lac operator CAP protein binding site.

THE bending and flexibility of DNA are important in packaging, recombination and transcription1–3. Bending decreases electrophoretic mobility in a manner depending on bend position within a fragment (circular permutation4) and on the distance between bends (phasing analysis5,6). Bending can also affect DNA ring closure (cyclization7–10). The lack of a complete theory for the mechanism of gel retardation hampers measurement of bend magnitudes by electrophoresis, whereas cyclization is done entirely in solution and is well understood theoretically9. Disagreements between bend angles estimated by the two electrophoretic assays have been ascribed to DNA flexibility11. Here we test this interpretation using an internal loop as a model flexible locus. Whereas the circular permutation and helical phasing experiments are only subtly affected by the loop, DNA cyclization kinetics detects and quantifies substantial increases in torsional and bending flexibility. Furthermore, the results support a functional role for the stress of DNA bending in inducing base-pair opening12.

The double-helical DNA biopolymer is particularly resistant to bending and twisting deformations. This property has important implications for DNA folding in vitro and for the packaging and function of DNA in living cells. Among the outstanding questions in the field of DNA biophysics are the underlying origin of DNA stiffness and the mechanisms by which DNA stiffness is overcome within cells. Exploring these questions requires experimental methods to quantitatively measure DNA bending and twisting stiffness both in vitro and in vivo. Here, we discuss two classical approaches: T4 DNA ligase-mediated DNA cyclization kinetics and lac repressor-mediated DNA looping in Escherichia coli. We review the theoretical basis for these techniques and how each can be applied to quantitate biophysical parameters that describe the DNA polymer. We then show how we have modified these methods and applied them to quantitate how apparent DNA physical properties are altered in vitro and in vivo by sequence-nonspecific architectural DNA-binding proteins such as the E. coli HU protein and eukaryotic HMGB proteins.

The lac operon and its repressor (LacI) are the prototype model for gene regulation. LacI negatively regulates lacZYA by binding a primary DNA operator site overlapping the promoter, and repression is enhanced by secondary operators that deliver LacI via looping intervening DNA. LacI's ability to form stable loops with a variety of DNA lengths has been attributed to protein flexibility and/or to multiple loop topologies. Previously developed DNA constructs in which looping is hyperstabilized by an A-tract bend placed between two operators provide different loop shapes depending on the operator/bend helical phasing. Here, FRET is used to characterize the sequence/structure landscape of a set of related constructs with systematically varied operator/bend phasings. Donor and acceptor fluorophores positioned on either side of the operator provide multiple distance constraints on the orientations of the LacI-DNA loop. The results suggest that LacI can form many different looped states whose relative energetics can be measured. Also, IPTG addition demonstrates that inducer-bound LacI still forms stable loops, probably with different geometries relative to the repressed state. This comprehensive looping landscape should allow determination of whether protein flexibility is necessary to explain the results.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Small DNA minicircles are useful for characterizing protein-induced DNA bending and twisting, because obfuscating effects of DNA flexibility are less important than in larger DNA. Our work on DNA geometry and flexibility in protein-DNA complexes has employed T4 ligase-mediated DNA cyclization to make minicircles. Experiments can be carried out as forward ligations, or equivalently protein binding to minicircles can be characterized. In every case we have studied, topological characterization of minicircle synthesis or properties has led to unexpected geometric or mechanistic conclusions. Examples concerning the catabolite activator protein, E. coli RNA polymerase, the Lac repressor, and the TATA-box binding protein are discussed. Topological results have the experimental advantages that they are qualitatively unmistakable and internally controlled: new topoisomers are readily identified even in small amounts, and they are formed in the same reaction as relaxed products. Simulations of topoisomer distributions are quite sensitive to geometrical and flexibility parameters, which helps set stringent constraints on possible structural/dynamic models. However, the disadvantage of a topological measurement is it is consistent with any combination of writhe and twist that sums to the observed ΔLK, so it is difficult to be confident that a structural/dynamic model is a unique solution.

DNA looping is important for gene repression and activation in Escherichia coli and is necessary for some kinds of gene regulation and recombination in eukaryotes. We are interested in sequence-nonspecific architectural DNA-binding proteins that alter the apparent flexibility of DNA by producing transient bends or kinks in DNA. The bacterial heat unstable (HU) and eukaryotic high-mobility group B (HMGB) proteins fall into this category. We have exploited a sensitive genetic assay of DNA looping in living E. coli cells to explore the extent to which HMGB proteins and derivatives can complement a DNA looping defect in E. coli lacking HU protein. Here, we show that derivatives of the yeast HMGB protein Nhp6A rescue DNA looping in E. coli lacking HU, in some cases facilitating looping to a greater extent than is observed in E. coli expressing normal levels of HU protein. Nhp6A-induced changes in the DNA length-dependence of repression efficiency suggest that Nhp6A alters DNA twist in vivo. In contrast, human HMGB2-box A derivatives did not rescue looping.

