Differences in secondary structure between packaged and unpackaged single-stranded DNA of bacteriophage phi X174 determined by Raman spectroscopy: a model for phi X174 DNA packaging.

The single-stranded packaged genome (ssDNA) of bacteriophage phi X174 is shown by Raman spectroscopy to lack both the ordered phosphodiester backbone and base stacking, which are demonstrated for unpackaged, protein-free ssDNA. In solutions of moderate ionic strength, unpackaged ssDNA contains 36 +/- 7% of deoxyribosyl phosphate groups with conventional B-type backbone geometry [i.e., gauche- and trans orientations, respectively, for the 5'O-P (alpha) and 3'O-P (zeta) torsions], indicative of hairpin formation and intramolecular base pairing. Additionally, the bases of unpackaged ssDNA are extensively stacked. Estimates from Raman band hypochromic effects indicate that unpackaged ssDNA contains approximately 70% of the maximal base stacking exhibited in the linear, double-stranded, replicative form III of phi X174 DNA. Conversely, for the packaged phi X174 genome, ordered (B-type) phosphodiester groups are not present, and only 40% of the base stacking in RFIII DNA is observed. These results are interpreted as evidence that the substantial hairpin-forming potential of ssDNA is eliminated by specific and extensive ssDNA-protein interactions within the phi X174 virion. Comparison of the present results with studies of other packaged single-stranded nucleic acids suggests that proteins of the capsid shell (gpF + gpG + gpH) do not fully account for the conformational constraints imposed on ssDNA of phi X174. Accordingly, we propose a model for ssDNA packaging in which the small basic gpJ protein, which is packaged along with the genome, is involved stoichiometrically in binding to the ssDNA (approximately 90 nucleotides per subunit). The proposed gpJ-DNA interactions could prevent helical hairpin formation, restrict base stacking, and disfavor fortuitous base pairing within the capsid. The present analysis is based upon use of model nucleic acids of known conformation for calibration of the Raman intensity in the region 810-860 cm-1 in terms of specific secondary structures. The calibration curve allows quantitative determination of the percentage of ssDNA nucleotides for which the 5'O-P-O3' group is configured (g-,t) as in the B-form of DNA. The method proposed here is analogous to that employed by Thomas and Hartman (1973) for ssRNA and should be applicable to single-stranded DNA and to partially denatured forms of double- and multiple-stranded DNAs.     

The binding affinity of Ff gene 5 protein depends on the nearest-neighbor composition of the ssDNA substrate

The Ff gene 5 protein (g5p) is considered to be a nonspecific single-stranded DNA binding protein, because it binds cooperatively to and saturates the Ff bacteriophage single-stranded DNA genome and other single-stranded polynucleotides. However, the binding affinity Komega (the intrinsic binding constant times a cooperativity factor) differs by over an order of magnitude for binding to single-stranded polynucleotides such as poly[d(A)] and poly[d(C)]. A polynucleotide that is more stacked, like poly[d(A)], binds more weakly than one that is less stacked, like poly[d(C)]. To test the hypothesis that DNA base stacking, a nearest-neighbor property, is involved in the binding affinity of the Ff g5p for different DNA sequences, Komega values were determined as a function of NaCl concentration for binding to six synthetic sequences 48 nucleotides in length: dA48, dC48, d(AAC)16, d(ACC)16, d(AACC)12, and d(AAACC)9A3. The binding affinities of the protein for these sequences were indeed found to be related to the nearest-neighbor compositions of the sequences, rather than to simple base compositions. That is, the g5p binding site, which is spanned by four nucleotides, discriminates among these sequences on the basis of the relative numbers of nearest neighbors (AA, CC, and AC plus CA) in the sequence. The results support the hypothesis that the extent of base stacking/unstacking of the free, nonbound ssDNA plays an important role in the binding affinity of the Ff gene 5 protein.     

