Loop Contributions to the Folding Thermodynamics of DNA Straight

Jan 13, 2015 - Pseudoknots have diverse and important roles in many biological functions. We used a combination of UV spectroscopy and differential ...
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Loop Contributions to the Folding Thermodynamics of DNA Straight Hairpin Loops and Pseudoknots Calliste Reiling, Irine Khutsishvili, Kai Huang, and Luis A. Marky* Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, Nebraska 68198-6025, United States S Supporting Information *

ABSTRACT: Pseudoknots have diverse and important roles in many biological functions. We used a combination of UV spectroscopy and differential scanning calorimetry to investigate the effect of the loop length on the unfolding thermodynamics of three sets of DNA stem-loop motifs with the following sequences: (a) d(GCGCTnGCGC), where n = 3, 5, 7, 9; (b) d(CGCGCGT4GAAATTCGCGCGTnAATTTC), where n = 4, 6, and 8; and (c) d(TCTCTTnAAAAAAAAGAGAT5TTTTTTT), where n = 5, 7, 9, and 11. The increase in loop length of the first set of hairpins yielded decreasing TM’s and constant unfolding enthalpies, resulting in an entropy driven decrease in the stability of the hairpin (ΔG° = −7.5 to −6.1 kcal/mol). In the second set, the increase in the length of the loops yielded similar TM’s and slight increases in the unfolding enthalpies. This translated into more stable pseudoknots with an increasing ΔG° from −13.2 to −17.1 kcal/mol. This effect can be rationalized in terms of the increased flexibility of the pseudoknot with larger loops optimizing base-pair stacking interactions. In the last set of molecules, the increase in the length of one of the loops yielded an increase in the TM’s and larger increases in the enthalpies, which stabilize the pseudoknot significantly increasing the ΔG° from −8.5 to −16.6 kcal/mol. In this set, the thymine loop is complementary to the stem of A·T base pairs and the longer loops are able to form T*A·T base triplets due to the partial folding of the thymine loop into the ceiling of the major groove of the duplex, thus yielding a net formation of 1−3 T*AT/T*AT base-triplet stacks at the middle of its stem, depending on the loop length.

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INTRODUCTION The secondary structure of a specific nucleic acid can be predicted in a precise way on the basis of the sequence. A single stranded DNA molecule that has a sequence partially complementary to itself is able to fold into an intramolecular structure. A complete physical description of how this folding takes place is essential to understand how nucleic acids carry out their biological function. This physical description is dependent on the contributions from base pairing, base stacking, as well as ion binding and hydration. Hairpin structures are commonly found in RNA molecules and have important functional roles. RNA hairpins are important in controlling gene expression as shRNA.1 Not only can RNA form hairpins, but it has also been shown that ssDNA can form hairpin structures recognized by proteins.2,3 ssDNA is also involved in site-specific recombination, transcription, and replication.4−6 Due to variation in their loop length, stems and interactions between them, pseudoknots (Scheme 1) belong to an interesting and diverse RNA structural motif. Pseudoknots have various roles in biological function. Examples include selfsplicing introns,7 forming the catalytic core of various ribozymes,8−10 telomerase,11,12 riboswitches,13 and ribosomal frameshifting.14,15 Ribosomal frameshifting, a common mechanism found in viruses, is when there is a change in the reading frame which allows for different mRNAs to be translated. This event occurs when a pseudoknot initiates the frameshift at the slippery sequence.14 The high efficiency of frameshifting may © 2015 American Chemical Society

Scheme 1. Cartoon of the Sequences of the DNA Stem-Loop Motifsa

a

The n in Tn refers to the number of thymines in each loop.

be attributed to the formation of a local triplex within the pseudoknot increasing its interaction with the ribosome.15 In typical targeting reactions of nucleic acids with partially complementary strands, the unpaired bases in the loop drive the reaction because they are able to form additional base pairs providing favorable targeting free energies. In the targeting reactions of stem-loop motifs, the targeting reactions of pseudoknots with partially complementary strands are less favorable due to their compactness.16,17 For this reason, we want to investigate the folding/unfolding thermodynamics of DNA pseudoknots to determine their overall stability as a function of loop length. Currently, our laboratory is interested in both the putative structure and overall physical properties for the folding (and unfolding) of nucleic acid stem-loop motifs. Our current Received: November 20, 2014 Revised: January 12, 2015 Published: January 13, 2015 1939

DOI: 10.1021/jp5116417 J. Phys. Chem. B 2015, 119, 1939−1946

Article

The Journal of Physical Chemistry B Table 1. Sequence, Name, and Extinction Coefficient of Oligonucleotidesa

a

sequence

name

ε260 (mM−1 cm−1)

d(GCGCT3GCGC) d(GCGCT5GCGC) d(GCGCT7GCGC) d(GCGCT9GCGC) d(CGCGCGT4GAAATTCGCGCGT4AATTTC) d(CGCGCGT6GAAATTCGCGCGT6AATTTC) d(CGCGCGT4GAAATTCGCGCGT6AATTTC) d(CGCGCGT4GAAATTCGCGCGT8AATTTC) d(TCTCTT5AAAAAAAAGAGAT5TTTTTTT) d(TCTCTT7AAAAAAAAGAGAT5TTTTTTT) d(TCTCTT9AAAAAAAAGAGAT5TTTTTTT) d(TCTCTT11AAAAAAAAGAGAT5TTTTTTT)

