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Structural Insight on the Unbound State of the DNA Analog of the preQ Riboswitch: A Thermodynamic Approach 1

Calliste Reiling-Steffensmeier, and Luis A. Marky Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00596 • Publication Date (Web): 27 Oct 2017 Downloaded from http://pubs.acs.org on November 1, 2017

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Biochemistry

Structural Insight on the Unbound State of the DNA Analog of the preQ1 Riboswitch: A Thermodynamic Approach

Calliste Reiling-Steffensmeier and Luis A. Marky* Dept. of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, NE 68198-6025, United States

*Corresponding author: [email protected]

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ABSTRACT The preQ1 riboswitch aptamer domain is very dynamic in its unbound state with the ability to form multiple structures: a hairpin, kissing hairpins, and pseudoknot-like structure. The aim of this study is to determine whether or not the DNA analog (PreQ1) is able to form similar structures as the reported RNA aptamer. Using a thermodynamic approach, we report on structural determination using differential scanning calorimetry at different salt conditions. Further analysis of the primary sequence allowed us to design modified molecules to determine what potential structures are forming in this single stranded DNA analog. We found, in 16 mM Na+ solution, PreQ1 has three transitions with TMs of 14.8, 19.4 and 26.2 °C and total ∆H of -44.7 kcal/mol. With the increase in salt to 116 mM, there are TMs of 22.3, 28.7 and 38.9°C and ∆H of -69.1 kcal/mol, while at 216 mM, the three transitions have TMs of 24.4, 31.6 and 42.9°C with a total ∆H of -71.5 kcal/mol. Therefore, the increase in enthalpy is due to the formation of additional base-pair stacks. The modified molecules, which would inhibit pseudoknot formation, kissing hairpins, and internal loop interactions, were fully characterized and compared to the native DNA analog. The analysis of the enthalpy and differential binding of counterions, allows us to conclude this single stranded DNA analog under physiological conditions is not forming a pseudoknot-like structure. Instead, two potential structures: a Compact-Hairpin and a Kissing-Complex are more likely and could be in equilibrium.

Keywords: DNA Secondary Structures; Hairpin; Kissing Thermodynamics; Differential Scanning Calorimetry

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Hairpins;

Pseudoknots;

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INTRODUCTION In principle, the folding of nucleic acids is controlled by the local base sequence in a precise and potentially predictable way.

Knowledge of the structure of a particular duplex alone cannot

provide an understanding of the forces responsible for maintaining the distinct structures in nucleic acids. The folding of nucleic acids is dependent on the contributions from base pairing, base stacking, ion binding and hydration.1–5 In nature DNA usually exists in a double stranded helical state, while RNA is found in a single-stranded state of shorter lengths.6,7 The discovery of chromosomal sequences have led scientists to postulate the formation of non-canonical DNA structures in the duplex state, examples include: telomeres,8–10 centromeres11–14 and tribase repeats.15,16 Telomeres and centromeres are required for the proper replication and segregation of eukaryotic chromosomes through the cell cycle;8,9 telomeres are specialized structures that stabilize the ends of linear chromosomes9 while centromeres play a fundamental role in chromosome movement during mitosis and meiosis.11,12 On the other hand, RNA molecules, like tRNA, mRNA and rRNA fold into a variety of secondary and tertiary structures.6,7,17 The formation and biological functions of RNA molecules is well documented;10,17–23 for example, short hairpin RNA (shRNA) is used to control gene expression.24–27 Riboswitches are also involved in the control gene expression and in monitoring the cellular environment by the interaction of their Aptamer with the particular ligand, which has downstream effects on the expression platform.28–31 It it has been shown that single stranded DNA (ssDNA) can form hairpin structures that are involved in site-specific recombination, transcription, and replication.32–34 The formation of other secondary structures are also possible which has been studied extensively by our laboratory; some examples include: small hairpin loops, pseudoknots, and hairpins containing internal loops

