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Incorporation of a cationic aminopropyl chain in DNA hairpins: thermodynamics and hydration.
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PMID:  11522834     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
We report on the physicochemical effects resulting from incorporating a 5-(3-aminopropyl) side chain onto a 2'-deoxyuridine (dU) residue in a short DNA hairpin. A combination of spectroscopy, calorimetry, density and ultrasound techniques were used to investigate both the helix-coil transition of a set of hairpins with the following sequence: d(GCGACTTTTTGNCGC) [N = dU, deoxythymidine (dT) or 5-(3-aminopropyl)-2'-deoxyuridine (dU*)], and the interaction of each hairpin with Mg(2+). All three molecules undergo two-state transitions with melting temperatures (T(M)) independent of strand concentration that indicates their intramolecular hairpin formation. The unfolding of each hairpin takes place with similar T(M) values of 64-66 degrees C and similar thermodynamic profiles. The unfavorable unfolding free energies of 6.4-6.9 kcal/mol result from the typical compensation of unfavorable enthalpies, 36-39 kcal/mol, and favorable entropies of approximately 110 cal/mol. Furthermore, the stability of each hairpin increases as the salt concentration increases, the T(M)-dependence on salt yielded slopes of 2.3-2.9 degrees C, which correspond to counterion releases of 0.53 (dU and dT) and 0.44 (dU*) moles of Na(+) per mole of hairpin. Absolute volumetric and compressibility measurements reveal that all three hairpins have similar hydration levels. The electrostatic interaction of Mg(2+) with each hairpin yielded binding affinities in the order: dU > dT > dU*, and a similar release of 2-4 electrostricted water molecules. The main result is that the incorporation of the cationic 3-aminopropyl side chain in the major groove of the hairpin stem neutralizes some local negative charges yielding a hairpin molecule with lower charge density.
Authors:
A M Soto; B I Kankia; P Dande; B Gold; L A Marky
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Nucleic acids research     Volume:  29     ISSN:  1362-4962     ISO Abbreviation:  Nucleic Acids Res.     Publication Date:  2001 Sep 
Date Detail:
Created Date:  2001-08-27     Completed Date:  2001-09-20     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  0411011     Medline TA:  Nucleic Acids Res     Country:  England    
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Languages:  eng     Pagination:  3638-45     Citation Subset:  IM    
Affiliation:
Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA.
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MeSH Terms
Descriptor/Qualifier:
Base Sequence
Calorimetry
DNA / chemistry*,  metabolism
Magnesium / chemistry
Nucleic Acid Conformation*
Nucleic Acid Denaturation
Oligonucleotides / chemistry
Spectrophotometry, Ultraviolet
Temperature
Thermodynamics
Ultrasonics
Water / chemistry
Grant Support
ID/Acronym/Agency:
CA36727/CA/NCI NIH HHS; CA76049/CA/NCI NIH HHS; GM42223/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Oligonucleotides; 7439-95-4/Magnesium; 7732-18-5/Water; 9007-49-2/DNA
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Journal ID (nlm-ta): Nucleic Acids Res
ISSN: 0305-1048
ISSN: 1362-4962
Publisher: Oxford University Press, Oxford, UK
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Copyright ? 2001 Oxford University Press
Received Day: 25 Month: 4 Year: 2001
Revision Received Day: 18 Month: 7 Year: 2001
Accepted Day: 18 Month: 7 Year: 2001
Print publication date: Day: 1 Month: 9 Year: 2001
Volume: 29 Issue: 17
First Page: 3638 Last Page: 3645
ID: 55897
Publisher Id: gke505
PubMed Id: 11522834

Incorporation of a cationic aminopropyl chain in DNA hairpins: thermodynamics and hydration
Ana Maria Soto1
Besik I. Kankia1
Prasad Dande12
Barry Gold12
Luis A. Marky12a
1Department of Pharmaceutical Sciences and 2Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198, USA
aTo whom correspondence should be addressed at: Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198, USA. Tel: +1 402 559 4628; Fax: +1 402 559 9543; Email: lmarky@unmc.edu