Hp-3 Hp-5 Hp-7 Hp-9 PsK-4,4 PsK-6,6 PsK-4,6 PsK-4,8 PsK-5 PsK-7 PsK-9 PsK-11

93.2 107.9 129.8 139.0 292.8 330.6 307.7 322.2 331.1 344.3 366.6 375.5

Subscripts in the sequence are for the loops in either the hairpins or pseudoknots.

temperature ramp of approximately 0.6 °C min−1. The analysis of the shape of the melting curves yielded transition temperatures, TM, which corresponds to the inflection point of the helix−coil transitions. To determine the molecularity of the transition(s) of each DNA stem-loop motif, we investigated the dependence of TM over at least a 10-fold range of total strand concentration. If the TM remains constant in this range of strand concentration, it indicates a monomolecular or intramolecular transition.28 Additional UV melting curves were obtained as a function of salt concentration to determine the differential binding of ions. Differential Scanning Calorimetry (DSC). The total heat required for the unfolding of each oligonucleotide (hairpin or pseudoknot) was measured with a VP-DSC differential scanning calorimeter from Microcal (Northampton, MA). These thermograms were obtained with a temperature ramp of ∼0.75 °C min−1 with oligos ranging in concentration from 0.13 to 0.050 mM. Standard thermodynamic profiles and TM’s are determined from the DSC experiments using the following relationships:28,29 ΔHcal = ∫ ΔCp(T) dT and ΔScal = ∫ ΔCp(T)/ T dT, and the Gibbs equation: ΔG°(T) =ΔH − TΔS, where ΔCp is the anomalous heat capacity of the oligonucleotide solution during the unfolding process, ΔHcal and ΔScal are the unfolding enthalpy and entropy, respectively, both assumed to be temperature-independent, and ΔG°(T) is the free energy at a temperature T. Determination of the Differential Binding of Counterions. Additional UV melting curves were obtained as a function of salt concentration to determine the differential binding of counterions, ΔnNa+. This ΔnNa+ linking number is measured experimentally using the following relationship:30−33

understanding has been enhanced by thermodynamic investigations of the helix−coil transitions of model oligonucleotide compounds of known sequence, on both the stability and structure of DNA and RNA.18−22 Our laboratory is primarily focused on understanding the folding/unfolding of singlestranded DNA oligomers that are designed specifically to adopt intramolecular structures.23−25 An investigation of the physical properties of their 100% helical conformations over a wider temperature range is due to a lower entropy penalty, yielding transition temperatures higher than their bimolecular counterparts.25 In this work, we present a thermodynamic description of the unfolding of DNA intramolecular stem-loop motifs. Specifically, we used a combination of spectroscopic and calorimetric techniques to investigate the unfolding thermodynamics of two sets of pseudoknots with and without stem-loop complementarity and the unfolding of straight hairpins as a control set of molecules. The overall results show that the second set of pseudoknots is more stable, i.e., more favorable folding free energy terms. This folding is enthalpy driven, and the higher enthalpy terms imply the formation of base triplets (or basetriplet stacks) between the helical stem and complementary loop bases. This is consistent with the pseudoknot targeting reactions that resulted with lower interacting free energies.16

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MATERIALS AND METHODS Materials. All DNA molecules were synthesized by IDT, reverse-phase HPLC purified, desalted on a G-10 Sephadex column, and lyophilized to dryness prior to experiments. The sequences of the 12 stem-loop DNA oligonucleotides, Scheme 1, and their designations are reported in Table 1. The concentration of each oligomer solution was determined from absorbance measurements at 260 nm at 90 °C using the molar absorptivities, in mM−1 cm−1 of strands, reported in the last column of Table 1. These values were calculated by extrapolation of the tabulated values of the dimers and monomer bases from 25 °C to high temperatures, using procedures reported earlier.26,27 Buffer solutions consisted of 10 mM sodium phosphate buffer and pH 7.0, adjusted with different salt concentrations up to 0.2 M NaCl. All chemicals used in this study were reagent grade. Temperature-Dependent UV Spectroscopy (UV Melting Curves). Absorbance versus temperature profiles (UV melting curves) were measured at 260 nm with a thermoelectrically controlled Aviv 14 DS UV/vis spectrophotometer (Lakewood, NJ). The absorbance was scanned with a

Δn Na+ = (ΔHcal /RTM 2)[∂TM /∂ ln(Na +)]

(1)

2

The (ΔHcal/RTM ) term of eq 1 is determined directly from DSC experiments, whereas the term in brackets is determined from the slopes of the plots of TM as a function of the activity of salt, ranging from 16 to 216 mM. The activity term of eq 1 is converted to its concentration term by multiplying it by the 0.9 constant over this range of salt concentration.