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and bulges.20,21,35–38 There are also molecular dynamic studies showing DNA is able to form pseudoknots39 and structural studies showing DNA hairpins have kissing interactions, similar to RNA.40,41 The purpose of this study is to use unfolding thermodynamics as a tool for determining the ability of ssDNA to form different secondary structures. We have investigated a DNA oligonucleotide with the same sequence of the preQ1 riboswitch aptamer domain. The reason for choosing this riboswitch is due to its conformational flexibility, which is able to form multiple structures that have been structurally characterized. These structures include: a pre-folded like state similar to that of the H-type pseudoknot31 and a hairpin/kissing hairpin complex.42 Therefore, the objective is to determine if this DNA analog has the conformational plasticity to form any or all of these secondary conformations. Ultimately, we would like to determine from a thermodynamic prospective which structures are the most favorable for this ssDNA to form: a pseudoknot, hairpin, and/or kissing hairpins. In this work, we used temperature-dependent UV spectroscopy and differential scanning calorimetry to determine the folding/unfolding thermodynamics of a DNA analog (PreQ1), derived from the sequence of the preQ1 riboswitch aptamer domain and several single stranded DNA control molecules. We found that this DNA analog is likely forming an intramolecular hairpin with internal loop interactions and/or a bimolecular complex of kissing hairpins.

MATERIALS AND METHODS Materials. All oligonucleotides were synthesized by the Integrated DNA Technologies (IDT) (Coralville IA), HPLC purified and desalted by column chromatography using G-10 Sephadex exclusion chromatography. The sequences of oligonucleotides used in this work and their

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designation are shown in Figure 2. The concentrations of the oligomer solutions were determined at 260 nm and 90°C using an Aviv Spectrophotometer Model 14DS UV-Vis using the molar extinction coefficients: 376 mM-1 cm-1 (PreQ1), 305 mM-1 cm-1 (PreQ1-T5), 361mM-1 cm-1 (PreQ1-ACCC) 366 mM-1 cm-1 (PreQ1-TT-Loop), 366 mM-1 cm-1 (PreQ1-C-Loop). These values were obtained by extrapolation of the tabulated values for dimers and monomeric bases43,44 at 25°C to 90°C using procedures reported previously.38,45 Inorganic salts from Sigma were reagent grade and used without further purification. All oligonucleotide solutions were prepared by dissolving the dry and desalted oligonucleotides in buffer. Temperature-Dependent UV Spectroscopy. Absorbance versus temperature profiles were measured at 260 nm with a thermoelectrically controlled Aviv Spectrophotometer Model 14DS UV-Vis (Lakewood, NJ). The temperature was scanned at a heating rate of 0.6 °C/min, and shape analysis of the melting curves yielded transition temperatures, TMs.46 The transition molecularity for the unfolding of a particular complex was obtained by monitoring TM as a function of the strand concentration. Intramolecular complexes show a TM-independence on strand concentration, while the TM of intermolecular complexes does depend on strand concentration.38 Differential Scanning Calorimetry. The total heat required for the unfolding of each oligonucleotide was measured with a VP-DSC differential scanning calorimeter from Microcal (Northampton, MA). Standard thermodynamic profiles and TMs are obtained from the DSC experiments using the following relationships38,46: ∆H = ∫∆Cp(T)dT; ∆Scal = ∫∆Cp(T)/TdT, and the Gibbs equation, ∆G°(T) =∆H - T∆S; where ∆Cp is the anomalous heat capacity of the ODN solution during the unfolding process, ∆H and ∆S are the unfolding enthalpy and entropy,

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respectively, assumed to be temperature-independent. ∆G°(T) is the free energy at temperature T, 5 °C. Determination of the Differential Binding of Counterions. Additional DSC melting curves, as a function of salt concentrations, were performed to determine the differential binding of counterions, ∆nNa+, between the folded and coil states. The ∆nNa+ linking number represent the uptake (or release) of counterions, for the helix-coil transition of each complex, and are measured experimentally using the equation: +

∆nNa+=1.11 [∆H/RTM2] (∂TM/∂ln [Na ])

(1)

The 1.11 value is a constant which is used to convert solution activities into concentrations and ∆H/RTM2 is an average between the values obtained from DSC in three salt conditions.