INTRODUCTION

DNA is normally considered a stiff rod-like molecule with a persistent end-to-end distance of >100 bp (1). Despite this apparent rigidity, many DNA affinity binding proteins, including histones, can introduce very significant non-linear distortions in DNA (2,3). Several distinct mechanisms have been proposed to account for the distortion induced by DNA binding proteins and clearly different proteins can utilize different bending pathways. A feature of some DNA bending proteins is the introduction of cationic side chains, e.g. Arg and Lys, at the center of the bend or kink. A well-studied DNA bending protein is the catabolite activating protein (CAP) transcription factor, which induces a 45? kink in the DNA at the TG step in its TGTGA conserved binding sequence (4). These TG steps are well conserved on both sides of the dyad axis in DNA binding sites so the CAP dimer bends the DNA by 90?. Based on the final structure, it is reasonable to assume that the initial event in the binding of CAP to DNA involves H-bonds between Arg and Glu residues on the protein and G and C nucleotides at the TG step that is at the center of the kinked region. As a consequence of the kinking, salt bridges and additional H-bonds can then form at sequences distal to the bent?region. In an effort to understand the effects of basic?amino?acid side chains in protein-mediated bending, 5-(?-aminoalkyl)-2?-deoxyuridine substitutions were used to mimic the interactions of basic amino acids with DNA (5,6). Using electrostatic footprinting and molecular modeling, the location of the cationic sidechain has been determined to be in the major groove toward the 3?-side (7,8). Significantly, when 5-(?-aminoalkyl)-2?-deoxyuridine substitutions were appropriately phased with an intrinsically bent A-tract, they caused aberrant gel mobility, presumably due to the induction of non-linear DNA regions (5,6).

In order to specifically understand how the positioning of cationic charge in the major groove affects local DNA structure, a detailed thermodynamic characterization of DNA containing a single 5-(3-aminopropyl)-2?-deoxyuridine (dU*) residue is necessary. In this work, we are incorporating specific chemical groups in the DNA bases of short oligonucleotides that form intramolecular hairpins. Single-stranded hairpins are good thermodynamic models to mimic the pseudo-first-order helix?coil transition of natural DNA polymers. The main advantage for using these molecules is that they are small with an ellipsoidal shape and their helix?coil transitions are unimolecular that take place in a two-state fashion at convenient experimental temperatures. In addition, the small size of these molecules allows us to exclusively investigate the local contributions of the chemical incorporation of cationic chains in DNA, excluding the contributions of adjacent long double helical arms.

Specifically, we have used a combination of temperature-dependent UV spectroscopy and differential scanning calorimetry (DSC) techniques to investigate the helix?coil transition of a set of hairpins with the following sequence: d(GCGACTTTTTGNCGC), where N represents 2?-deoxyuridine (dU), deoxythymidine (dT) or dU* (Fig. 1). We also used density and ultrasonic techniques to characterize the overall hydration of each hairpin and their interaction with Mg2+ ions. The comparison of thermodynamic profiles yields the specific energetic and hydration contribution for placing a 3-aminopropyl side chain or methyl group, on a deoxyuridine position located in the major groove and at the stem of a small DNA hairpin loop. The results show that the main effect of the amino charge at the end of this chain is to neutralize local negative charge.


MATERIALS AND METHODS
Materials

The phosphoramidite derivative of dU* was prepared as previously described (9,10). All oligodeoxyribonucleotides were synthesized in University of Nebraska Medical Center, the Eppley Institute Molecular Biology Shared Resource, purified by reverse phase HPLC, desalted on a G-10 Sephadex column using a triethylammonium carbonate buffer and lyophilized to dryness. The dry oligomers were then dissolved in the appropriate buffer. For simplicity, each hairpin is designated as follows: ?dU? for the control hairpin, d(GCGACTTTTTGUCGC), ?dT? for d(GCGACTTTTTGTCGC) and ?dU*? for the hairpin containing the 5-(3-aminopropyl)dU or d(GCGACTTTTTGU*CGC). The extinction coefficients of oligonucleotides were calculated in water from the tabulated values of the monomer and dimer nucleotides (11) at 260 nm and 25?C. These values were then estimated in the random coil state at high temperatures using a procedure reported earlier (12). The molar extinction coefficient for each oligonucleotide is 1.32 ? 105 M?1 cm?1 at 90?C. The buffer solutions used consisted of 10 mM sodium phosphate pH 7.0, adjusted to the desired ionic strength with NaCl, or 10 mM HEPES pH 7.5 for the density and ultrasound experiments. MgCl2 and all other chemicals were reagent grade.