Hairpin structures in synthetic oligodeoxynucleotides: sequence effects on the duplex-to-hairpin transition

We have synthesized and examined a number of fully and partly self-complementary palindromic oligodeoxynucleotides for their ability to assume in solution a unimolecular hairpin structure. The main results obtained by a combined optical and electrophoresis investigation show that: (i) DNA folding needs not be driven by mismatched base pairings over the dyad; fully self-complementary palindromic duplexes, comprising regular (CG)n DNA fragments, possess a considerable intrinsic propensity to make intramolecular base pairings; (ii) The duplex-hairpin interconversion is, in general, a slow process independent of the length and base composition of the palindrome; (iii) The palindromic sequences energetically least favored to form hairpin structures consist of C:G base pairs around the dyad axis and of T:A blocks in the arms of the inverted repeat; (IV) The base composition of the stem strongly influences the hairpin thermal stability. For instance, the substitution of one C:G with one A:T base pair in the stem helix of d(CG)7 diminishes the stability of the hairpin by 9 degrees C. It is found that the stability of the stem helix, in hairpins of defined sequence and with the same loop length, decreases in the order alternating-CG greater than homo-CG greater than AC(GT) greater than alternating-AT, i.e. as in polynucleotides. The thermodynamic parameters for the hairpin-coil transition are reported.     

The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy

Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC–dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC–dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC–dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides.     

Elongation of tandem repetitive DNA by the DNA polymerase of the hyperthermophilic archaeon Thermococcus litoralis at a hairpin-coil transitional state: a model of amplification of a primordial simple DNA sequence

DNA is replicated by DNA polymerase semiconservatively in many organisms. Accordingly, the replicated DNA does not become larger than the original DNA (template DNA), implying that replicative synthesis by DNA polymerase alone cannot explain the diversification of primordial simple DNA. We demonstrate that a single-stranded tandem repetitive oligodeoxyribonucleic acid (oligoDNA) composed of a palindromic or quasi-palindromic motif sequence and 25-50% GC content is elongated in vitro to more than 20,000 bases at 70-74 degrees C by the DNA polymerase of the hyperthermophilic archaeon Thermococcus litoralis without a bimolecular primer-template complex. The efficiency of elongation decreased when the palindromic structure of the oligoDNA was destroyed or when the GC content of the oligoDNA was outside the range of 25-50%. The thermal melting transition profile of the oligoDNA, as observed by ultraviolet spectroscopy, exhibited a biphasic curve, reflecting a duplex-hairpin transition at 31-40 degrees C and a hairpin-coil transition at 70-77 degrees C. The optimal reaction temperature for the elongation, for instance, of oligoDNA (AGATATCT)(6) (72 degrees C) was very close to its hairpin-coil transition melting temperature (70.4 degrees C), but was markedly higher than the temperature at which duplex oligoDNA can exist stably (<35.9 degrees C). These results suggest that a hairpin-based "intramolecular primer-template structure" is formed transiently in the oligoDNA, and it is elongated by the DNA polymerase to long DNA through repeated cycles of folding and melting of the hairpin structure. We discuss the implication of this phenomenon, "hairpin elongation", from the standpoint of potential amplification of simple DNA sequences during the evolution of the genome.     

Recognition and cleavage of hairpin structures in nucleic acids by oligodeoxynucleotides.