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RESULTS AND DISCUSSION We are investigating the thermodynamic stability of three sets of DNA stem-loop motifs (Scheme 1) with a variable loop length. The first set are straight hairpins, the second set are pseudoknots with loops noncomplementary to the stem, while the third one consists of pseudoknots with one of the loops 1940

DOI: 10.1021/jp5116417 J. Phys. Chem. B 2015, 119, 1939−1946

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The Journal of Physical Chemistry B

Figure 1. UV melting curves and concentration dependence. All experiments were performed in 10 mM NaPi buffer pH 7.0; TM (±0.5 °C) using oligonucleotide concentrations ranging from 2 to 75 μM.

hyperchromicities of 15−17% at 260 nm. Our main observation is larger thymine loops increase the TM of the pseudoknots while they decrease the TM of straight hairpins. The general effects can be rationalized in terms of the location of the loop relative to the duplex stem. In the case of the pseudoknots, the loop bases constitute the ceiling of the nearby major groove, making the helical stems less accessible to the solvent, and, therefore, more temperature stable, whereas, in the straight hairpins, the loops are adjacent to the stems and, therefore, more exposed to the solvent. To determine the transition molecularity, we follow the TM as a function of strand concentration at different salt concentrations. These TM dependences are shown in Figure 1B in low salt; we obtained similar TM’s over a 10-fold increase in strand concentration, indicating that each molecule forms intramolecularly at all salts. DSC Unfolding of DNA Stem-Loop Motifs. DSC profiles are shown in Figure 2; the transitions of each stem-loop motif are clearly defined and take place without heat capacity effects. The resulting thermodynamic profiles are shown in Table 2. In this section, the main parameter obtained from DSC is the unfolding enthalpy (ΔHcal); this is discussed in terms of the effect of the loop length, Figure 3A. The unfolding of the straight hairpins (set 1) shows a monophasic transition with ΔHcal’s ranging from 29.2 kcal/mol (Hp-3) and leveling off to 38.9 kcal/mol for the other hairpins with longer loops. This is in excellent agreement with the enthalpy of 38.4−40.9 kcal obtained from nearest-neighbor (N-N) contributions at 1 M NaCl concentration;18,34 these estimated values include two

complementary to the stem. Initially, we use UV melting techniques to follow the temperature unfolding of each molecule, by measuring its TM. We test if this unfolding takes place intramolecularly, by following the dependence of TM on strand concentration. Then, we use DSC to obtain thermograms for the unfolding of each molecule. The UV and DSC results are combined to measure the differential binding of ions for the folding/unfolding of these molecules. The comparison of the thermodynamic results within each set and among all three sets yielded the contributions of varying the loop length. All Hairpins and Pseudoknots Form Intramolecularly at Convenient TM’s. The helix−coil transition of each hairpin molecule is initially characterized by temperature-dependence spectroscopy, Figure 1A. All curves follow the characteristic sigmoidal behavior for the unfolding of a nucleic acid helix. The melting curves for the straight hairpins are monophasic with TM’s decreasing from 81 to 56 °C as the loop length increases from 3 to 9 thymines. The overall effect is a TM decrease of 4.2 °C per loop thymine. This set has hyperchromicities of 20% at 275 nm with the exception of Hp-3, which has a hyperchromicity of 14%. The melting curves for the first type of pseudoknots (set 2) are biphasic having a broad transition with TM’s ranging from 48 to 52 °C and a sharper transition with similar TM’s of 70 °C. This set has hyperchromicities of 20− 23% at 275 nm. The melting curves for the second type of pseudoknots (set 3) are monophasic with TM’s increasing from 53 to 59 °C as the loop increases from 5 to 9 thymines; the effective increase is 1 °C per loop thymine. PsK-11, however, is biphasic with TM’s of 41 and 59 °C. This set also has similar 1941

DOI: 10.1021/jp5116417 J. Phys. Chem. B 2015, 119, 1939−1946

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The Journal of Physical Chemistry B Table 2. Thermodynamic Profiles for the Folding of Hairpins and Pseudoknotsa Set 1 TM (°C)

ΔHcal (kcal/mol)

ΔG°(5) (kcal/mol)

80.7

−29.2

−6.2

TΔS (kcal/mol)

ΔnNa+ (mol of Na+/phosphate)

Hp-3 −23.0

−0.030 (−0.041)

−30.6

−0.033 (−0.054)

−32.6

−0.041 (−0.076)

−33.2

−0.053 (−0.11)

TΔS (kcal/mol)

ΔnNa+ (mol of Na+/phosphate)

Hp-5 73.3

−38.1

−7.5 Hp-7

62.8

−39.4

−6.8 Hp-9

56.2

−39.3

−6.1 Set 2

TM (°C)

ΔHcal (kcal/mol)

ΔG°(5) (kcal/mol)

