What aspects of DNAs structure contribute to the stability of the molecule?

Nucleic Acids Res. 2006; 34(two): 564–574.

Base-stacking and base-pairing contributions into thermal stability of the DNA double helix

Received 2005 October 28; Revised 2006 Jan four; Accustomed 2006 Jan 4.

Supplementary Materials: [Supplementary Material]

GUID: F1938898-9D66-45C8-A70A-D1140875FB35

GUID: 1E16E7DF-A0E1-4EA9-A76C-C19F46258232

Abstract

Two factors are mainly responsible for the stability of the DNA double helix: base of operations pairing between complementary strands and stacking between adjacent bases. By studying Deoxyribonucleic acid molecules with solitary nicks and gaps we measure temperature and salt dependence of the stacking energy of the Deoxyribonucleic acid double helix. For the beginning time, Dna stacking parameters are obtained directly (without extrapolation) for temperatures from below room temperature to close to melting temperature. We also obtain DNA stacking parameters for different salt concentrations ranging from fifteen to 100 mM Na⁺. From stacking parameters of individual contacts, we calculate base-stacking contribution to the stability of A•T- and G•C-containing Dna polymers. We observe that temperature and table salt dependences of the stacking term fully determine the temperature and the common salt dependence of Deoxyribonucleic acid stability parameters. For all temperatures and salt concentrations employed in present study, base-stacking is the main stabilizing cistron in the Dna double helix. A•T pairing is e'er destabilizing and G•C pairing contributes almost no stabilization. Base-stacking interaction dominates not only in the duplex overall stability but also significantly contributes into the dependence of the duplex stability on its sequence.

INTRODUCTION

Stability of the DNA double helix with respect to separation of complementary strands is known to depend on the base limerick of the duplex (1–4). A classical study of Marmur and Doty (1) on Dna polymer stability gives a linear relationship betwixt the G•C content of the polymer and its melting temperature. The simplest explanation of the linear dependence is that A•T- and Grand•C-pairs differ in stability independently of their neighbors. Accordingly, base-stacking interactions accept been thought to found merely a pocket-size correction to the major effect of the differences in G•C and A•T pair stabilities (5). Nearest-neighbor stability parameters take been introduced to account for sequence effects in Dna stability. These parameters are obtained from the analysis of the melting data for DNA polymers (5), DNA oligomers (6–eight) and Deoxyribonucleic acid dumbbells (ix,10), and present the cumulative (base pairing and stacking) contribution of each dinucleotide stack to the overall stability of the molecule. In fact, DNA melting experiments practice not permit separation of the two contributions.

Partition of base pairing and stacking contributions to DNA stability not simply delivers a new aspect in the fundamental understanding of Deoxyribonucleic acid construction and energetics, but also information technology has significant implications in a number of biological processes. Fluctuations in local helical conformation of DNA, the miracle known as DNA breathing, pb to exceptional events of base pair opening thus making commonly buried groups available for modification and interaction with proteins (11,12). Fluctuational base of operations pair opening implies disruption of hydrogen bonds between the complementary bases and flipping the base out of the helical stack disrupting ii contacts. Heterogeneous stacking at these contacts make up one's mind sequence dependence of the base pair fluctuational movement. Moreover, single-stranded break (a nick) in the DNA double helix is stabilized by stacking interactions betwixt base pairs flanking the lesion; these interactions are sequence-dependent (13). In the jail cell, DNA nicks are substrates for Dna damage-detecting and Deoxyribonucleic acid-repair proteins (fourteen–17). In vitro, Deoxyribonucleic acid nick-based approaches are often used in Deoxyribonucleic acid detection and amplification protocols (18,19). Also, stabilization achieved through coaxial stacking interactions has been put into result to improve the efficiency of short primer hybridization for standard sequencing protocols (20) and within the format of sequencing by hybridization arroyo (21). By using partially double-stranded probe with a 5 nt overhang, the latter method takes advantage of the enhancement of the stability of the brusk duplex formed between the probe overhang and single-stranded target past coaxial stacking with the duplex interface of the probe. Another biotechnology application involving nicked DNA is the germination of padlock probes via ligation of a nick formed past two termini of bogus oligonucleotide hybridized to single-stranded or locally opened duplex Deoxyribonucleic acid (22–24).

A number of theoretical studies addressed label of stacking and base pairing contributions to the duplex stability (25–30). Experimentally, energetics of base of operations-stacking interactions in nucleic acids has been evaluated by studying the upshot of dangling (unpaired) terminal bases on the overall stability of duplexes (31–35) and in the coaxial stacking hybridization experiments where binding of a short oligonucleotide to the single-stranded Deoxyribonucleic acid template is assisted past stacking interaction with the nearby duplex interface (36–41). Both of these approaches rely on thermal denaturation measurements of brusk duplex molecules.

We accept recently introduced an entirely new approach for characterization of stacking interactions in the DNA double helix (13). For this purpose nosotros study Deoxyribonucleic acid molecules with lone nicks positioned in a strictly divers sequence context. We bailiwick these molecules to Page. Structurally, introduction of a suspension to one of the strands of DNA perturbs just slightly the double helix conformation with stacking and base of operations pairing at the nick site remaining intact (42–49). Nicked molecules take been shown to move somewhat slower during Page than intact molecules of the same size; this retardation is enhanced at college temperatures (14,50,51).