We have used a self-cleaving RNA molecule related to a subsequence of plant viroids (a "hammerhead") to study the length-dependent folding of RNA produced during transcription by Escherichia coli RNA polymerase. Transcript elongation is arrested at defined positions using chain-terminating ribonucleoside triphosphate analogues (3'-deoxyNTP's or 3'-O-methylNTP's). When the transcript can form the "hammerhead" structure it self-cleaves to give a truncated product. The experiment yields an RNA sequencing ladder which terminates at the length at which cleavage becomes possible; the sequencing ladder is compared to those generated by using a noncleaving transcript or by using [alpha-thio]ATP in place of ATP. We have shown that 15-18 nucleotides (nt) of RNA past the cleavage point must be synthesized before the transcript can self-cleave within a ternary complex, whereas RNA freed from the complex by heating can cleave with only 3 or more nt present beyond the cleavage point. There are sequence-dependent as well as length-dependent effects. The results suggest that 12 +/- 1 nt are sequestered within the ternary complex and are consistent with the presence of a DNA-RNA hybrid within the transcription bubble, as proposed by others. The results indicate that the "hammerhead" structure does not disrupt the hybrid. It appears that the RNA beyond the hybrid is not restrained by interactions with the enzyme, since the last stem of the self-cleaving structure forms as soon as the RNA composing it emerges from the DNA-RNA hybrid. Self-cleaving of the transcript offers a simple structural probe for studying less well-characterized transcription complexes. The relevance of the results to models for transcription termination is discussed.

We use cryo-electron microscopy to compare 3D shapes of 158 bp long DNA minicircles that differ only in the sequence within an 18 bp block containing either a TATA box or a catabolite activator protein binding site. We present a sorting algorithm that correlates the reconstructed shapes and groups them into distinct categories. We conclude that the presence of the TATA box sequence, which is believed to be easily bent, does not significantly affect the observed shapes.

Synthetic histidine-lysine (HK) peptides, comprised of histidine and lysine, have been shown to be effective carriers of nucleic acids. We compared the transfection ability of one these HK peptides, H2K4b, in complex with plasmids prepared under three conditions: 1) water, 2) water and then Opti-MEM (or 150 mM NaCL), or 3) Opti-MEM (or 150 mM NaCL). When H2K4b in complex with a luciferase-containing plasmid complex was first formed in water before Opti-MEM was added, luciferase activity increased significantly in most cell lines compared to when the polyplex was formed in Opti-MEM; in two cell lines, this sequential change in salt (e.g., water/ then Opti-MEM) increased transfection activity by as much as five-fold. Furthermore, H2K4b: luciferase-containing plasmid complex formed only in water resulted in a marked decrease in luciferase activity compared to when the complex was formed in Opti-MEM or water/then Opti-MEM. Similarly, sequential changes in salt conditions (e.g., water/Opti-MEM) during the preparation of the H2K4b: |[beta]|-galactosidase plasmid complex augmented galactosidase expression. H2K4b polyplexes, formed first in water and then in Opti-MEM were intermediate in size (761 |[plusmn]| 292 nm) compared to polyplexes formed in water (81.1 |[plusmn]| 25.6 nm) or Opti-MEM (1729 |[plusmn]| 559nm); atomic force microscopy confirmed these findings. Nevertheless, the surface charge on the H2K4b/plasmid DNA complex changed minimally despite different conditions in which the polyplex was formed. Variation in the salt conditions during formation of the HK:DNA complexes may increasetransfection due to the size and DNA release from the polyplexes.

The Escherichia coli lac operon provides a classic paradigm for understanding regulation of gene transcription. It is now appreciated that lac promoter repression involves cooperative binding of the bidentate lac repressor tetramer to pairs of lac operators via DNA looping. We have adapted components of this system to create an artificial assay of DNA flexibility in E. coli. This approach allows for systematic study of endogenous and exogenous proteins as architectural factors that enhance apparent DNA flexibility in vivo. We show that inducer binding does not completely remove repression loops but it does alter their geometries. Deletion of the E. coli HU protein drastically destabilizes small repression loops, an effect that can be partially overcome by expression of a heterologous mammalian HMG protein. These results emphasize that the inherent torsional inflexibility of DNA restrains looping and must be modulated in vivo.