48.1 70.2

−28.4 −49.4 −77.8

−3.8 −9.4 −13.2

−31.4 −51.9 −83.3

−4.5 −9.9 −14.4

−33.4 −63.0 −96.4

−4.9 −12.0 −16.9

−38.1 −63.2 −101.3

−5.1 −12.0 −17.1

TM (°C)

ΔHcal (kcal/mol)

ΔG°(5) (kcal/mol)

52.8

−60.1

−8.5

56.2

−86.4

−13.2

59.3

−87.5

−14.2

41.2 58.7

−26.4 −83.6 −110

−3.0 −13.6 −16.6

52.0 70.4

52.6 70.7

Figure 2. DSC unfolding curves for each set of molecules. DSC experiments were performed in 10 mM NaPi, 100 mM NaCl buffer pH 7.0 using oligonucleotide concentrations ranging from 50 to 130 μM.

48.3 70.2

thymines stacked at the 3′ end of the d(GCGC)2 stem. The sudden increase in the enthalpy (9.1 kcal/mol) between Hp-3 and Hp-5 has to do with improved base-pair stacking of the stem, due to the less constrained loop of five thymines, Figure 3A. The first set of pseudoknots (set 2) showed biphasic transitions, characteristic of the unfolding of stems with different sequences. The GAAATT/AATTTC stem unfolds first followed by the CGCGCG/CGCGCG stem. The increase in the length of both loops from 4 to 6 thymines yielded total ΔHcal’s of 77.8 kcal/mol (PsK-4,4) and 83.3 kcal/mol (PsK6,6). This enthalpy increase (∼6 kcal) can be attributed to better base-pair stacking of each stem, as seen with the straight hairpins when the length of their loops changes from 3 to 5 thymines. However, these enthalpy values are much lower than the calculated N-N enthalpy of 97.1 kcal/mol34 or 106 kcal/ mol18 for the stem of these pseudoknots, which assumes that the central base pairs are forming a complete base-pair stack. The lower enthalpy values imply poor base-pair stacking of the stems due to the induced effects of the constrained loops. In addition, the difference in the experimental and predicted values is the salt concentration used. The experimental enthalpy values are obtained in 116 mM Na+, while the predicted value from N-N is in 1 M NaCl. When the increase in the length of the left side loop (Scheme 1) was considered, we obtained total enthalpies of 77.8 kcal/mol (Psk-4,4), 96.4 kcal/mol (Psk-4,6), and 101.3 kcal/mol (Psk-4,8); see Figure 3A. The sudden increase in the enthalpy (18.6 kcal/mol) between Psk-4,4 and Psk-4,6 yielded an improvement of the base-pair stacking in the stem, due to the less constrained loop of six thymines. This

PsK-4,4 −24.6 −40.0 −64.6 PsK-6,6 −26.9 −42.0 −68.9 PsK-4,6 −28.5 −51.0 −79.5 PsK-4,8 −33.0 −51.2 −84.2 Set 3 TΔS (kcal/mol) PsK-5 −51.6 PsK-7 −73.2 PsK-9 −73.3 PsK-11 −23.4 −70.0 −93.4

−0.071 (−0.10)

−0.069 (−0.11)

−0.087 (−0.13)

−0.087 (−0.14) ΔnNa+ (mol of Na+/phosphate) −0.073 (−0.11) −0.085 (−0.14) −0.079 (−0.14)

−0.098 (−0.18)

a

All experiments were done in 10 mM NaPi buffer, 100 mM NaCl at pH 7. Experimental errors are as follows: TM (±0.5 °C), ΔHcal (±5%), TΔScal (±5%), ΔG°(5) (±7%). ΔnNa+ (±12%) values in parentheses are in mol of Na+/helical phosphate.

effect is followed by a leveling off of the enthalpy with Psk-4,8, resulting in a value consistent with the average enthalpy value of 102 kcal obtained from the N-N parameters. Most pseudoknots in set 3 unfold through asymmetric monophasic transitions due to the similar TM’s of the two stems; however, PsK-11 unfolds through a biphasic transition. The increase in the length of the right side loop (Scheme 1) from 5 to 11 thymines yielded total ΔHcal’s of 60.1 kcal/mol (PsK-5), 86.4 kcal/mol (PsK-7), 87.5 kcal/mol (PsK-9), and 110 kcal/mol (PsK-11). The predicted enthalpies from N-N parameters are 87.3 kcal/mol34 and 90.5 kcal/mol18 for the duplex stem of this set of pseudoknots; however, we obtained 1942

DOI: 10.1021/jp5116417 J. Phys. Chem. B 2015, 119, 1939−1946

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Figure 3. Enthalpy and ΔnNa+ as a function of the size of the thymine loop. All experiments were done in 10 mM NaPi buffer and 100 mM NaCl at pH 7.0; ΔHcal (±5%), ΔnNa+ (±12%).