The model of the DNA nick underlying our approach involves an equilibrium between the two conformations as shown in Figure 1a (xiii). One conformation is very close to that of the intact double helix where stacking between the base of operations pairs flanking the nick is conserved. We assume that no optimization of stacking interactions—similar in case of duplexes with dangling nucleotides (52)—occurs at the site of the nick in DNA. The other conformation corresponds to consummate loss of stacking at the nick site thus inducing a kink in DNA. The fast equilibration between stacked/straight and unstacked/bent conformations of the nick direct affects the mobility of DNA molecule during Page leading to a differential retardation characteristic to a particular dinucleotide carrying the nick, i.e. KL. For each contact, stacking free energy parameter, $Δ G_{KL}^{ST}$ , is ascribed governing the distribution of the molecules betwixt the two conformations. Using Page mobility information of DNA molecules with solitary nicks flanked by all possible combinations of the base pairs nosotros extract stacking parameters of the DNA double helix (13). Using heterogeneous stacking parameters and a well-known Marmur–Doty (1) dependence of Dna stability on its content, nosotros have previously arrived at a revised set of the nearest-neighbor stability parameters which is in total agreement with the Deoxyribonucleic acid melting data (thirteen).

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Urea-enhanced gel electrophoresis of nicked and gapped Dna. (a) Nicked DNA molecule undergoes stacked-unstacked transition governed by stacking parameter $Δ G_{KL}^{ST}$ . Stacked country is close to intact molecule; unstacked state is approximated by a molecule with a gap. (b) Dna molecules with solitary nicks and gaps in forward (F) or reverse (R) strand were obtained past enzymatic digestion. Nicking enzymes and their recognition sites are shown in one color; cleavage sites are indicated by arrowheads. In KL/F and KL/R fragments nick is positioned within KL/Chiliad′L′ dinucleotide stack. Fragments with 2 nt-long gaps, G2/F and G2/R, are the result of sequential digestion by ii nicking enzymes as shown. (c) Effect of temperature of PAGE on the separation of intact (I), nicked (N) and gapped (1000) DNA fragments. Nicked stacks are shown at the top of each lane; arrowheads signal to the location of the nick. Gapped and nicked fragments resolved in i lane have lesions in the same strand. PAGE was conducted in the presence of 2.3 M urea at temperatures indicated to the left of each console.

Hither we measure out, for the first time, temperature dependence and salt dependence of base of operations-stacking contribution to the Deoxyribonucleic acid duplex stability. Contributions of A•T and G•C pairing are estimated from the comparison of A•T- and Grand•C-containing polymer stability parameters with the stacking terms. We find that throughout the temperature range employed, base-stacking interactions stabilize DNA double helix. Temperature dependence of the base-stacking term fully determines the temperature dependence of the Dna stability parameter. Base pairing term is destabilizing in case of A•T and somewhat stabilizing in case of G•C pairs. Differential contribution of base-stacking in A•T- and M•C-containing contacts is responsible for 50% of the dependence of Dna stability on its G•C content. Salt weather affect stacking parameters leading to stabilization upon an increase in sodium concentration. In this case, nosotros as well discover that salt dependence of stacking term determines the salt dependence of the DNA stability term.

MATERIALS AND METHODS

DNA

The 300 bp-long DNA molecules used in this study are PvuII/PvuII (all enzymes used in this study were purchased from New England Biolabs, Ipswich MA) restriction fragments of pUC19 derivatives described before (13). Briefly, pKL (were K and L are DNA bases) carries recognition sites for nicking endonucleases each of which introduces a single nick to the forrard (N.BstNB I) or reverse (Due north.Alw I) strand of KL/Yard′L′ dinucleotide stack (or just KL; K′ and 50′ are complementary to K and 50) yielding KL/F and KL/R fragments, respectively (Effigy 1b). The nick is located 104 bp apart from the terminus of PvuII/PvuII fragment. Molecules with a single 2 nt gap in place of a nick are obtained by digesting pG2F and pG2R by N.BbvC IA followed by Northward.BstNBI digestion. As shown in Figure 1b, nicking sites of these nicking endonucleases are ii nt apart and located on the frontwards strand of G2/F and reverse strand of G2/R. To remove 5′ phosphoryl groups, nicked and gapped molecules were incubated with Alkaline metal Phosphatase (CIP). Following treatment with enzymes, all DNA samples were purified past phenol extraction, precipitated with ethanol and resuspended in TE buffer [x mM Tris (pH 7.4) and one mM EDTA].

Gel electrophoresis

Nicked, gapped and intact Dna fragments 300 bp in length were meantime subjected to electrophoresis through 6.seven% (w/five) polyacrylamide gels run at 17 5/cm with one× TBE [ninety mM Tris (pH 8.0), 90 mM boric acid and i mM EDTA] every bit a running buffer. We accept shown before that under this set of PAGE weather stacked-unstacked equilibrium at the nick site of DNA is not affected past specific experimental design (13). Specifically, Figure five of (13) demonstrates that the presence of the gel matrix and force practical to the molecule during Page practise non touch on apparent complimentary free energy parameters of stacked-unstacked equilibrium at the DNA nick. We also conducted Page in the presence of increasing concentrations of sodium by adding NaCl up to 85 mM to the gel and to the running buffer; the forcefulness of electrical field for these experiments was reduced to 12 V/cm. The gel electrophoresis was performed using Protean II xi Cell (BioRad, Hercules, CA) where the gel-plate sandwich is mounted against the temperature-controlling core continued to a circulating water bath set to a desired temperature. The temperature during PAGE was measured by a thermocouple (Oakton, Vernon Hills, IL) with a type T wire probe inserted directly into the gel. DNA band patterns were visualized past ethidium bromide staining and detected by the CCD camera.

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Effect of phosphorylation state of 5′ nt at the nick site on stacked-unstacked equilibrium. (a) Stacking parameters measured in 1× TBE at 37°C of A•T-containing (elevation panel) and G•C-containing (lesser console) nicked dinucleotide stacks before (circles) and after (triangles) dephosphorylation are compared. White and gray fills are used for fragments with the nick in the forwards and opposite strand, respectively. Nicked dinucleotide stacks with a purine at a 5′-side of the nick are underscored (see text). (b) Common salt dependence of ΔG ^ST values of nicked contacts indicated to the right of each console earlier (circles) and after (triangles) dephosphorylation. Total concentration of sodium assuming 1× TBE to be equivalent to 15 mM Na⁺ is indicated, see Figure iv.