Temperature-dependent UV spectroscopy

Absorption versus temperature profiles (melting curves) for the helix?coil transition of DNA hairpins were obtained with a thermoelectrically controlled UV/Vis Aviv 14DS spectrophotometer, interface to a PC for data acquisition and analysis. The temperature was scanned at a heating rate of ?0.6?C/min. Analysis of melting curves yields the helix?coil transition temperature, TM, and van?t Hoff enthalpies, ?HvH (13). Melting curves as a function of strand and salt concentrations were?obtained to confirm the intramolecular formation of each?hairpin and to determine the thermodynamic release of counterions, ?nNa+, accompanied their helix?coil transition.

Differential scanning calorimetry

The heat of unfolding of each molecule was measured with the MC-2 differential scanning calorimeter from Microcal Inc. (Northampton, MA). A typical experiment consists of obtaining a heat capacity profile of a DNA solution against a buffer solution. For the analysis of the experimental data, a buffer versus buffer scan is subtracted from the sample versus buffer scan; both scans in the form of mcal/s versus temperature were converted to mcal/?C versus temperature by dividing each experimental data point by the corresponding heating rate. Integration of the resulting curve, ??CpdT, and normalization for the number of moles yields the molar unfolding enthalpy, ?Hcal, which is independent of the nature of the transition; the molar entropy, ?Scal is obtained by a similar procedure, ?(?Cp/T)dT. The free energy at any temperature T is then obtained with the Gibbs equation: ?G?cal(T) = ?Hcal ? T?Scal, which assumes similar heat capacities for the hairpin and random coil states. Furthermore, the nature of the helix?coil transition is obtained by inspecting the ?HvH/?Hcal ratio; a value of nearly 1 indicates a two-state transition (13).

Measurement of hydration parameters

Ultrasonic velocity measurements were made with a home-built instrument, based on the resonator method (14,15), using two cells differentially and in the frequency range of 7?8 MHz. The molar increment of ultrasonic velocity, A, is defined by the relationship: A = (U ? Uo)/(UoC), where U and Uo are the ultrasonic velocities of the solution and solvent, respectively, and C is the concentration of solute. In the acoustical titrations of each hairpin with Mg2+, 2?5 ?l aliquots of MgCl2 are added stepwise to 700 ?l hairpin solution. Mixing was performed directly with a built-in magnetic stirrer (15). The resulting A?values were corrected for the contribution of salt concentration by carrying out similar titrations of the buffer. The ?A values are calculated with the relationship: ?A = A ? A0, where A and A0 are the molar increment of ultrasonic velocity of the Mg2+?hairpin and hairpin solutions, respectively.

The density of each hairpin solution was measured with a DMA-602 densimeter (Anton Paar, Graz, Austria) with two 200 ?l cells. The apparent molar volume, ?V, is calculated using the equation (16): ?V = M/? ? (? ? ?o)/(?oC), where ?o and ? are the density of the solvent and solution, respectively, and M is the molecular weight of the hairpin. The molar volume change, ??V, accompanying the interaction of Mg2+ with each hairpin is calculated using the equation ??V = ?V ? ?Vo, where ?V and ?Vo are the apparent molar volume of the Mg2+?hairpin and hairpin solutions, respectively. All solutions were prepared by weight using a microbalance and special precautions were taken to prevent solvent evaporation. The molar adiabatic compressibility, ?KS, is obtained from the relationship (17): ?KS = 2?o(?V ? A ? M/2?o), where ?o is the adiabatic compressibility coefficient of the solvent. Similarly, the change in the molar adiabatic compressibility, ??KS, is determined from the ?A and ??V according to the relationship: ??KS = 2?o(??V ? ?A).