experimentally a ΔHcal value of 69.4 kcal/mol in 116 mM Na+ for this stem duplex with two dangling thymines at each end (TCTCTTTTTTTT/TTAAAAAAAAGAGATT), in order to prevent fraying35 (data not shown). The unfolding enthalpy of 69.4 kcal/mol, actually 65.2 kcal/mol if the middle AA/TT is considered as partially stacked, indicates that in PsK-5 there is either some constraint on the stem and/or fraying at the 3′ end of the AT base pairs. In this second set of pseudoknots, it is important to emphasize that the loop is complementary to the corresponding stem and an increased unfolding enthalpy would correspond to formation of a local triplex. The loop is also nearby the major groove of the stem and is able to form TAT base triplets. For instance, when the length of the loop on the right side (Scheme 1) is 7, 9, or 11 thymines, higher enthalpy terms are observed. Relative to the control duplex and using an enthalpy of 65.2 kcal/mol, we obtained enthalpy increases of 21.2 kcal/mol (PsK-7), 22.3 kcal/mol (PsK-9), and 44.8 kcal/ mol (PsK-11), respectively. These enthalpies can be attributed to improving base-pair stacking of the stem, closing the fraying of the ends, and formation of base triplets. We estimated an enthalpy of 5.1 kcal/mol, by subtracting the enthalpy of PsK-5 (60.1 kcal/mol) from the corrected enthalpy of the stem duplex (65.2 kcal/mol), for these first two contributions, yielding excess enthalpies of 16.1 kcal/mol (PsK-7), 17.2 kcal/mol (PsK9), and 39.7 kcal/mol (PsK-11). The enthalpy of a TAT/TAT base-triplet stack has been determined to be equal to 24.0 kcal/ mol,36 and the enthalpy of the AA/TT base-pair stack is considered to be ∼8.5 kcal/mol.18,37,34 This means that an enthalpy of 15.5 kcal/mol is needed to form a single base-triplet stack from the addition of the loop to complement the duplex stem. By dividing the excess enthalpy of each pseudoknot by 15.5 kcal/mol, we estimate PsK-7 and PsK-9 form one TAT base-triplet stack, while PsK-11 forms 2−3 TAT/TAT basetriplet stacks. Differential Binding of Counterions in the Folding of DNA Stem-Loop Motifs. We obtained UV melting curves as a function of salt concentration (data not shown), 16−216 mM of Na+, to determine the slope of the TM dependences on salt concentration. This data in combination with the DSC thermograms allows us to determine the differential binding of ions according to eq 1. Generally, as the concentration of sodium increases, the helical−coil transitions are shifted to higher temperatures; these TM dependences are shown in Figure S1 (Supporting Information). This is consistent with the stabilizing effect of cations on nucleic acid duplexes due to their larger charge density parameter.38 The resulting ΔnNa+ values

are summarized in the last column of Table 2. These values were calculated first in terms of the total number of phosphates. We obtained values ranging from 0.030 to 0.053 (set 1), 0.071 to 0.087 (set 2), and 0.073 to 0.098 (set 3); these values are small but consistent with counterion uptake by short DNA duplexes of 4−10 base pairs.39,40 In general, the ΔnNa+ values increase with the increase in the length of the thymine loop: specifically, we obtained a net uptake of 0.004 mol of Na+/ phosphate-thymine loop for all three sets of stem-loop motifs. However, a better assessment for the differential binding of counterions is to consider just the phosphates associated with the helical stems; this will include two extra loop phosphates for each molecule and considers the other loop phosphates to behave like random coil phosphates, i.e., 8 helical phosphate groups (set 1) and 22 helical phosphate groups for sets 2 and 3. The resulting values are shown in parentheses in the last column of Table 2, and Figure 3B shows their dependence on loop length. The increase in the loop length yielded a net uptake in mol of Na+ per helical phosphate-per thymine of the following: 0.011 (set 1), 0.075 (set 2), and 0.011 (set 3). The molecules begin to approach polymer behavior. Furthermore, the molecules with larger loops, Hp-9, PsK-4,8, and PsK-11, show higher counterion uptake of 0.11, 0.14, and 0.18, respectively. PsK-11 shows the largest uptake of ions, a value in excess when compared to a phosphate in the duplex state of a DNA polymer, ∼0.17 mol of Na+/mol of Pi. This can be rationalized in terms of some loop thymines forming Hoogsteen base pairing with the stem, which positions their phosphates in a triple helical state. This speculation is consistent with the formation of base-triplet stacks that was observed from the analysis of the folding enthalpies, in spite of ion binding being a long-range interaction as opposed to the short-range interactions of base-pair stacking. Thermodynamic Profiles for the Folding of DNA Hairpins and Pseudoknots. Standard folding thermodynamic profiles at 5 °C for each transition of each stem-loop motif are summarized in Table 2 and are shown as bar graphs, Figure 4. Inspection of this table indicates that the folding of each molecule is accompanied by favorable free energy terms resulting from the compensation of favorable enthalpy and unfavorable entropy terms, which is characteristic for the folding of a nucleic acid. In general, these favorable enthalpy terms correspond mainly to the formation of base pairs, and base-triplet stacks (for some pseudoknots of set 3), as discussed in the previous sections. The unfavorable entropy terms arise from contributions of the ordering of strands and the uptake of 1943