Relative mobilities of Dna fragments with a nick, μ, were calculated as a ratio of the distance migrated by the nicked fragments to the distance migrated by the intact fragment during Folio. All experiments were conducted at least in triplicates. To enhance the separation of the molecules with lonely nicks from the intact molecules we added diverse concentrations of urea (0.4 to 8 Yard) to the gel thus assuasive subsequent extrapolation of measured parameters to zero urea (xiii). We also conducted PAGE in the presence of dimethylformamide (DMF) in place of urea. In this case, a smaller range of concentrations was used since the gels became fragile at concentrations of DMF above 1.five G.

Melting experiments

Two constructed 65 nt-long complementary Dna strands (MWG Biotech Inc., High Point, NC) were annealed in TE buffer appended with a desired concentration of NaCl (from 20 to 200 mM) at 0.5 µM of each strand. The same duplex was also prepared in the i×TBE buffer. The purity of the duplex was checked by PAGE. Melting curves of Deoxyribonucleic acid duplexes were collected using a CARY Bio100 spectrophotometer equipped with the peltier thermocontroller (Varian Instruments, Walnut Creek, CA). Both denaturation and renaturation curves were collected at slow heating/cooling rate of at 0.08 °/min; no hysteresis betwixt heating and cooling curves has been observed. Melting temperature, T _G, was estimated equally a temperature at the midpoint of the duplex-to-curlicue transition.

In melting studies we determined stabilities of iv Deoxyribonucleic acid duplexes, which differed in G•C content, χ (DNA sequences are listed in Supplementary Tabular array i). Common salt dependence of A•T-containing duplex stability (χ = 0%) was measured directly for 22 mM < [Na⁺] < 202 mM (i.e. 20–200 mM NaCl plus 1 mM EDTA). Salt dependence of more than stable G•C-containing duplex was obtained by extrapolating T _M's measured for four duplexes with χ = 0%, χ = fourteen%, 32% and 41.5% to χ = 100%. Corresponding plots are shown in Figure 4a.

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Stability of DNA under different table salt weather. (a) Dependence of T _M of 65 bp-long DNA duplexes on their Thousand•C content in 1× TBE (triangles, dashed line) and in 10 mM TE (pH 7.4) (circles, solid lines) appended with NaCl at 20 mM (line one), 50 mM (line 2), 100 mM (line 3) and 200 mM (line 4). Linear extrapolation to χ = 100% is used to estimated T _M of M•C-containing Deoxyribonucleic acid. (b) Stability of A•T-containing (open symbols) and G•C-containing (closed symbols) 65 bp-long duplex Dna every bit a function of sodium concentration (circles) and in one×TBE (triangles). T _M's of A•T-containing DNA were measured directly; T _{One thousand}'due south of M•C-containing DNA were estimated by extrapolation shown in (a). For both duplexes, stability in one× TBE corresponds to the stability in the presence of fifteen mM Na⁺ (as plotted).

RESULTS

Stacked-unstacked equilibrium at Dna nick in the presence of a denaturant

During gel electrophoresis, a 300 bp-long Deoxyribonucleic acid molecule with a alone nick migrates slower with respect to the Dna fragment without the lesion (Figure 1c). Retardation of nicked fragments depends on the identity of the base of operations pairs flanking the nick like in case of the molecules with nicks flanked by Equally and Ts shown in Effigy 1c. Differential retardation of nicked Deoxyribonucleic acid is due to specific interactions characteristic to each nicked dinucleotide stack. Quantitatively, equilibrium between stacked/closed and unstacked/open conformations at the nick site is governed by stacking free energy, ΔK ^ST, so that:

$\frac{N_{closed}}{{Due north}_{open}} = exp (- \frac{Δ G^{ST}}{R T}),$

where N _airtight and N _{open up} are occupancies of stacked and unstacked conformations at the Dna nick, respectively, R is the universal gas abiding and T is the accented temperature.

At a given temperature, due to fast equilibration between the two states, mobility of nicked Dna is a weighted average of the mobility of molecules in closed state and mobility of molecules in open up state (xiii). Since stacked conformation of the Dna nick is very close to the DNA molecule without the lesion μ_airtight is given by the mobility of the intact Deoxyribonucleic acid fragment. The presence of a single-stranded stretch in a Deoxyribonucleic acid molecule with a short gap prevents stacking interactions betwixt the base pairs flanking the gap thus forcing the molecule into the open state. We use the mobility of molecules with a single 2 nt-long gap to gauge μ_open. Thus stacking parameter ΔK ^ST describing stacked-unstacked equilibrium at a nick site can exist calculated directly from the mobility data using Equations one and two (13):

$\frac{{Northward}_{airtight}}{{Due north}_{open}} = \frac{μ - μ_{open}}{μ_{closed} - μ}$

Since the perturbations brought nearly by the nick are often minor we introduce urea to the gel in order to heighten the separation of nicked molecules from intact molecules (Figure 2c) (13,51). Data obtained in urea-enhanced PAGE is then extrapolated to zero urea concentration resulting in stacking parameters for each nicked dinucleotide stack in the absence of urea, $Δ {Yard}_{KL}^{ST}$ (Figure 2d). The apply of denaturant during Folio is an imperative office of our experimental arroyo since it allows characterization of even subtle differences in nicked molecule mobilities. To ensure that the nature of the denaturant does not affect the resulting stacking parameters we also conducted Page in the presence of varying concentrations of DMF (Figure 2nd). Additionally, for the least stable stacks, we can measure stacking parameters directly at zero urea without extrapolating the mobility information collected in the presence of denaturant; this parameter is also plotted in Figure 2d. DMF-enhanced and urea-enhanced Page yield quantitatively similar dependencies of apparent stacking parameters on the concentration of the denaturant. This finding is quite unexpected and is nigh likely due to a narrow range of DMF concentrations used in Page; an increase in DMF concentration could yield more significant differences in apparent $Δ G_{KL}^{ST}$ values measured in the presence of the 2 denaturants. Nosotros are unable to behave these experiments since the gels containing more than than ane.5 1000 DMF are too fragile to handle. Extrapolation of the information obtained in DMF-enhanced and urea-enhanced PAGE experiments to cypher denaturant concentration is consistent with $Δ G_{KL}^{ST}$ values measured directly in the absence of denaturant.