Background of hydration parameters

The following relationships (18): ?V = Vm + ?Vh and ?KS?= Km + ?Kh, provide a molecular interpretation for the absolute measurement of ?V and ?KS, as follows. The Vm and Km terms are the intrinsic molar volume and intrinsic molar compressibility, respectively, of the solute. The ?Vh term represents the hydration contribution of the change in the volume of water that surrounds a solute. The ?Kh term is the hydration contribution of the change in the compressibility of water around a solute and the compressibility of the void volumes between the solute and of the surrounding water. In the ?KS values of oligodeoxynucleotides, the contribution of Km is small relative to their hydration terms. Therefore, the ?KS value simply reflects the hydration contribution, i.e. ?KS?= ?Kh. In a similar way, the ??V and ??KS parameters of Mg2+ binding to oligonucleotides can be expressed in terms of their sole hydration contributions: ??V = ??Vh and ??KS = ??Kh, this assumes that the DNA conformation does not change.


RESULTS
UV and calorimetric unfolding of hairpin loops

For the exclusive formation of single-stranded hairpins, the TM value is expected to be independent of strand concentration, which would mean that intramolecular hairpins form at low temperatures. In addition, intramolecular hairpin loops will have low hyperchromicity and high TM values. To confirm the molecularity of these hairpins, the helix?coil of each molecule was investigated as a function of strand concentration. The melting curves were performed in the strand concentration range of 5?250 ?M. The UV melting of all three molecules at 260 nm occurs in broad monophasic transitions with average hyperchromicities of ?8.7% (Fig. 2A). Typical excess heat capacity versus temperature profiles (Fig. 2B) indicate that all transitions are monophasic and take place without changes in the heat capacity of the initial and final states. The TM value dependence on strand concentration of these techniques is shown in Figure 2C. Similar TM values are obtained in this concentration range for each oligodeoxynucleotide, confirming the formation of single-stranded hairpins at low temperatures. Furthermore, the thermal stability of the hairpins follows the order: dU (63.7?C) < dU* (64.3?C) < dT (65.1?C), and similar ?Hcal values of 36?39 kcal/mol are obtained (Table 1). The comparison of ?HvH, determined from the shape of the calorimetric melting curves, and ?Hcal allows us to draw conclusions about the nature of the transitions (13). We obtained ?HvH/?Hcal ratios of 1.05?1.11 that indicate all three hairpins melt in two-state transitions.

Thermodynamic release of counterions

For each hairpin, increasing the concentration of Na+ from 16 to 200 mM resulted in the shift of the melting curves to higher temperatures (Fig. 3A). The increase in salt concentration shifted the hairpin?random coil equilibrium towards the conformation with higher charge density parameter. The TM?dependence on salt concentration for each hairpin is shown in Figure 3B. A linear dependence was obtained with slope values in the range of 2.3?2.9?C. The differential binding of ions for the helix?coil transition of a hairpin, ?nNa+, was calculated from the relationship (19): ?(ln K)/?[ln (Na+)] = ?nNa+, where K is the equilibrium constant and the (Na+) term is the ionic activity of sodium. Using the chain rule, [?(ln K)/?TM][?TM/?[ln (Na+)] = ?nNa+, and the van?t Hoff equation, ?(ln K)/?T = ?H/RT2, we obtained: ?TM/?(ln [Na+]) = 0.9(RTM2/?Hcal)?nNa+. The ?TM/?(ln [Na+]) term in this relationship corresponds to the slope of the lines in Figure 3B, and 0.9 is a proportionality constant of converting mean ionic activities into concentrations. The term in parenthesis was obtained from DSC experiments, and R is the gas constant. We obtained ?nNa+ values or counterion releases of 0.44 mol Na+/mol hairpin for the dU* hairpin and an average of ?0.53 mol Na+/mol hairpin for the dU and dT hairpins.

Thermodynamic profiles for the formation of hairpins

The free energy values were calculated at 20?C from the Gibbs equation, using the enthalpies and entropies obtained in calorimetric melting experiments: ?G?cal = ?Hcal ? 293.15 ?Scal. In the Gibbs equation, both the enthalpy and entropy are assumed independent of temperature. Table 1 lists standard thermodynamic profiles for the formation of each hairpin at 20?C, and the data indicate that at this temperature all three oligodeoxynucleotides are ordered and exist as hairpins. The favorable ?G?cal value for the formation of each hairpin resulted from the characteristic partial compensation of a favorable enthalpy and unfavorable entropy terms. The favorable ?Hcal values result from the formation of H-bonds and base pair stacks in the stem, whereas the unfavorable entropy values indicates an ordering of the systems due to an uptake of counterions and water molecules (see below).