The possibility of designing antisense oligodeoxynucleotides complementary to non-adjacent single-stranded sequences containing hairpin structures was studied using a DNA model system. The structure and stability of complexes formed by a 17mer oligonucleotide with DNA fragments containing hairpin structures was investigated by spectroscopic measurements (melting curves) and chemical reactions (osmium tetroxide reaction, copper-phenanthroline cleavage). A three-way junction was formed when the oligonucleotide was bound to both sides of the hairpin structure. When the complementary sequences of the two parts of the oligonucleotide were separated by a sequence which could not form a hairpin, the oligonucleotide exhibited a slightly weaker binding than to the hairpin-containing target. An oligodeoxynucleotide-phenanthroline conjugate was designed to form Watson-Crick base pairs with two single-stranded regions flanking a hairpin structure in a DNA fragment. In the presence of Cu2+ ions and a reducing agent, two main cleavage sites were observed at the end of the duplex structure formed by the oligonucleotide-phenanthroline conjugate with its target sequence. Competition experiments showed that both parts of the oligonucleotide must be bound in order to observe sequence-specific cleavage. Cleavage was still observed with target sequences which could not form a hairpin, provided the reaction was carried out at lower temperatures. These results show that sequence-specific recognition and modification (cleavage) can be achieved with antisense oligonucleotides which bind to non-adjacent sequences in a single-stranded nucleic acid.     

The formation of adjacent triplex-duplex domainsin DNA.

The ability of single-stranded DNA oligomers to form adjacent triplex and duplex domains with two DNA structural motifs was examined. Helix-coil transition curves and a gel mobility shift assay were used to characterize the interaction of single-stranded oligomers 12-20 nt in length with a DNA hairpin and with a DNA duplex that has a dangling end. The 12 nt on the 5'-ends of the oligomers could form a triplex structure with the 12 bp stem of the hairpin or the duplex portion of the DNA with a dangling end. The 3'-ends of the 17-20 nt strands could form Watson-Crick pairs to the five base loop of the hairpin or the dangling end of the duplex. Complexes of the hairpin DNA with the single-stranded oligomers showed two step transitions consistent with unwinding of the triplex strand followed by hairpin denaturation. Melting curve and gel competition results indicated that the complex of the hairpin and the 12 nt oligomer was more stable than the complexes involving the extended single strands. In contrast, results indicated that the extended single-stranded oligomers formed Watson-Crick base pairs with the dangling end of the duplex DNA and enhanced the stability of the adjacent triplex region.     

The effect of hydrostatic pressure on the thermal stability of DNA hairpins

DNA hairpins consist of two distinct structural domains: a double stranded stem and a single-stranded loop that connect the two strands of the stem. Previous studies of short DNA hairpins have revealed that loop and stem sequences can significantly affect the thermodynamic stability of short DNA hairpins. In this work we present the effect of hydrostatic pressure on the helix-coil transition temperature (T_M) for 11 16-base, hairpin-forming DNA oligonucleotides. All of the samples form a hairpin with a 6-base pair stem and a four-base loop. In addition, the four base pairs at the end of the stem distal from the loop are the same for every molecule. We have varied loop sequence and identity of the two duplex base pairs adjacent to the loop. Using the change in UV absorption to monitor the conformational state of the oligonucleotide the hairpin-coil transition temperature of these molecules was studied as a function of sodium ion concentration and pressure. From these data we calculated the volume change accompanying the transition. Model-dependent (van't Hoff) transition parameters such as ΔH_vH and transition volume (ΔV) were estimated from the analysis of conformational transitions. Experiments revealed that the ΔV for denaturation of these molecules range from − 2.35 to + 6.74 cm³ mol⁻¹. The expansibility (ΔΔV/ΔT) and the pressure dependence of cation release are also presented. The difference in the volume change for this transition is related to the differences in the hydration of these molecules.     

Electrical detection of the temperature induced melting transition of a DNA hairpin covalently attached to gold interdigitated microelectrodes