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Event of ambient atmospheric condition on PAGE of nicked (open circles) and gapped (airtight circles) molecules. Relative mobilities of AA/F and G2/F fragments are measured (a) at different temperatures, PAGE weather: one× TBE, 2.3 K urea; (b) equally a part of ionic strength, Page atmospheric condition: 37°C, 3.5 M urea, 1× TBE appended with NaCl (total concentration of sodium assuming 1× TBE to be equivalent to 15 mM Na⁺ is indicated, run into Figure 4); (c) in the presence of urea, Folio conditions: 37°C, 1× TBE. (d) Effect of denaturant concentration on stacking parameters of AA/F calculated from mobility data using Equations one and ii. PAGE was carried out in 1× TBE at 42°C in the presence of varying concentrations of urea (closed diamonds), DMF (open diamonds) or in the absence of the denaturant (dotted diamond).

Event of temperature on stacked-unstacked equilibrium

Our farther experiments involve conducting urea-enhanced PAGE at different temperatures to obtain temperature dependence of DNA stacking parameters. Molecules with solitary nicks flanked by A•T pairs shown in Effigy 1c, display a defined dependence of the electrophoretic shift towards the molecules with a ii nt gap when temperature increases from 22 to 52°C. Relative electrophoretic mobilities of nicked AA/F fragment and gapped G2/F fragment are plotted every bit a role of temperature in Figure 2a. A thirty degrees increase in temperature leads to gradually decreasing Folio mobility of the nicked fragment while mobility of the gapped fragment remains largely unaffected. In terms of occupancies of the 2 states of the nicked site, the shift in mobility is equivalent to the shift in the equilibrium towards the open conformation of the nicked stack. Similar results were obtained for the K•C-containing contacts. More narrow temperature range has been employed in this case since molecules with nicks located in more stable G•C-containing stacks do not resolve well enough from intact molecules at temperatures under xxx°C.

Stacking free energies were calculated from the mobility data for all four A•T- and four G•C-containing nicked stacks using Equations 1 and ii. For each stack, molecules with solitary nicks located on the forwards strand, KL/F and on the reverse strand, KL/R, were examined. Our farther analysis is grounded on the assumption that the stacking free energies of the stacked-unstacked equilibrium at the site of the Deoxyribonucleic acid nick are close to the stacking parameters of the intact Dna double helix (thirteen). In this example we calculate the $Δ G_{KL}^{ST}$ by averaging (i) the parameters obtained for KL/F and KL/R and (2) the parameters obtained for the stacks equivalent due to dyad symmetry of the double helix (in our example there are ii pairs of stacks: AA/TT and TT/AA pair and GG/CC and CC/GG pair). Individual stacking parameters are listed in Supplementary Tabular array ii and are plotted every bit a role of temperature in Figure 3a. Fault bars on this plot stand for to the besprinkle of individual stacking parameters around the hateful. Equally expected, throughout the temperature range employed, $Δ G_{KL}^{ST}$ values for both A•T- and G•C-containing contacts increase (go less stable) gradually with increasing temperature.

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Effect of ambient conditions—temperature (a) and ionic force (b)—on DNA stacking parameters for A•T- and G•C-containing contacts. Dinucleotide stacks are shown at the top of each panel. Error bars represent the scatter range of the experimentally determined $Δ G_{KL}^{ST}$ values of nicked stacks (see text). This data is tabulated in Supplementary Tables 2 and 3.

Effect of table salt concentration on stacked-unstacked equilibrium

To address the effect of ionic force on stacked-unstacked equilibrium at the site of DNA nick we conducted urea-enhanced PAGE at dissimilar salt concentrations by adding NaCl to the gel and to the running buffer. Table salt dependence of relative PAGE mobilities of AA/F nicked fragment and corresponding G2/F gapped fragment is shown in Figure 2b. Unlike in case of increasing temperature (Figure 2a), contrary tendencies are displayed past the two molecules with increasing table salt concentration. Mobility of the fragment with a gap decreases. The decrease is likely due to more efficient screening of phosphate charges reducing electrostatic repulsion between the two double-stranded segments on either side of the gap thus making the gap more flexible (53). Like effect of the salt concentration is expected at the nick site in open conformation. Mobility of the nicked fragment increases corresponding to the salt-assisted shift towards stacked state of the nick.

To facilitate the comparison of Dna stacking parameters we determine experimentally, with the DNA stability parameters we have to business relationship for the difference in salt weather. Our PAGE-based experiments were conducted in a standard gel electrophoresis buffer 1× TBE. In order to detect concentration of NaCl equivalent to Page conditions with respect to Deoxyribonucleic acid stability, we conducted melting experiments, i.east. we determined T _Thou of duplex Dna in 1× TBE and in the presence of diverse concentrations of NaCl (Figure 4). For A•T-containing DNA, melting temperatures under different salt conditions were determined direct. A Deoxyribonucleic acid molecule consisting just of guanines and cytosines is much more than stable and melts around or in a higher place 100°C depending on the concentration of NaCl. Melting temperatures of G•C-containing Deoxyribonucleic acid were obtained past extrapolating T _M's measured for four Dna duplexes with dissimilar G•C-contents (χ = 0, xiv, 32 and 41.5%) to χ = 100% (Effigy 4a). Thermal stabilities of A•T- and G•C-containing Deoxyribonucleic acid duplexes in the presence of NaCl and in i× TBE are compared in Figure 4b. Melting temperatures of both molecules in ane× TBE correspond to the T _M of these molecules in the presence of fifteen mM [Na⁺].