Overall hydration properties of hairpins

We used density and ultrasonic techniques to measure the volume and compressibility parameters that characterize the hydration of each hairpin. The absolute values of ?V and ?KS are shown in the last two columns of Table 1. All three ?V?values are positive, which is expected because the value corresponds to the sum of two contributions: the intrinsic volume or van der Waals volume of each molecule and its associated volume of hydration. The magnitude of these ?V?values follows the order: dU* > dT > dU, and the differences were small but significant (Table 1). However, this trend is consistent with the size of the uridine modifications in the 5?position: 3-aminopropyl > methyl > hydrogen. On the other hand, the ?KS values were negative and corresponded to the much larger contribution of water in the hydration shell of these hairpins. These values followed a similar trend as the ?V?values (Table 1) but their magnitudes were within experimental uncertainties. Therefore, these compressibility parameters reflect a similar hydration level for all three hairpins; and in the absence of compensating effects, if any, indicate that the chemical substitutions in these hairpins have similar hydration contributions.

Hydration effects accompanying the binding of Mg2+ to hairpins

Acoustical titrations for the addition of Mg2+ to each hairpin are shown in Figure 4. The resulting curves are actually Mg2+-binding isotherms and their shapes were very similar. However, some small differences occur in the initial slopes and overall amplitude of the curves. The initial slopes were proportional to the Mg2+?nucleic acid binding affinities (KMg2+), although the overall amplitude at [Mg2+]/[Phosphate] saturation levels were ?0.6, and correspond to the total dehydration effect of Mg2+ binding, ?A (Fig. 4). The interaction of Mg2+ with each hairpin yielded negative values of ?A, which indicate a release of water molecules. This water release corresponds to the net hydration changes of the Na+?Mg2+ exchange that takes place in the ionic atmosphere of the hairpin. A quantitative evaluation of both KMg2+ and ?A values is determined from fitting the Mg2+-binding isotherms interactively using a binding model where Mg2+ interacts with each hairpin containing one type of identical and independent binding sites (20). The fitting procedure is characterized by three parameters: KMg2+, ?AMg2+, and the apparent number of Mg2+ sites, nMg2+. This procedure is simplified by obtaining the nMg2+ values from the X-intercept of the experimental ultrasonic titrations (20). We obtained an average ?AMg2+ value of ?10.5?ml/mol of Mg2+ and KMg2+ values of ?9.5 ? 103 M?1. The strength of Mg2+ binding to the hairpins is lowest with the dU*?hairpin and consistent with its lower release of Na+ ions. These effects suggest a lower charge density parameter for the dU* hairpin. The hydration parameters, ??V and ??KS, of Mg2+ binding to hairpins are shown in Table 2. All values were positive (Table 2) and consistent with the reported effects derived from the values of ?A. Mg2+ binding releases water molecules from the hydration shells of the free Mg2+ ions and/or interacting nuclei acid atomic groups. The similarity of the ??V and ??KS values, among the three hairpins, indicates a similar release of water molecules.


DISCUSSION
Formation of intramolecular hairpins at low temperatures

The exclusive formation of monomolecular hairpin loops at low temperatures for all three sequences is confirmed with the following experimental observations. First, their experimental TM values are ?65 and ?20?C higher than the estimated TM?values of the corresponding bimolecular duplexes with an internal loop of 10 thymine residues. Secondly, the TM value of each molecule remains the same over a 40-fold change in strand concentration and has a slight dependence on salt?concentration. The average release of counterions is 0.50?mol Na+/mol hairpin, which is indicative of their low charge density. Thirdly, the calorimetric unfolding enthalpy is 37.0 kcal/mol in low salt, which is characteristic of the melting behavior of short double helical stems. This is in good agreement with the predicted enthalpy values of 35.1?36.8 kcal/mol, obtained from DNA nearest-neighbor parameters (21,22).