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energy term decreased, making the formation of the hairpin more unfavorable. In set 2, the first set of pseudoknots, PsK-4,4, yielded a ΔG° value of −13.3 kcal/mol, while we measured ΔG° values of −16.9 and −17.1 kcal/mol for PsK-4,6 and PsK4,8, respectively. This initial increase in stability (−1.2 kcal/ mol) can be attributed to a more relaxed molecule with better base-pair stacking. Thus, when looking at the pseudoknots with a loop large enough to release a constraint on the stem, a ΔG° value of −0.1 kcal/mol per thymine in the loop is obtained. The increase in the loop length increases the formation of the pseudoknot with a slightly more favorable ΔG° term. In the third set of molecules, ΔG° ranges from −13.2 to −16.6 kcal/mol; however, the stem in PsK-5 is not formed completely and only has a ΔG° value of −8.5 kcal/mol. The effect of increasing the length of the loop is −0.9 kcal/mol per thymine, indicating as the loop length increases the formation of the pseudoknot in which the loop is complementary to the stem is much more favorable. This increase in free energy can be attributed to the loop being in the major groove and forming base-triplet stacks. This increase in stability has been confirmed in a targeting reaction, where the targeting of PsK-5 is more favorable than PsK-9 even though PsK-9 has a larger number of bases in the loop, which typically drives the reaction.16 In summary, the effect of increasing the length of the loops of straight hairpins is entropy driven, yielding unfavorable free energy terms, while the effect is enthalpy driven for the pseudoknots, yielding favorable free energy terms. Furthermore, this favorable enthalpy contribution was larger for the pseudoknots forming base-triplet stacks.

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CONCLUSIONS We have investigated the unfolding thermodynamics of DNA straight hairpins and pseudoknots with and without sequences complementary to the stem to determine the effect of loop length on the stability of DNA stem-loop molecules. Specifically, we were interested in whether or not formation of a local and short triplex in pseudoknots with appropriate loop length and sequence is favorable. We determined the unfolding thermodynamics of three sets of stem-loop motifs: hairpin loops, control pseudoknots, and triplex forming pseudoknots using melting techniques. The favorable folding of DNA molecules results from the typical compensation of favorable enthalpy and unfavorable entropy terms. This confirms the flexibility of DNA oligonucleotides being able to form hairpins and pseudoknots; therefore, they can be used to mimic known RNA secondary structures. The folding thermodynamic data shows that pseudoknots with the appropriate sequence and loop length are more stable due to a more favorable enthalpic contribution. This enthalpy value is due to the partial folding of the thymine loop (third strand) on the major groove of the duplex, forming one to three TAT/ TAT base-triplet stacks at the middle of its stem, depending on the loop length.

Figure 4. Thermodynamic profiles for DNA stem-loop motifs. All experiments were done in 10 mM NaPi buffer and 100 mM NaCl at pH 7.0. Experimental errors are as follows: ΔG°(5) (±7%), ΔHcal (±5%), TΔScal (±5%).

counterions and the putative immobilization of water molecules. In all sets, the entropy term becomes more unfavorable as the loop length increases, which is due to a counterbalance between the increased disorder of the loop and the higher uptake of ions. If the first molecule with shorter loops in each set was not considered, we obtained an entropic (TΔS) effect on the loop length of 0.7 kcal/mol (set 1), 2.5 kcal/mol (set 2), and 5.1 kcal/mol (set 3). This shows that the pseudoknots are more ordered with a higher uptake of counterions, meaning that these molecules most likely are more compact. In set 3, there is a greater effect in the unfavorable entropy term due to the loop thymines coming into the ceiling of the major groove, forming base-triplet stacks and creating additional ordering in the system. In terms of the free energy contributions for the folding of each molecule at 5 °C and neglecting the first molecule with shorter loops in each set (Table 2), we obtained a ΔG° value ranging from −7.5 to −6.1 kcal/mol (set 1), corresponding to ∼0.4 kcal/mol difference in free energy per thymine in the loop. Meaning, as the length of the loop increased the free

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ASSOCIATED CONTENT

S Supporting Information *

Figure showing TM dependences on salt concentration for each molecule. The increase in the concentration of sodium shifts the helical−coil transitions to higher temperatures; i.e., salt favors the helical state, which has the higher charge density parameter. This material is available free of charge via the Internet at http://pubs.acs.org. 1944