We studied eight A•T-containing and 8 G•C-containing nicked dinucleotide stacks. Like in the experiments on the temperature dependence of stacking parameters described in a higher place, for each KL contact we examined KL/F and KL/R molecules and the stacking parameter was obtained as their average in case of unique contacts (AT, TA, GC and CG). In case of stacks with dyad symmetry, nosotros too average $Δ G_{KL}^{ST}$ values of equivalent stacks. Private stacking parameters are plotted in Figure 3b and listed in Supplementary Table iii. Note that all $Δ {Chiliad}_{KL}^{ST}$ values are plotted as a function of the 'effective' concentration of sodium, i.e. the fist signal at [Na⁺] = 15 mM corresponds to the experiment conducted in ane× TBE, the final betoken at [Na⁺] = 100 mM corresponds to 1× TBE appended with 85 mM of NaCl. All $Δ G_{KL}^{ST}$ values become more negative gradually every bit concentration of sodium increases.

Effect of phosphate at the nick site on stacked-unstacked equilibrium

Site-specific nicks in KL/F and KL/R fragments were introduced enzymatically. Nicking enzymes, like whatsoever brake enzymes, cleave DNA strand producing a 5′ phosphate and a iii′ hydroxyl termini, which translate into two negative charges at the nick site of the double helix. To gauge the effect of the phosphate negative charges on the stacked-unstacked equilibrium at the Deoxyribonucleic acid nick we considered Dna molecules where the phosphate has been removed from the nick site. Dephosphorylated nicks carry no charge.

The molecules with dephosphorylated nicks and gaps were studied by urea-enhanced Page in the aforementioned manner equally the molecules with phosphates. Equally expected, treatment of DNA fragments with Alkali metal Phosphatase, which results in the removal of terminal phosphates as well as dephosphorylation of the gap, had no upshot on Folio mobility of intact and gapped fragments (data not shown). ΔG ^ST values obtained for molecules with nicked stacks before and after dephosphorylation are compared in Figure 5a. A defined trend is observed for both A•T- and G•C-containing contacts—at that place is a significant ∼0.4–0.6 kcal/mol shift towards more than negative energies upon removal of a phosphate from the nick site only if phosphorylated v′ nt is a purine nucleotide, like in example of AA/F and TT/R, but not in case of AA/R or TT/F. When the phosphate is located on a pyrimidine nucleotide, its removal has no effect on ΔG ^ST values. This trend holds for 15 out of 16 nicked fragments shown in Effigy 5a; the only exception is GC/F. The origin of such various behavior cannot be addressed past our approach and information technology deserves a separate detailed study. We tin can simply suppose that removal of the phosphate from purine nucleotides allows rearrangement—in a sequence-independent manner—of the local structure benefiting stacked conformation. Alternatively, differential effect of phosphate positioning on purines or pyrimidines might be entropic in origin due to larger conformational flexibility of purine nucleotides without 5′ phosphates (54).

Notation that ΔThousand ^ST values of nicked stacks with and without phosphate display like dependences on salt concentration (Figure 5b). For nicks flanked by A•T pairs, this is the case for AA/R, TT/F, AT/F and AT/R fragments which brandish no change in ΔChiliad ^ST values upon removal of the phosphate from 5′-T nt and for AA/F, TT/R, TA/F and TA/R fragments which shift towards more negative ΔG ^ST values upon dephosphorylation of 5′-A nt. This consequence indicates that the phosphate charge at the nick does not contribute to the salt dependence of the measured stacking component; differences seen in Figure 5a between the nicked stacks with or without phosphate on 5′-purine nucleotides are not due to electrostatic interactions. In our further analysis we consider but molecules carrying phosphates at the nicks.

DISCUSSION

The nature of the free free energy parameters governing stacked-unstacked equilibrium at DNA nick

We obtain stacking free energy parameters by studying the equilibrium between stacked and unstacked class of DNA nick schematically presented in Figure 1a. The Dna molecule in the stacked state is very close the molecule without the lesion (42–49). Strictly speaking, the conformation of the unstacked land is unknown and our analysis is based on the assumption that unstacked land is close to the molecule with a brusk gap in identify of a nick. Indeed, presence of a unmarried-stranded gap precludes stacking interactions between 2 helical interfaces. Note, that this is non the case for ane nt gaps—it appears that stacking betwixt the base of operations pairs flanking the gap is restored to some degree leading to anisotropic, directional angle of the molecule reducing the size of the gapped crenel (48,55). Molecules with longer gaps however, have been shown to possess isotropic angle flexibility which manifests itself in the absence of helical periodicity in electrophoretic mobility and cyclization kinetics measurements (fifty,53). Molecules with gaps two, 3 and 4 nt in length drift very closely during Page revealing similarity of their effective conformations (13,50). Additional factors [i.e. sequence of single-stranded linker (56)] come into effect in one case the gap size is longer than persistent length of unmarried chains—in this case, the gap is probable to act as a hinge.

Thus, we use molecules with ii nt-long gaps positioned in forward or reverse strand to estimate the open state of the nick; the site of the gap is thought of every bit a bespeak of increased isotropic bending flexibility, which leads to retardation of the molecule during PAGE (57). Perturbations of DNA fragment at the gap sites are largely unaffected by ambient atmospheric condition—temperature, salt and denaturant concentration (Figure 2a–c).