Thermodynamics of forming DNA hairpins with single base modifications

We have investigated three DNA hairpins containing specific substitutions at the 5 position of uridine: -H, -CH3 and -CH2CH2CH2N+H3, and complete thermodynamic and hydration profiles for the formation of each hairpin at 20?C are listed in Table 1. For a better understanding of these profiles, it is important to emphasize the physical factors involved in their favorable formation, namely contributions from base pairing, base pair stacking, hydration and counterion binding. These non-covalent interactions depend on the specific chemical composition of the strands used and overall conformation. The formation of each hairpin is accompanied by favorable free energy terms, which results from an enthalpy?entropy compensation, the uptake of ions and immobilization of water molecules (Table 1). The resulting enthalpies correspond to the net balance of the following contributions: base pairing and base pair stacking interactions of the stem and stacking of the loop bases at the stem?loop interface. The uptake of electrostricted water molecules by the hairpin stem and the release of structural water from the thymine bases prior to forming constrain loops may also contribute favorably while the uptake of counterions has a negligible heat contribution (23). The unfavorable entropy terms correspond to contributions of forming a higher ordered hairpin?loop structure, resulting from the intramolecular association of a random coil, and a net uptake of water and counterions. Therefore, the similar standard thermodynamic profiles (?G?, ?H and ?S) for hairpin folding indicate similar base pairing and base pair stacking contributions in all hairpins. This suggests that all three hairpins have a similar conformation and the incorporation of a methyl group or of an aminopropyl group on uracil has a negligible effect on their conformation.

Incorporation of the charged aminopropyl chain yields a lower uptake of counterions

The combined UV and calorimetric melting experiments yielded, in the formation of hairpins, counterion uptakes of 0.44 mol Na+/mol hairpin for the dU* hairpin, and an average of ?0.53 mol Na+/mol hairpin for the dU and dT hairpins. Using these values, we estimate 0.055 mol Na+/mol phosphate and 0.066 mol Na+/mol phosphate, respectively, if only the eight phosphate groups of the stem are taken into account. Relative to the value of 0.17 mol Na+/mol phosphate for longer duplex DNA molecules, these values are low but consistent with the formation of 5 bp in the hairpin stems (24). The significant observation is the lower uptake of counterions for the hairpin containing the dU* modification. This shows that the positively charged aminopropyl chain at pH 7 is partially neutralizing the negative charges in the stem of the dU*?hairpin. The actual degree of neutralization of negative phosphate charge by the aminopropyl group may be estimated from the 0.09 mol Na+/mol hairpin differential uptake of counterions, and assuming all three hairpins have similar conformation and base pair stacking interactions. We estimate an ?14% higher phosphate neutralization for the local placement of the aminopropyl chain in the major groove of the stem of the dU*?hairpin. We stressed that the aminopropyl group does not form a salt bridge with the phosphate backbone (7,8,25). The charge neutralization might take place by the proposed electrostatic mechanism of Rouzina and Bloomfield (26). In this mechanism, the localization of the cationic aminopropyl group in the major groove of DNA electrostatically repels sodium counterions associated with the anionic phosphates yielding an unscreened backbone. In turn, the weakly screened phosphates then interact strongly with the tethered cation causing a collapse of the major groove. This scenario is consistent with our thermodynamic results and nicely also explains how the ?-aminoalkyl side chains induce DNA bending.