DOI: 10.1021/jp5116417 J. Phys. Chem. B 2015, 119, 1939−1946

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(18) Breslauer, K. J.; Frank, R.; Blocker, H.; Marky, L. A. Predicting DNA duplex stability from base sequence. Proc. Natl. Acad. Sci. U. S. A.. 1986, 83, 3746−3750. (19) Xia, T.; SantaLucia, J., Jr.; Burkard, M. E.; Kierzek, R.; Schroeder, S. J.; Jiao, X.; Cox, C.; Turner, D. H. Thermodynamic parameters for an expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick base pairs. Biochemistry 1998, 37, 14719−14735. (20) SantaLucia, J., Jr.; Allawi, H. T.; Seneviratne, P. A. Improved nearest-neighbor parameters for predicting DNA duplex stability. Biochemistry 1996, 35, 3555−3562. (21) Sugimoto, N.; Nakano, S.; Katoh, M.; Matsumura, A.; Nakamuta, H.; Ohmichi, T.; Yoneyama, M.; Sasaki, M. Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. Biochemistry 1995, 34, 11211−11216. (22) Rentzeperis, D.; Shikiya, R.; Maiti, S.; Ho, J.; Marky, L. A. Folding of intramolecular DNA hairpin loops: Enthalpy-entropy compensations and hydration contributions. J. Phys. Chem. B 2002, 106, 9945−9950. (23) Marky, L. A.; Maiti, S.; Olsen, C. M.; Shikiya, R.; Johnson, S. E.; Kaushik, M.; Khutsishvili, I. Building Blocks of nucleic acid nanostructures: unfolding thermodynamics of intramolecular DNA complexes. In Biomedical Applications of Nanotechnology; Labhasetwar, V., Leslie-Pelecky, D., Eds.; John Wiley & Sons, Inc.: Hoboken, NJ, 2007; pp 191−226. (24) Lee, H.-T.; Olsen, C. M.; Waters, L.; Sukup, H.; Marky, L. A. Thermodynamic contributions of the reactions of DNA intramolecular structures with their complementary strands. Biochimie 2008, 90, 1052−1063. (25) Lee, H.-T.; Carr, C.; Siebler, H.; Waters, L.; Khutsishvili, I.; Iseka, F.; Domack, B.; Olsen, C. M.; Marky, L. A. A thermodynamic approach for the targeting of nucleic acid structures using their complementary single strands. Methods Enzymol. 2011, 492, 1−26. (26) Cantor, C. R.; Schimmel, P. R. The conformation of biological macromolecules. Biophysical Chemistry; W.H. Freeman: San Francisco, CA, 1980. (27) Marky, L. A.; Blumenfeld, K. S.; Kozlowski, S.; Breslauer, K. J. Salt-dependent conformational transitions in the self-complementary deoxydodecanucleotide d(CGCAATTCGCG): evidence for hairpin formation. Biopolymers 1983, 22, 1247−1257. (28) Marky, L. A.; Breslauer, K. J. Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves. Biopolymers 1987, 26, 1601−1620. (29) Marky, L. A.; Maiti, S.; Olsen, C. M.; Shikiya, R.; Johnson, S. E.; Kaushik, M.; Khutsishvili, I. Building blocks of nucleic acid nanostructures: unfolding thermodynamics of intramolecular DNA complexes. In Biomedical Applications of Nanotechnology; Labhasetwar, V., Leslie-Pelecky, D., Eds.; Wiley-Interscience: Hoboken, NJ, 2007; pp 191−225. (30) Kaushik, M.; Suehl, N.; Marky, L. A. Calorimetric unfolding of the bimolecular and i-motif complexes of the human telomere complementary strand, d(C3TA2)4. Biophys. Chem. 2007, 126, 154− 164. (31) Khutsishvili, I.; Zhang, N.; Marky, L. A.; Crean, C.; Patel, D. J.; Geacintov, N. E.; Shafirovich, V. Thermodynamic profiles and nuclear magnetic resonance studies of oligonucleotide duplexes containing single diastereomeric spiroiminodihydantoin Lesions. Biochemistry 2013, 52, 1354−1363. (32) Lee, H.-T.; Khutsishvili, I.; Marky, L. A. DNA complexes containing joined triplex and duplex motifs: melting behavior of intramolecular and bimolecular complexes with similar sequences. J. Phys. Chem. B 2011, 114, 541−548. (33) Khutsishvili, I.; Johnson, S.; Lee, H.-T.; Marky, L. A. Unfolding thermodynamics of DNA intramolecular complexes involving joined triple- and double-helical motifs. Methods in Enzymology; Academic Press: San Diego, CA, 2009; Vol. 466, Chapter 20, pp 477−502. (34) SantaLucia, J., Jr. A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Biochemistry 1998, 95, 1460−1465.

AUTHOR INFORMATION

Corresponding Author

*Phone: (402) 559-4628. Fax: (402) 559-9543. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants MCB-0616005 and MCB1122029 from the National Science Foundation and GAANN grant P200A120231 (C.R.) from the U.S. Department of Education.