Nicked Deoxyribonucleic acid undergoes transition between the stacked (closed or intact-like) and unstacked (open or gapped-similar) states (13). Note, that like in instance of brusque gaps, perturbation at the nick site has been shown to be isotropic with no preferential directionality (50,58). The partitioning of molecules between these conformations determines the electrophoretic mobility of the nicked fragment and is the basis for measuring stacking parameters using Equations one and 2.

For further analysis nosotros assume that the parameters describing the equilibrium at the nick site of DNA that we measure are in fact the aforementioned stacking parameters that stabilize intact double helix and determine the commencement term on the right-hand side of Equation 3:

$Δ {Thou}_{KL} = Δ G_{KL}^{ST} + \frac{1}{ii} Δ {Thou}_{1000}^{BP} + \frac{one}{ii} Δ G_{L}^{BP},$

where ΔG _KL is the total melting free energy parameter (per 1 base of operations pair) of a long Deoxyribonucleic acid molecule (neglecting the end effects) and ΔG ^BP terms are the gratuitous energy contributions due to pairing betwixt complementary bases (5). In that location are 2 ΔG ^BP parameters $Δ G_{A • T}^{BP}$ and $Δ {One thousand}_{G • C}^{BP}$ for A•T and Thousand•C pairs, respectively.

How accurate is the above supposition? Experimentally, the assumption proves to stand a critical test when we compare the $Δ {Yard}_{KL}^{ST}$ values obtained for KL/F and KL/R series: these values indeed are very shut [see Figure 4b in ref. (13)]. The $Δ G_{KL}^{ST}$ values for dinucleotide stacks equivalent due to dyad axis of symmetry of the double helix (e.g. AA/TT and TT/AA) are shut as well.

Let u.s. examine the stacked-unstacked equilibrium at the nick of the dinucleotide stack and compare information technology to the helix-to-coil transition of a DNA doublet. The schematic in Figure 6 depicts these two transitions. Hydrogen bonding between complementary bases and stacking of pairs forth the helical centrality are disrupted upon melting; duplex melting to single strands is accompanied by the gain in conformational entropy, release of counterions and change in interactions with the solvent. Strictly speaking, segmentation of these furnishings betwixt ΔG ^ST and ΔG ^BP terms is not known. For example, base of operations pairing terms in Equation 3 include breakage of hydrogen bonds between bases and formation of hydrogen bonds with h2o molecules rearranging the water structure around Dna bondage. As well, base-stacking interactions have electrostatic and hydrophobic components.

An external file that holds a picture, illustration, etc. Object name is gkj454f6.jpg

Overall stability, stacking and base of operations pairing contributions for DNA polymers at different temperatures (a) Temperature dependence of the stacking contributions to the stability of A•T- (carmine circles) and G•C- (bluish circles) containing polymers. Straight solid line of the same color gives the temperature dependence of the stability parameter of corresponding polymer calculated using Equations 5 and vi at [Na⁺] = xv mM. (b) Temperature dependence of A•T (ruby) and K•C (blue) base pairing parameters calculated as a deviation between stability and stacking terms using information in (a). Horizontal broken lines correspond to mean values of $Δ G_{A • T}^{BP} = 0.57$ kcal/mol and $Δ G_{K • C}^{BP} = - 0.eleven$ kcal/mol.

The stacked-unstacked transition of a nicked DNA doublet involves loss of stacking interactions while hydrogen bonding between the complementary bases is preserved (Figure vi). It also involves removal of structural constraints imposed in the double helix making most of the conformational infinite of the intact saccharide-phosphate backbone accessible. This role of backbone conformational entropy proceeds is close to the gain per i strand upon duplex melting. The conformation about the glycosidic bond in an open stack, however, is preserved every bit in duplex class. Nicked strand is lacking O3′-P linkage; the absenteeism of this constraint allows for boosted conformations when 2nd-order interactions (interactions depending on two torsional angles) are repealed (59). Note that the backbone conformational entropy modify is independent of the particular sequence and does not affect heterogeneity of $Δ M_{KL}^{ST}$ values. Nosotros assume that conformation of base of operations pairs flanking the nick is preserved as in stacked duplex. Like in case of helix-to-coil transition, other factors contributing to transition parameters come from interactions with solvent and counterions.

We assign free energy difference to stacked-unstacked equilibrium at the nick site of Deoxyribonucleic acid—this parameter describes interaction of ii neighboring base pairs and is sequence-dependent. As noted above, it likewise includes a role of conformational entropy alter and other relevant interactions. These factors are universal for all neighboring pairs and practise not contribute to heterogeneity of measured $Δ {Yard}_{KL}^{ST}$ . We assume farther that this parameter represents the stacking term of intact Deoxyribonucleic acid duplex in Equation 3. Note that this assumption explicitly implies that stacked/closed state of the nicked stack is equivalent to the stack in duplex without the lesion and no optimization of the base pair stacking occurs upon introduction of the nick like in case of dangling unpaired nucleotides (52). If this assumption is valid, and then parameters of stacked-unstacked equilibrium at the nicks positioned on i or the other strand of the each dinucleotide stack volition be close. Indeed, we accept shown previously that stacking parameters for KL/F and KL/R molecules are similar (13). Parameters obtained for stacks equivalent due to dyad axis of symmetry, i.eastward. AA and TT, GG and CC, are also like. Note that marked difference is observed in case of coaxial stacking parameters determined in melting experiments (39).