The temperature induced melting transition of a self-complementary DNA strand covalently attached at the 5′ end to the surface of a gold interdigitated microelectrode (GIME) was monitored in a novel, label-free, manner. The structural state of the hairpin was assessed by measuring four different electronic properties of the GIME (capacitance, impedance, dissipation factor and phase angle) as a function of temperature from 25°C to 80°C. Consistent changes in all four electronic properties of the GIME were observed over this temperature range, and attributed to the transition of the attached single-stranded DNA (ssDNA) from an intramolecular, folded hairpin structure to a melted ssDNA. The melting curve of the self-complementary single strand was also measured in solution using differential scanning calorimetry (DSC) and UV absorbance spectroscopy. Temperature dependent electronic measurements on the surface and absorbance versus temperature values measured in solution experiments were analyzed assuming a two-state process. The model analysis provided estimates of the thermodynamic transition parameters of the hairpin on the surface. Two-state analyses of optical melting data and DSC measurements provided evaluations of the thermodynamic transition parameters of the hairpin in solution. Comparison of surface and solution measurements provided quantitative evaluation of the effect of the surface on the thermodynamics of the melting transition of the DNA hairpin.     

Electrostatic effects on the stability of condensed DNA in the presence of divalent cations.

Cylindrical cell model Poisson-Boltzmann (P-B) calculations are used to evaluate the electrostatic contributions to the relative stability of various DNA conformations (A, B, C, Z, and single-stranded (ss) with charge spacings of 3.38 and 4.2 A) as a function of interhelix distance in a concentrated solution of divalent cations. The divalent ion concentration was set at 100 mM, to compare with our earlier reports of spectroscopic and calorimetric experiments, which demonstrate substantial disruption of B-DNA geometry. Monovalent cations neutralize the DNA phosphates in two ways, corresponding to different experimental situations: 1) There is no significant contribution to the ionic strength from the neutralizing cations, corresponding to DNA condensation from dilute solution and to osmotic stress experiments in which DNA segments are brought into close proximity to each other in the presence of a large excess of buffer. 2) The solution is uniformly concentrated in DNA, so that the neutralizing cations add significantly to those in the buffer at close DNA packing. In case 1), conformations with lower charge density (Z and ssDNA) have markedly lower electrostatic free energies than B-DNA as the DNA molecules approach closely, due largely to ionic entropy. If the divalent cations bind preferentially to single-stranded DNA or a distorted form of B-DNA, as is the case with transition metals, the base pairing and stacking free energies that stabilize the double helix against electrostatic denaturation may be overcome. Strong binding to the bases is favored by the high concentration of divalent cations at the DNA surface arising from the large negative surface potential; the surface concentration increases sharply as the interhelical distance decreases. In case 2), the concentration of neutralizing monovalent cations becomes very large and the electrostatic free energy difference between secondary structures becomes small as the interhelical spacing decreases. Such high ionic concentrations will be expected to modify the stability of DNA by changing water activity as well as by screening electrostatic interactions. This may be the root of the decreased thermal stability of DNA in the presence of high concentrations of magnesium ions.     

Role of the closing base pair for d(GCA) hairpin stability: free energy analysis and folding simulations

Hairpin loops belong to the most important structural motifs in folded nucleic acids. The d(GNA) sequence in DNA can form very stable trinucleotide hairpin loops depending, however, strongly on the closing base pair. Replica-exchange molecular dynamics (REMD) were employed to study hairpin folding of two DNA sequences, d(gcGCAgc) and d(cgGCAcg), with the same central loop motif but different closing base pairs starting from single-stranded structures. In both cases, conformations of the most populated conformational cluster at the lowest temperature showed close agreement with available experimental structures. For the loop sequence with the less stable G:C closing base pair, an alternative loop topology accumulated as second most populated conformational state indicating a possible loop structural heterogeneity. Comparative-free energy simulations on induced loop unfolding indicated higher stability of the loop with a C:G closing base pair by ~3 kcal mol(-1) (compared to a G:C closing base pair) in very good agreement with experiment. The comparative energetic analysis of sampled unfolded, intermediate and folded conformational states identified electrostatic and packing interactions as the main contributions to the closing base pair dependence of the d(GCA) loop stability.     