RESULTS and DISCUSSION Examination of Potential Secondary Structures. Given the question of whether or not this DNA sequence has the ability to form multiple conformations, we have examined the primary sequence to determine potential secondary structures that may be forming. The sequence of PreQ1 seems to be straight forward when it comes to the structure of the unbound state, a hairpin with a 5’ dangling end and long 3’ tail (Figure 1A). Using nearest-neighbor values determined at 1 M NaCl,47 we have calculated a nearest-neighbor enthalpy (ΔHNN) of -31.8 kcal/mol. This however, may not be what is actually forming. When considering the formation of both intramolecular and intermolecular structures there are other structures to consider besides this simple hairpin. First, the intramolecular structures will be explained followed by the intermolecular ones.

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ΔHNN = -31.8 kcal/mol






ΔHNN = -142.2 kcal/mol Figure 1. Potential Structures of PreQ1 Based on Its Sequence. These structures have been determined after further analysis of the PreQ1 sequence. They include both monomolecular (A-E) and bimolecular (F and G) structures.

The first intramolecular structure is extending off of the hairpin shown in Figure 1A, with the 5’ dGG end forming a strong mismatch with the first flanking 3’ dA in the 3’ tail (GA) and a dG•dT wobble base-pair with the following dT (Figure 1B) yielding a ΔHNN value of -47.1 kcal/mol. Building upon this structure, the 3’ tail has the potential to form an dA•dT base-pair

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with two AA mismatches when folding back onto itself (Figure 1C), increasing the ΔHNN to -54.5 kcal/mol. Inclusion of the internal loop interactions would increase the ΔHNN to -69.9 or -62.5 kcal/mol (with and without tail interaction, respectively) (Figure 1D). Finally, a possibility for this analog is to form a pseudoknot with a ΔHNN of -69.8 kcal/mol (Figure 1E). Two potential intermolecular structures are considered. The first one is a bimolecular kissing complex with 6 base-pairs formed between the loop bases of two hairpins (Figure 1F), which contribute with a ΔHNN of -39.8 kcal/mol; this kissing complex would have a total ΔHNN of -94.3 or -86.9 kcal/mol, depending on whether the 3’ tail is forming the dA•dT base-pair as shown in Figure 1C. The final structure is a bimolecular duplex containing 10 dA•dT and 8 dG•dC base-pairs, two dG•dT wobble base-pairs, and 6 interspaced mismatches (Figure 1G). When considering all of these base-pair stacking interactions a ΔHNN of -142.2 kcal/mol is predicted. Due to the above structural manifold, multiple molecules have been designed (see Figure 2) and studied to determine the structure/structures that are forming in this DNA analog. For instance, PreQ1-T5 should show the enthalpic contribution from the stem of the hairpin and any tail interactions from the dangling end, while PreQ1-ACCC would inhibit pseudoknot formation due to the modifications in the 3’ tail that are no longer complementary to the hairpin loop. Furthermore, PreQ1-TT-Loop would inhibit the formation of the kissing complex because the loops are no longer complementary to each other. Finally, PreQ1-C-Loop would minimize the closing of the hairpin loop due to unfavorable internal loop interactions. These structures have been examined in the same way as the native sequence to rule out any potential structures other than the ones intended and no other structures were determined.

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Biochemistry

Figure 2. Sequence, Structures and Designation of the Molecules Investigated. Modifications were made to the PreQ1 sequence at both the 3’ tail and loop sequence to help determine the structure of PreQ1.