Stability of DNA polymer nether different ambient atmospheric condition

Allow u.s.a. consider a Deoxyribonucleic acid polymer comprised of adenines and thymines. The set of nearest-neighbor stability parameters of all A•T-containing dinucleotides, i.e. ΔThou _AT, ΔG _AA = ΔChiliad _TT, ΔThou _TA, fully describes the contacts establish in this polymer. According to Equation 3, stability parameter per 1 bp, ΔG _A•T, of the A•T-polymer with a random sequence is given by [run into ref. (13)]:

$\begin{matrix} Δ {Yard}_{A • T} = \frac{one}{4} \sum_{AT, AA, TT, TA} Δ G_{KL} \\ = \frac{1}{4} \sum_{AT, AA, TT, TA} Δ {Yard}_{KL}^{ST} + Δ G_{A • T}^{BP} \end{matrix}$

four

Stacking contribution to the polymer stability is the starting time term of the right-hand side of Equation 4; the second term presents the base pairing contribution. Experimentally determined stacking term is the mean of stacking parameters nosotros obtained for all A•T-containing contacts. Stability parameter for G•C-containing polymer, ΔGrand _Yard•C, is calculated analogously from stacking, $Δ G_{GC}^{ST}, Δ {Thou}_{GG}^{ST} = Δ {One thousand}_{CC}^{ST}, Δ G_{CG}^{ST}$ and base of operations pairing $Δ G_{G * C}^{BP}$ contributions:

$\begin{matrix} Δ G_{K • C} = \frac{one}{4} \sum_{GC, GG, CC, CG} Δ G_{KL} \\ = \frac{1}{4} \sum_{GC, GG, CC, CG} Δ G_{KL}^{ST} + Δ G_{G • C}^{BP} \end{matrix}$

Deoxyribonucleic acid stability parameters in terms of melting temperatures, $T_{M}^{A • T}$ and $T_{M}^{G • C}$ , are salt-dependent and are given by the following empirical equations for A•T- and G•C-containing polymers, respectively (60):

$\begin{array}{l} T_{Thousand}^{A • T} = 355.55 + 7.95 ln N a^{+} and \\ T_{M}^{G • C} = 391.55 + 4.89 ln {Na}^{+}, \end{array}$

where $T_{M}^{A • T}$ and $T_{Chiliad}^{GC}$ are in degrees Kelvin. Equation half dozen translates into gratis energy difference stability parameters using ΔS° = −24.85 cal/mol One thousand (61) and Equation vii.

Stability parameters of A•T-containing and G•C-containing polymers calculated for [Na⁺] = fifteen mM using Equations 6 and vii are plotted in Figure 7a. Salt dependences of these parameters calculated at 37°C are presented in Figure 8a.

An external file that holds a picture, illustration, etc. Object name is gkj454f7.jpg

Overall stability, stacking and base pairing contributions for Dna polymers at different salt weather. (a) Salt dependence of the stacking term in the stability of A•T- (ruby circles) and G•C- (blue circles) containing polymers. Straight solid lines of the same color stand for to the salt dependence of the Dna polymer stability calculated using Equations 5 and vi at 37°C. (b) Salt dependence of the base pairing parameters for A•T (red) and M•C (bluish) pairs calculated at each concentration equally a difference betwixt stability term and stacking term using information from (a). Horizontal broken lines correspond to hateful values of $Δ G_{A • T}^{BP} = 0.61 kcal / mol$ and $Δ {One thousand}_{One thousand • C}^{BP} = - 0.01 kcal / mol$ .

An external file that holds a picture, illustration, etc. Object name is gkj454f8.jpg

Schematic representation of (a) helix-to-roll transition of a DNA dinucleotide stack and (b) stacked-unstacked equilibrium at a nick site.

Temperature dependence of stacking and base of operations pairing contributions to DNA stability

Stability of a Deoxyribonucleic acid polymer comprised only of A'south and T's arranged in a random order is given by two terms—the stacking term and the base of operations pairing term—that appear on the right-mitt side of Equation iv. Stacking term is calculated as an average of stacking parameters of A•T-containing contacts, which we determine in PAGE experiments conducted under certain set of ambient atmospheric condition (temperature and salt concentration). Stacking term of the G•C-containing polymer is obtained analogously.

Stacking contributions to the stability of A•T- and G•C-containing polymers for unlike temperatures are plotted in Figure 7a. Comparison of the temperature dependence of stacking terms with the temperature dependence of stability terms for both polymers reveals remarkable similarity between the two. Linear regression analysis of the temperature dependences of ΔG ^ST values gives mean slope of $\frac{d Δ {Thousand}^{ST}}{d T} = 0.026$ kcal/molK, which is very close to the slope of temperature dependence of stability parameters described by Equations 6 and 7. It is axiomatic that temperature dependence of stacking component fully determines the temperature dependence of DNA stability parameters throughout the temperature range employed in our experiments.

Co-ordinate to Equations 4 and v, the difference between the melting stability of the polymer and the stacking term gives the contribution of A•T or G•C base of operations pairing. The plots shown in Effigy 7b nowadays the base of operations pairing term calculated for different temperatures by subtracting the experimentally determined stacking term from the polymer stability parameter. With good accuracy, the base pairing term is contained of temperature within the temperature range used in our experiments. The horizontal lines give the mean values of base pairing parameters at 15 mM Na⁺ of $Δ M_{A • T}^{BP} = 0.57 kcal / mol$ and $Δ {Grand}_{G • C}^{BP} = - 0 . eleven kcal / mol$ . Nosotros conclude that stability of the Dna polymer with respect to strand separation is mainly determined by stacking interactions with base pairing being destabilizing (A•T pairs) or contributing nigh nothing (G•C-pairs) (Table one).