Effect of base-pair sequence on the conformations and thermally induced transitions in oligodeoxyribonucleotides containing only AT base pairs

Tm curves, CD spectra, and kinetics results of the self-complementary DNA dodecamers d(A6T6), d(A3T3A3T3), d(A2T2A2T2A2T2), d(ATATATATATAT), and d(T6A6) demonstrate that the thermal transitions of these oligomers at low salt concentration involve a hairpin intermediate. At high salt concentrations (greater than 0.1 M Na+) only a duplex to denatured-strand transition appears to occur. The temperature and salt-concentration regions of the transitions are very sequence dependent. Alternating-type AT sequences have a lower duplex stability and a greater tendency to form hairpins than sequences containing more nonalternating AT base pairs. Of the two nonalternating sequences, d(T6A6) is significantly less stable than d(A6T6). Both oligomers have CD curves that are very similar to the unusual CD spectrum of poly(dA).poly(dT). The Raman spectra of these two oligomers are also quite similar, but at low temperature, small intensity differences in two backbone modes and three nucleoside vibrations are obtained. The hairpin to duplex transition for the AT dodecamers was examined by salt-jump kinetics measurements. The transition is faster than transitions for palindromic-sequence oligomers containing terminal GC base pairs. Stopped-flow kinetics studies indicate that the transition is second order and has a relatively low activation energy. The reaction rate increases with increasing ionic strength. These results are consistent with a three-step mechanism for the hairpin to duplex reaction: (i) fraying of the hairpin oligomers' terminal base pairs, (ii) a rate-determining bimolecular step involving formation of a cruciform-type intermediate from two hairpin oligomers with open terminal base pairs, and (iii) base-pair migration and formation in the intermediate to give the duplex.     

Thermodynamics of single strand DNA base stacking

The thermodynamics of the stacking to unstacking transitions of 24 single-stranded DNA sequences (ssDNA), 10-12 bases in length, in sodium phosphate buffer were determined from 10 to 95 degrees C, using differential scanning calorimetry (DSC). An additional 22 ssDNA sequences did not exhibit an S<-->U transition in this temperature range. The transition properties of the ssDNA sequences with 相似文献   

DNA microarrays with unimolecular hairpin double-stranded DNA probes: fabrication and exploration of sequence-specific DNA/protein interactions

We have fabricated double-stranded DNA (dsDNA) microarrays containing unimolecular hairpin dsDNA probes immobilized on glass slides. The unimolecular hairpin dsDNA microarrays were manufactured by four steps: Firstly, synthesizing single-stranded DNA (ssDNA) oligonucleotides with two reverse-complementary sequences at 3' hydroxyl end and an overhang sequence at 5' amino end. Secondly, microspotting ssDNA on glutaraldehyde-derived glass slide to form ssDNA microarrays. Thirdly, annealing two reverse-complementary sequences to form hairpin primer at 3' end of immobilized ssDNA and thus to create partial-dsDNA microarray. Fourthly, enzymatically extending hairpin primer to convert partial-dsDNA microarrays into complete-dsDNA microarray. The excellent efficiency and high accuracy of the enzymatic synthesis were demonstrated by incorporation of fluorescently labeled dUTPs in Klenow extension and digestion of dsDNA microarrays with restriction endonuclease. The accessibility and specificity of the DNA-binding proteins binding to dsDNA microarrays were verified by binding Cy3-labeled NF-kappaB to dsDNA microarrays. The dsDNA microarrays have great potential to provide a high-throughput platform for investigation of sequence-specific DNA/protein interactions involved in gene expression regulation, restriction and so on.     