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REFERENCES

(1) Moore, C. B.; Guthrie, E. H.; Huang, M.T-H.; Taxman, D. J. Short hairpin RNA (shRNA): design, delivery, and assessment of gene knockdown. Methods Mol. Biol. 2010, 629, 141−158. (2) Rich, A. DNA comes in many forms. Gene 1993, 135, 99−109. (3) Bikard, D.; Loot, C.; Baharoglu, Z.; Mazel, D. Folded DNA in action: hairpin formation and biological functions in prokaryotes. Microbiol. Mol. Biol. Rev. 2010, 74, 570−588. (4) Son, Y. M.; Lee, J. H.; Kim, D. R. Modulation of RAG/DNA complex by HSP70 in V(D)J recombination. Biochem. Biophys. Res. Commun. 2008, 365, 113−117. (5) Brown, M. L.; Schroth, G. P.; Gottesfeld, J. M.; Bazett-Jones, D. P. Protein and DNA requirements for transcription factor IIIA-induced distortion of the 5 S rRNA gene promoter. J. Mol. Biol. 1996, 262, 600−614. (6) Carr, K. M.; Kaguni, J. M. Escherichia coli DnaA protein loads a single DnaB helicase at a DnaA box hairpin. J. Biol. Chem. 2002, 277, 39815−39822. (7) Adams, P. L.; Stahley, M. R.; Kosek, A. B.; Wang, J.; Strobel, S. A. Crystal structure of a self-splicing group I intron with both exons. Nature 2004, 430, 45−50. (8) Rastogi, T.; Beattie, T. L.; Olive, J. E.; Collins, R. A. A long-range pseudoknot is required for activity of the Neurospora VS ribozyme. EMBO J. 1996, 15, 2820−2825. (9) Ke, A.; Zhou, K.; Ding, F.; Cate, J. H.; Doudna, J. A. A conformational switch controls hepatitis delta virus ribozyme catalysis. Nature 2004, 429, 201−205. (10) Isambert, H.; Siggia, E. D. Modeling RNA folding paths with pseudoknots: application to hepatitis delta virus ribozyme. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 6515−6520. (11) Huard, T. S.; Autexier, C. Targeting human telomerase in cancer therapy. Curr. Med. Chem.: Anti-Cancer Agents 2002, 2, 577−587. (12) Theimer, C. A.; Blois, C. A.; Feigon, J. Structure of the human telomerase RNA pseudoknot reveals conserved tertiary interactions essential for function. Mol. Cell 2005, 17, 671−682. (13) Souliere, M. F.; Altman, R. B.; Micura, R. Tuning a riboswitch response through structural extension of a pseudoknot. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 3256−3264. (14) Huang, X.; Yang, Y.; Wang, G.; Cheng, Q.; Du, Z. Highly conserved RNA pseudoknots at gag-pol junction of HIV-1 suggest a novel mechanism for −1 ribosomal frameshifting. RNA 2014, 20, 587−593. (15) Chen, G.; Chang, K. Y.; Chou, M. Y.; Bustamante, C.; Tinoco, I., Jr. Triplex structures in an RNA pseudoknot enhance mechanical stability and increase efficiency of −1 ribosomal frameshifting. Proc. Natl. Acad. Sci. U. S. A. 2009, 106, 12706−12711. (16) Reiling, C.; Marky, L. A. Loop contributions on the stability and targeting of DNA pseudoknots. Biochem. Compd. 2014, 2, 10.7243/ 2052-9341-2-3. (17) Khutsishvili, I.; Johnson, S. E.; Reiling, C.; Prislan, I.; Lee, H.-T.; Marky, L. A. Interaction of DNA intramolecular structures with their complementary strands: a thermodynamic approach for the control of gene expression. Chemical Biology of Nucleic Acids; Springer-Verlag: Berlin, Heidelberg, 2014; Chapter 20, pp 367−383. 1945

DOI: 10.1021/jp5116417 J. Phys. Chem. B 2015, 119, 1939−1946

Article

The Journal of Physical Chemistry B (35) Senior, M.; Jones, R. A.; Breslauer, K. J. Influence of dangling thymidine residues on the stability and structure of two DNA duplexes. Biochemistry 1988, 27, 3879−3885. (36) Soto, A. M.; Loo, J.; Marky, L. A. Energetic contributions for the formation of TAT/TAT, TAT/CGC(+), and CGC(+)/CGC(+) base triplet stacks. J. Am. Chem. Soc. 2002, 124, 14355−14363. (37) Alessi, K. Thermodynamic contributions of deoxyoligonucleotides containing A-T base-pairs. Ph.D. dissertation, New York University, 1995. (38) Nakano, S.-I.; Fujimoto, M.; Hara, H.; Sugimoto, N. Nucleic acid duplex stability: influence of base composition on cation effects. Nucleic Acids Res. 1999, 27, 2957−2965. (39) Manning, G. S. The molecular theory of polyelectrolyte solutions with applications to the electrostatic properties of polynucleotides. Q. Rev. Biophys. 1978, 11, 179−246. (40) Record, M. T., Jr.; Anderson, C. F.; Lohman, T. M. Thermodynamic analysis of ion effects on the binding and conformational equilibria of proteins and nucleic acids: The roles of ion association or release, screening, and ion effects on water activity. Q. Rev. Biophys. 1978, 11, 103−178.

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DOI: 10.1021/jp5116417 J. Phys. Chem. B 2015, 119, 1939−1946