All three hairpin molecules have a similar hydration state

We showed that each hairpin yielded negative compressibility parameters, which indicate an uptake of water molecules. The overall effects are small but similar and consistent with the similar conformation of each hairpin with small chemical modifications, i.e. incorporation of additional 3?12 atomic groups out of ?420 atomic groups of a hairpin molecule. The magnitude of the molar volume of a hairpin molecule is attributed to both its van der Waals volume and the volume of its hydration shell. The magnitude of the molar compressibility is proportional only to the compressibility of the hydration shell because the van der Waals volume is considered incompressible. The contribution of water in the hydration shell of any molecule depends on the type of hydrating water, and this could be structural (or hydrophobic) around organic moieties and non-polar atomic groups or electrostricted around charges and polar atomic groups. In the case of DNA hairpin loops containing a stem of stacked base pairs and a loop of constrained thymines, the stem will have a higher immobilization of electrostricted water due to its increased charged density parameter while the loop immobilizes a higher degree of structural water due to a larger exposure of bases to the solvent. Hydration contributions related to the interaction of sodium ions with B-DNA are considered negligible, their deep occupancy in the minor groove has been considered very low (27) and, on average, the ions keep their hydration shells (28). The type of water may be estimated from the slope of the line of a plot of ?V versus ?KS, which yielded a slope of 20 ? 104 bar (data not shown). The magnitude of this slope is very large because of the small trends obtained with both parameters but it is in the direction that suggests a higher uptake of structural water. However, the overall uptake of water molecules by these hairpin molecules is supported by density investigations of similar DNA hairpins (29).

Hydration effects accompanying the binding of Mg2+ to hairpins

The interaction of Mg2+ with a nucleic acid duplex has been considered to be electrostatic (28,30,31). For this reason, Mg2+ is used to probe the effective charge density of each hairpin and its overall hydration. The magnitudes of the KMg2+ values are consistent with its electrostatic nature. The lower KMg2+ value for the dU* hairpin indicates that this hairpin has a lower charge density parameter, consistent with the additional neutralization of negative charge by the aminopropyl chain.

For the interpretation of the hydration effects of Mg2+ binding, it is important to take into account contributions from the effective charge density, conformation, sequence and overall hydration of the hairpin molecule. In addition, specific effects such as the formation of inner-sphere complexes when Mg2+ chelates specific atomic groups of the oligonucleotide should be considered. For the latter case, the resulting ??V and ??KS values of Mg2+ binding are compared to similar parameters for the formation (or dissociation) of complexes of metal ions with charged molecules with known three-dimensional structures. The similarity of the ??V (and ??KS) values of Mg2+ binding among the three hairpins indicates a similar release of water molecules from the hydration shells of the participating atomic groups. The type of hydrating water, structural or electrostricted, responsible for the hydration effects in the process of Mg2+ binding may be characterized empirically by their absolute value of k (equal to ??V/??KS) (32?34). Examples are the formation of cation?EDTA complexes, characterized by k values of 0.34?0.39 ? 104 bar (33). The formation of nucleic acid duplexes with higher non-polar character, are characterized with k values of 0.75 ? 104 bar (34). In this work, Mg2+ binding yields ??V/??KS ratios of 0.53 ? 104 bar, indicating the participation of electrostricted water. Furthermore, we used the following parameters, ?V = 2.5 cm3/mol and ??KS = 8.2 ? 10?4 cm3/bar-mol (35,36), to estimate the number of electrostricted water molecules involved in this interaction. The resulting ?V and ??KS values are divided by these parameters to yield a release of 2?4 electrostricted water molecules for the condensation of one Mg2+ ion. The hydration contribution of the displaced Na+?ions is considered negligible. This release is the result of removing water molecules from polar and charged groups of the nuclei acid and from the Mg2+ ions. On the other hand, the magnitude of these hydration parameters may well suggest the formation of one Mg2+?DNA inner-sphere complex in the stem. This is due to helical stems adopting the B-conformation, which renders them more hydrated, and their high content of dG?dC base pairs favors the formation of inner-sphere complexes (20). Alternatively, these hydration parameters are probably not indicative of a singly-coordinated inner-sphere contact complex, but instead support the idea that Mg2+ binding to the stem of each hairpin only slightly relaxes the electrostricted region lying beyond the inner layers. Thus, the main contribution to the volume change of ion pairing is likely from the relaxation of the outer-electrostricted region, with the removal of contact waters giving only a relatively small contribution to the volume and compressibility parameters.