Sequence-Dependent Elasticity and Electrostatics of Single-Stranded DNA: Signatures of Base-Stacking

Base-stacking is a key factor in the energetics that determines nucleic acid structure. We measure the tensile response of single-stranded DNA as a function of sequence and monovalent salt concentration to examine the effects of base-stacking on the mechanical and thermodynamic properties of single-stranded DNA. By comparing the elastic response of highly stacked poly(dA) and that of a polypyrimidine sequence with minimal stacking, we find that base-stacking in poly(dA) significantly enhances the polymer’s rigidity. The unstacking transition of poly(dA) at high force reveals that the intrinsic electrostatic tension on the molecule varies significantly more weakly on salt concentration than mean-field predictions. Further, we provide a model-independent estimate of the free energy difference between stacked poly(dA) and unstacked polypyrimidine, finding it to be ∼−0.25 k_BT/base and nearly constant over three orders of magnitude in salt concentration.     

Sequence-Dependent Elasticity and Electrostatics of Single-Stranded DNA: Signatures of Base-Stacking

Base-stacking is a key factor in the energetics that determines nucleic acid structure. We measure the tensile response of single-stranded DNA as a function of sequence and monovalent salt concentration to examine the effects of base-stacking on the mechanical and thermodynamic properties of single-stranded DNA. By comparing the elastic response of highly stacked poly(dA) and that of a polypyrimidine sequence with minimal stacking, we find that base-stacking in poly(dA) significantly enhances the polymer’s rigidity. The unstacking transition of poly(dA) at high force reveals that the intrinsic electrostatic tension on the molecule varies significantly more weakly on salt concentration than mean-field predictions. Further, we provide a model-independent estimate of the free energy difference between stacked poly(dA) and unstacked polypyrimidine, finding it to be ∼−0.25 k_BT/base and nearly constant over three orders of magnitude in salt concentration.     

Fluorescent Determination of Double-stranded DNA with an Anionic 1,1′-Azonaphthalene Derivative

Palatine chrome black 6BN (PCB6BN) is virtually non-fluorescent in an aqueous solution or in the presence of single-stranded DNA (ssDNA), whereas the fluorescence intensity of PCB6BN was linearly enhanced up to 300 μM of double-stranded DNA (dsDNA) base pairs. PCB6BN could be a useful fluorescent probe for quantifying dsDNA even when ssDNA is present for both heterogeneous and homogeneous assays.     

The spatial configuration of ordered polynucleotide chains. II. The poly(rA) helix.

Approximate details of the spatial configuration of the ordered single-stranded poly(rA) molecule in dilute solution have been obtained in a combined theoretical analysis of base stacking and chain flexibility. Only those regularly repeating structures which fulfill the criterion of conformational flexibility (based upon all available experimental and theoretical evidence of preferred bond rotations) and which also exhibit the right-handed base stacking pattern observed in nmr investigations of poly(rA) are deemed suitable single-stranded helices. In addition, the helical geometry of the stacked structures is required to be consistent with the experimentally observed dimensions of both completely ordered and partially ordered poly(rA) chains. Only a single category of poly(rA) helices (very similar in all conformational details to the individual chains of the poly(rA) double-stranded X-ray structure) is thus obtained. Other conformationally feasible polynucleotide helices characterized simply by a parallel and overlapping base stacking arrangement are also discussed.     

Influence of base sequence on the stability of the double helix of DNA.

On the basis of published measurements of the melting transitions of synthetic polydeoxyribonucleotides with known sequences we have determined the parameters of the interplane (stacking) interactions of base pairs in DNA over the range of ionic strengths from 0.01 to 0.1 M Na+. We found that deviations of the stacking-interaction energy from the mean value of 7-8 kcal/mole were extremely small and did not exceed 0.2 kcal/mole. We report an analysis of the influence of the heterogeneity of the stacking interactions on the melting parameters of polynucleotides with random sequences (models of natural DNA's). Inclusion of this effect does not significantly distort the linear dependence of the melting temperature on the relative content of G-C pairs and insignificantly affects the width of the helix-coil transition in DNA under normal conditions. However it is the heterogeneity of the stacking interactions that plays the crucial role in the melting of DNA under conditions where the difference between the relative stabilities of the A-T and G-C pairs tends to zero, as in concentrated solutions of tetraethylammonium and tetramethylammonium salts.