Table one

Stacking and base pairing contributions to DNA polymer stability under different ambience conditions^a

[Na⁺], mM	Temperature, °C	A•T-containing polymer		G•C-containing polymer
		$\frac{1}{4} \sum_{AT, AA, TT, TA} Δ {One thousand}_{KL}^{ST}$	$Δ {Thou}_{A • T}^{BP}$	$\frac{1}{4} \sum_{GC, GG, CC, CG} Δ {Chiliad}_{KL}^{ST}$	$Δ G_{M • C}^{BP}$
fifteen	32/52	−1.01/−0.36^b	0.57	−1.48/−1.02^b	−0.11
15/100	37	−0.92/−1.32^b	0.61	−one.44/−one.83^b	−0.01

Furthermore, throughout the temperature range used in our experiments, heterogeneity of base-stacking interactions of A•T- and Thousand•C-containing contacts, $δ Δ G^{ST} = \frac{1}{4} \sum_{AT, AA, TT, TA} Δ {Grand}_{KL}^{ST} - \frac{1}{4} \sum_{GC, GG, CC, CG} Δ G_{KL}^{ST}$ , is responsible for a substantial office of the difference in the stabilities of A•T- and G•C-polymers, δΔG = ΔG _A•T − ΔK _G•C (Figure 7a). In the presence of 15 mM Na⁺, heterogeneous stacking accounts for δΔYard ^ST = 0.six kcal/mol out of δΔ1000 = 1.2 kcal/mol stability difference of A•T- and K•C-containing polymers. Thus, the dependence of Dna stability on the G•C content is not merely due to the fact that One thousand•C pairs are stronger than A•T pairs (Figure 7b) simply besides is due to the stronger stacking component of G•C-containing contacts (Effigy 7a). This understanding of stacking domination constitutes a paradigm shift in the view on DNA stability.

Table salt dependence of stacking and base pairing contributions to Dna polymer stability

Stacking terms of A•T- and G•C-containing polymers are calculated from stacking parameters obtained in Folio experiments that have been carried out under different salt weather (Figure 8a). Salt dependences of the stability parameters of both polymers at 37°C calculated using Equation 6 and 7, are also shown in Effigy 8a. The 2 dependencies are very similar indicating that salt dependence of stacking term determines salt dependence of Dna stability parameters throughout the range of table salt concentration used in our experiments. In example of A•T-containing contacts, salt dependence of stacking component reproduces table salt dependence of DNA stability parameters with a smashing degree of accuracy. Salt dependences of stacking terms of A•T- and One thousand•C-containing contacts are similar to $\frac{d Δ G^{ST}}{d ln [North a^{+}]} = - 0.200 kcal / mol$ . The dependence of stacking parameters on salt concentration was unexpected. In fact, in our original publication nosotros assumed that base pairing term is fully responsible for ionic forcefulness dependence of DNA melting stability (13). Figure 8a shows that this supposition is incorrect and base of operations pairing term plotted in Effigy 8b is virtually independent of common salt concentration. Horizontal lines represent to the values of base pairing parameters averaged over the whole range of salt concentrations: $Δ {Yard}_{A * T}^{BP} = 0.61 kcal / mol$ and $Δ G_{G * C}^{BP} = - 0.01 kcal / mol$ (Table 1).

What is the origin of the ionic strength dependence of stacking parameters measured in Folio experiments? It is articulate that stacking is a cyberspace phenomenon involving hydrophobic, electrostatic and dispersion components (28,29,62–65). There is no understanding betwixt researchers, however, what is the dominant force in this interaction. Luo et al. (29) emphasize a ascendant function of nonelectrostatic interactions. Gellman and co-workers (65,66) report that neither solvophobic furnishings nor dispersion forces are important in aromatic stacking; stabilization is achieved through attractive interactions between positive and negative fractional charges on bases. Guckian et al. found that hydrophobic effects dominate stacking simply dispersion forces and electrostatic interaction between partial charges contribute substantially to the total base-stacking too. This is particularly truthful for natural bases, which accept meaning charge localization (28,67). In either instance, electrostatic component of base of operations-stacking interaction (substantial or not) will depend on the ambience ionic strength through local screening of partial charges at the solvent attainable positions. In the context of the DNA double helix, however, this effect is probable to be overwhelmed by the salt-dependent interactions betwixt highly negatively charged Dna backbones as described by the polyelectrolyte theory of DNA stability (68,69). Similarity between table salt dependences of Dna stacking parameters measured in PAGE experiments and DNA stability parameters indicates that electrostatic component contributes exclusively to $Δ 1000_{KL}^{ST}$ of Equation 3. Notation, that charge of the phosphate at the 5′ nt of the nicked stack does not affect salt dependence of measured $Δ {Thou}_{KL}^{ST}$ parameters.

CONCLUSIONS

Separation of the 2 contributions to thermal stability of the Deoxyribonucleic acid double helix is achieved by studying PAGE of DNA molecules with lonely nicks and gaps. For the starting time fourth dimension, the dependence of Dna stacking parameters on ambient conditions (temperature and salt concentration) is determined. Throughout the temperature and common salt concentration range of our experiments, base of operations-stacking interactions are always stabilizing for both A•T- and G•C-containing contacts in the Deoxyribonucleic acid double helix. In fact, DNA stability is mainly determined by base-stacking interactions. Thousand•C pairing does not contribute to stabilization of Dna duplex, while A•T pairing is e'er destabilizing. This finding presents a paradigm shift in the understanding of the interplay of the forces stabilizing Dna double helix. For all temperatures heterogeneity of stacking interactions in A•T- and 1000•C-containing contacts accounts for at least half of heterogeneity in the stability of A•T- and G•C-polymers; the other half is due to the difference in the energetics of A•T and Grand•C base pairing. The data on separation of stacking and base of operations pairing contributions accept made it possible to describe sequence-dependent fluctuational opening of the DNA double helix (70).

SUPPLEMENTARY Data

Supplementary Data are available at NAR Online.

Supplementary Material

Acknowledgments

The authors thank Dr R.Thousand. Georgiadis for providing access to spectrophotometer and Ms J. Ruemmele for technical help with melting experiments. The authors thank Dr Y. Mamasakhlisov and Dr P. E. Nielsen for fruitful discussions. Funding to pay the Open up Access publication charges for this article was provided by NIH.

Conflict of interest argument. None declared.

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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1360284/