Lac repressor (LacI) binds two operator DNA sites, looping the intervening DNA. DNA molecules containing two lac operators bracketing a sequence-directed bend were previously shown to form hyperstable LacI-looped complexes. Biochemical studies suggested that orienting the operators outward relative to the bend direction (in construct 9C14) stabilizes a positively supercoiled closed form, with a V-shaped LacI, but that the most stable loop construct (11C12) is a more open form. Here, fluorescence resonance energy transfer (FRET) is measured on DNA loops, between fluorescein and TAMRA attached near the two operators, approximately 130 basepairs apart. For 9C14, efficient LacI-induced energy transfer ( approximately 74% based on donor quenching) confirms that the designed DNA shape can force the looped complex into a closed form. From enhanced acceptor emission, correcting for observed donor-dependent quenching of acceptor fluorescence, approximately 52% transfer was observed. Time-resolved FRET suggests that this complex exists in both closed- and open form populations. Less efficient transfer, approximately 10%, was detected for DNA-LacI sandwiches and 11C12-LacI, consistent with an open form loop. This demonstration of long-range FRET in large DNA loops confirms that appropriate DNA design can control loop geometry. LacI flexibility may allow it to maintain looping with other proteins bound or under different intracellular conditions.

We are interested in how asymmetric charge neutralization of DNA by proteins results in DNA bending. We have previously reported electrophoretic phasing experiments utilizing homodimer peptides derived from GCN4, a yeast basic zipper (bZIP) transcription factor. Here we report the results of experiments that examine the importance of peptide sequence context in DNA bending and test the hypothesis that peptide structural asymmetry causes electrophoretic anomalies in the absence of DNA bending. We prepared two new series of bZIP peptides that differed dramatically in overall size, structure, and peptide sequence near the DNA. The magnitude of apparent DNA bending is independent of the structure of the protein. This result reduces the concern that bZIP protein structure causes electrophoretic anomalies in the absence of DNA bending. In all cases, both the magnitude and direction of the apparent DNA bend angle are strongly dependent on the local peptide charge. We attempted to validate independently our results with a minicircle competition binding assay. Binding preferences of severalfold for properly phased circular versus linear DNA templates were predicted. However, no binding preferences were observed. We propose that the minicircle binding assay may be intrinsically insensitive to DNA bending or flexibility induced by the bZIP peptides studied, and we provide a unifying explanation for the discrepancies between the cyclization and electrophoretic experiments.

DNA looping is fundamental to transcriptional regulation, and the archetypal loop is formed by the Lac repressor protein, LacI. Thermodynamic characterization of looping energetics with and without induction is a necessary input for quantitative modeling of gene expression, and the LacI system is also a test case for statistical mechanics and rod mechanics models of protein/DNA flexibility. We have systematically constructed a landscape of LacI-DNA looping variants patterned on previously developed molecules in which lac operators bracket a sequence-directed A-tract bend. The hyperstability of these loops enables study of LacI protein geometries that would otherwise be unstable with respect to unlooped single- or double-bound DNA. FRET donors and acceptors (Alexa 555 and 647) positioned on each side of both operators allow detection of all possible looping topologies (two parallel and two antiparallel trajectories). Comparisons among these fluorophore position variants allow measurement of the population of each loop state. Finally, we have studied the effect of IPTG on loop geometry and stability. Bulk and single-molecule FRET studies show that: (1) Remarkably, all the constructs on the landscape form stable loops. (2) Parallel and antiparallel topologies have different LacI geometries detected by different FRET efficiencies, probably due to differential effects of DNA stiffness in the two contexts. (3) Some constructs form an extended loop that can be identified by loss of FRET. (4) Induction affords a mixture of the initial loop, a state with decreased FRET, and additional FRET-silent states that we tentatively ascribe to specific-nonspecific loops. (5) States on the borders of the landscape regions comprising uniform loops are maximally sensitive to IPTG. The overall results do not agree with rod mechanics models that consider only DNA bending and flexibility, confirming that protein flexibility must be considered in modeling DNA loops.