CONCLUSION

We investigated the physicochemical effects of the attachment of a 3-aminopropyl group onto the 5-position of dU in a short DNA hairpin. The unfolding of three hairpins (two as control hairpins) and their interaction with Mg2+ ions were investigated using melting, density and acoustical techniques. All three intramolecular hairpins unfold in two-state transitions with similar TM values and similar thermodynamic profiles. The favorable free energies of 6.4?6.9 kcal/mol result from the typical compensation of favorable enthalpies (36?39 kcal/mol) and unfavorable entropies (110 cal/mol). All three hairpins have similar hydration levels; however, the stability of each hairpin has a weak dependence on salt concentration, which yielded a smaller counterion release for the hairpin with the cationic side chain. Therefore, the presence of this chain in the major groove of the hairpin stem effectively neutralizes a higher degree of negatively charged phosphate groups. The electrostatic interaction of Mg2+ with each hairpin yielded a similar release of 2?4 electrostricted waters and binding affinities consistent with the lower charge density parameter of the hairpin containing the cationic chain. The overall results will serve as thermodynamic baselines in future investigations of the effects of incorporating charge groups in long DNA oligonucleotide duplexes.



ACKNOWLEDGEMENTS

This work was supported by Grants GM42223 (LAM), CA76049 (BG) from the National Institutes of Health, and Cancer Center Support Grant CA36727 from the National Cancer Institute.


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Figures

[Figure ID: gke505f1]
Figure 1 

Sequence of deoxyribonucleotide and structure of base modifications.



[Figure ID: gke505f2]
Figure 2 

Thermodynamic characterization of the helix?coil transition of dU (circles), dT (squares) and dU* (triangles) in 10 mM sodium phosphate buffer at pH 7. (A) UV melting curves at oligonucleotide concentrations of 5 ?M. (B) DSC curves at oligonucleotide concentrations of ?225 ?M (in strands). (C) Dependence of TM on strand concentration.



[Figure ID: gke505f3]
Figure 3 

UV melting curves of oligonucleotides, at constant strand concentration of 5 ?M, as a function of salt concentration in 10 mM sodium phosphate buffer at pH 7. (A) Typical melting curves over a NaCl concentration range of 16?200 mM. (B) Dependence of TM on salt concentration, dU (circles), dT (squares) and dU* (triangles).



[Figure ID: gke505f4]
Figure 4 

Ultrasonic titration curves of DNA hairpins with MgCl2 in 10 mM HEPES buffer pH 7.5 and at oligonucleotide concentrations of ?150 ?M (in strands).



Tables
[TableWrap ID: gke505tb1] Table 1.  Complete thermodynamic parameters for the formation of hairpins at 20?C
Hairpin
Hyp.(%)
TM(?C)
?G?cal(kcal/mol)
?Hcal(kcal/mol)
T?Scal(kcal/mol)
?nNa+(per mol)
?V(cm3/mol)
?KS ? 104(cm3/bar-mol)
dU
8.9
63.7
?6.8
?39.0
?32.2
0.54
123
?13.0
dT
8.4
65.1
?6.4
?36.0
?29.6
0.52
127
?12.8
dU* 8.8 64.3 ?6.9 ?39.0 ?32.1 0.44 131 ?12.6

Standard thermodynamic parameters obtained in 10 mM sodium phosphate buffer pH 7, while the hydration parameters in 10 mM HEPES buffer pH 7.5. Experimental errors for each parameter are indicated in parenthesis as follows: TM (?0.5?C), ?G?cal (?5%), ?Hcal (?5% kcal/mol), T?Scal (?5%), ?nNa+ (?0.02), ?V (?1 cm3/mol) and ?KS.(?1.5 ? 10?4 cm3/bar-mol). ?V and ?KS are calculated per nucleotide units.


[TableWrap ID: gke505tb2] Table 2.  Binding and hydration parameters for the interaction of Mg2+ with hairpins at 20?C
Hairpin
?A (ml/mol)
nMg2+ (mol/mol P)
KMg2+ (M?1)
??V (cm3/mol)
??KS ? 104 (cm3/bar-mol)
k ? 10?4 (bar)
dU
?3.0
0.28
10.0 ? 103
9.7
18.3
0.53
dT
?2.8
0.28
9.7 ? 103
8.6
16.7
0.51
dU* ?3.3 0.27 8.7 ? 103 9.8 18.1 0.54

All experiments were done in 10 mM HEPES buffer pH 7.5. Experimental errors for each parameter are indicated in parenthesis as follows: ?A (?10%), nMg2+ (?10%), KMg2+ (?20%), ??V (?5%), ??KS.(?8%) and k (?13%).



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