DNA ring closure experiments on short restriction fragments ( approximately 160 bp) bound by the TATA box binding protein (TBP) have demonstrated the formation of negative topoisomers, consistent with crystallographically observed TBP-induced DNA untwisting but in contrast to most previous results on topological effects in plasmid DNA. The difference may be due to the high free energy cost of substantial writhe in minicircles. A speculative mechanism for the loss of TBP-induced writhe suggests that TBP is capable of inducing DeltaTw between 0 and -0.3 in minicircles, via loss of out-of-plane bending upon retraction of intercalating Phe stirrups, and that TBP can thus act as a "supercoil shock absorber". The proposed biological relevance of these observations is that they may model the behavior of DNA in constrained chromatin environments. Irrespective of the detailed mechanism of TBP-induced supercoiling, its existence suggests that chromatin remodeling and enhanced TBP binding are thermodynamically linked. Remodeling ATPases or histone acetylases release some of the negative supercoiling previously restrained by the nucleosome. When TBP takes up the supercoiling, its binding should be enhanced transiently until the unrestrained supercoiling is removed by diffusion or topoisomerases. The effect is predicted to be independent of local remodeling-induced changes in TATA box accessibility.

DNA ring closure methods have been applied to TATA box DNA and its complex with the TATA box-binding protein (TBP). The J factors for cyclization (effective concentrations of one DNA end about the other) have been measured using cyclization kinetics, with and without bound TBP, for 18 DNA constructs containing the adenovirus major late promoter TATA box (TATAAAAG) separated by a variable helical phasing adapter from sequence-induced A-tract DNA bends. Six phasing lengths were used at three overall DNA lengths each. Cyclization kinetics were also measured in the absence of protein for the same set of molecules bearing a mutant TATA box (TACAAAAG). The results suggest that the TATA box DNA itself is strongly bent and anisotropically flexible, in a direction opposite to the bend induced by TBP, and that the mutant TACA box is much less bent/flexible. The bending and flexibility of the free DNA may govern the energetics of recognition of different DNA sequences by TBP, and the intrinsic bend may act to repress transcription complex assembly in the absence of TBP. The cyclization kinetics of TBP-DNA complexes in solution predict a geometry generally consistent with crystal structures, which show dramatic bending and unwinding. The novel observation of TBP-induced topoisomers suggests that this minicircle approach is able to distinguish TBP-induced unwinding from writhe (these cancel out in larger DNA), and this in turn suggests that changes in supercoiling in small topological domains can control TBP binding.

A Monte Carlo simulation method for studying DNA cyclization (or ring-closure) has been extended to the case of protein-induced bending, and its application to experimental data has been demonstrated. Estimates for the geometric parameters describing the DNA bend induced by the catabolite activator protein (CAP or CRP) were obtained which correctly predict experimental DNA cyclization probabilities (J factors), determined for a set of 11 150 to 166 bp DNA restriction fragments bearing A tracts phased against CAP binding sites. We find that simulation of out-of-phase molecules is difficult and time consuming, requiring the geometric parameters to be optimized individually rather than globally. A wedge angle model for DNA bending was found to make reasonable predictions for the free DNA. The bend angle in the CAP-DNA complex is estimated to be 85 to 90 degrees, in agreement with estimates from gel electrophoresis and X-ray co-crystal structures. Since the DNA is found to have a pre-existing bend of 15 degrees, the change in bend angle induced by CAP is 70 to 75 degrees, in a agreement with an estimate from topological measurements. We find evidence for slight (approximately 10 degrees) unwinding by CAP. The persistence length and helical repeat of the unbound portion of the DNA are in accord with literature-cited values, but the best-fit DNA torsional modulus C is found to be 1.7 (+/- 0.2) x 10(-19) erg. cm, versus literature estimates and best-fit values for the free DNA of 2.0 x 10(-19) to 3.4 x 10(-19) erg.com. Simulations using this low value of C predict that cyclization of molecules with out-of-phase bends proceeds via undertwisting or overtwisting of the DNA between the bends, so as to align the bends, rather than through conformations with substantial writhe. We present experiments on the topoisomers formed by cyclization with CAP which support this conclusion, and thereby rationalize the surprising result that cyclization can actually be enhanced by out-of-phase bends if the twist required to align the bends improves the torsional alignment of the ends. The relationship between the present work and previous studies on DNA bending by CAP is discussed, and recommendations are given for the efficient application of the cyclization/simulation approach to DNA bending.

Positioned nucleosomes are believed to play important roles in transcriptional regulation and for the organization of chromatin in cell nuclei. Here, we have isolated the DNA segments in the mouse genome that form the most stable nucleosomes yet characterized. In separate molecules we find phased runs of three to four adenine nucleotides, extensive CA repeats, and in a few cases phased TATA tetranucleotides. The latter forms the most stable nucleosome yet characterized. One sequence with CAG repeats was also found. By fluorescence in situ hydridization the selected sequences are shown to be localized at the centromeric regions of mouse metaphase chromosomes.

The Staphylococcus aureus plasmid pT181 possesses a DNA replication enhancer element, called cmp, that is required in cis for optimal utilization of the initiator protein by the origin of replication. The minimal nucleotide sequence required for cmp activity was defined by testing progressively smaller DNA fragments for their ability to restore cmp activity in a plasmid mutant deleted for cmp. These experiments indicate that cmp is a sequence of 100 base pairs (bp) characterized by a loosely repeated sequence motif and phased oligo(dT) tracts. Intrinsic DNA bending at cmp was detected by a circular permutation assay of the locus using polyacrylamide-gel electrophoresis and by computer modeling. The cmp element was found to contain two loci of intrinsically bent DNA that confer an overall bent conformation to this replication enhancer.

We have applied T4 ligase-mediated DNA cyclization kinetics to protein-induced bending in DNA. The presence and direction of a static bend can be inferred from J factors for cyclization of 150- to 160-base-pair minicircles, which include a catabolite activator protein binding site phased against a sequence-directed bend. We demonstrate a quasi-thermodynamic linkage between cyclization and protein binding; we find that properly phased DNAs bind catabolite activator protein approximately 200-fold more tightly as circles than as linear molecules. The results unambiguously distinguish DNA bends from isotropically flexible sites and can explain cooperative binding by proteins that need not contact each other.

Publisher Summary This chapter discusses experimental approaches to the measurement of ring closure probability ( J factor or Jacobson–Stockmayer factor) and the theory used to analyze ring closure, and thereby obtain fundamental physical information from the J factor. It illustrates these procedures with examples from the cyclization of bent DNA molecules (A tract multimers) and recent work on the application of cyclization methods to protein–DNA complexes. Cyclization kinetics is unusual among molecular biological techniques in its requirement for computation of the properties of models to interpret the experimental results in anything more than a highly qualitative way. The theory of cyclization kinetics can also be applied to other systems where DNA assumes conformations and where its ends are fixed in a defined spatial orientation. In vivo and in vitro results on lac repressor-mediated DNA loops have been interpreted using the cyclization paradigm, and the enhancement of cyclization has been used to provide evidence for looping. The chapter also highlights the analysis of topoisomer distributions established by ligation under various conditions that can provide complementary information to that available from cyclization kinetics.

The Escherichia coli LacI protein represses the lac operon by blocking transcription. Tetrameric LacI binds simultaneously to a promoter-proximal DNA operator and an auxiliary operator, and the resulting DNA loop increases the efficiency of repression. A hyperstable closed-form LacI-DNA loop was previously shown to be formed on a DNA construct (9C14) that includes a sequence-directed bend flanked by operators. Previous bulk and single molecule fluorescence resonance energy transfer (SM-FRET) experiments on dual fluorophore-labeled 9C14-LacI loops demonstrate that LacI-9C14 adopts a single, stable, rigid DNA loop conformation, despite the presence of flexible linkers in LacI. Here, we characterize the LacI-9C14 loop by SM-FRET as a function of inducer isopropyl-β,D-thiogalactoside (IPTG) concentration. Energy transfer measurements reveal partial but incomplete destabilization of loop formation by IPTG, with no change in the energy transfer efficiency of the remaining looped population.View Large Image | View Hi-Res Image | Download PowerPoint SlideModels for the regulation of the lac operon often assume complete disruption of LacI-operator complexes upon inducer binding to LacI. Our work shows that even at saturating IPTG there is still a significant population of LacI-DNA complexes in a looped state, in accord with previous in vivo experiments that show incomplete induction.

With the continued development of pulsed x‐ray sources, it may in time be possible to use transient x‐ray diffraction to follow the molecular dynamics of chemical reactions in the liquid and solid states. To explore this possibility from the theoretical side, we have calculated, using classical molecular dynamics, the picosecond time‐resolved x‐ray scattering of a simplified model for a liquid state chemical reaction of substantial interest: the photodissociation of I2 molecules in rare gas and hexane solvents. The time scale of the separation of the I atoms and the effect of the solvent on their motion are observed in the computed transient x‐ray diffraction patterns, and such effects might also be observed in a suitably designed experiment. This illustrates that transient x‐ray diffraction might be an experimental tool for discovering the molecular dynamics of chemical reactions, with the advantage over transient optical spectroscopies such as infrared, electronic, and Raman that the connections between dynamics and diffraction can be precisely computed without uncertainties due to imprecise knowledge of dipole moments, transition dipole moments, and polarizabilities.

Les ions les plus abondants correspondent aux tetrameres, hexameres ou agregats plus grands; ces derniers sont stabilises par les coordinats hydroxo