UV Melts Showed all Modified Oligonucleotides Form Intramolecular Complexes at Low Salt. Typical UV melting curves of PreQ1 and modified molecules at 260 nm and 10 mM NaPi, pH 7 are shown in Figure 3A. All curves are sigmoidal with hyperchromicities ranging from 10% to 15%. The objective is to determine the molecularity of each transition by following the TM as a function of strand concentration (Figure 3B). If the TM remains constant then it is considered intramolecular, otherwise complex formation is intermolecular.46 In this case, all molecules formed intramolecularly due to similar TMs over a 10-fold increase in strand concentration as shown in Figure 3B. Analysis of the UV melting curves and the average of the van’t Hoff enthalpies indicate PreQ1 has a TM of 20.3°C and ΔHvH of 25 kcal/mol. When

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changing the loop to five thymines (PreQ1-T5), there is an increase in the TM to 37.6°C and ΔHvH of 28 kcal/mol. This increase in the TM is an effect of the loop length, as the loop length of the hairpins decreases the TM increases, previously shown to be 4.2°C per loop thymine in straight hairpin loops.36 If the loop of PreQ1 behaved as a 12 thymine base loop, one would expect the TM of PreQ1 to be 29.4°C lower than that of PreQ1-T5. The observation that it is only 17.3°C lower indicates that there are interactions occurring in the loop of PreQ1, because of its higher TM. Modification of the 3’ tail (PreQ1-ACCC) yielded a TM = 18.3°C with a ΔHvH of 30 kcal/mol. The loop modifications, replacing a GC for a TT (PreQ1-TT-Loop) results in a TM of 19.8°C and ΔHvH of 32 kcal/mol, while replacement of the right side of the loop to cytosines (PreQ1-C-Loop) results in a TM of 23.5°C and ΔHvH of 33 kcal/mol. Overall, from UV melts it appears that there is little effect on the TM and ΔHvH as modifications are made to the primary sequence, the exception is the effect seen by decreasing the loop length. Therefore, it appears as though a similar structure is forming for all of the molecules at this lower concentration of salt. Furthermore, even though these molecules show TM-independence, the long duplex (Figure 1G) cannot be fully eliminated as a possible structure due to its length of 20 base-pairs, which would have reached polymer melting behavior, i.e., pseudo first order transition.48 Also, the kissing complex (Figure 1F) cannot be ruled out yet because the formation of 6 base-pairs at low salt conditions is unfavorable.49 Therefore, the next experiments performed with DSC have been carried out as a function of salt concentration to help determine if intermolecular interactions take place and to determine if salt further stabilizes these structures.

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Figure 3. UV Melting Curves and TM Dependence on Concentration. All molecules under low salt conditions were found to be intramolecular due to the TM being independent of strand concentration. All experiments were carried out in 10 mM Sodium Phosphate buffer at pH 7.0; TM (±0.5°C). Concentrations of total strands ranged between 1-16 µM

DSC Unfolding of PreQ1 and Modified Molecules to Determine Structure Formation.

The

DSC unfolding for PreQ1 and modified molecules are shown in Figure 4 at three different salt conditions (Fits for all DSC curves are shown in S1) and the resulting thermodynamic profiles are reported in Table 1. We used different salt concentrations to stabilize and distinguish potential structures that may or may not be forming. For instance, PreQ1 has three transitions under low salt (16 mM Na+) which is different than what is observed by the UV melt. The TM determined from UV is an average of the two well defined DSC transitions. Under higher salt concentration the number of transitions remains at three (116 and 216 mM Na+), however, there

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is a large change in the overall total enthalpy from -44.7 to -71.5 kcal/mol (Table 1), which confirms the formation of additional stacking interactions or much better base-pair stacking. To determine how PreQ1 is folding, the modified molecules were characterized by DSC. PreQ1-T5 shows one transition with enthalpies ranging from -44.9 to -48.7 kcal/mol (Table 1). Based on the observed enthalpy and sequence, PreQ1-T5 likely forms the hairpin stem with an GG/AT and GA/TA base-pair stacks (Figure 1B) and a dA•dT base-pair in the 3’ tail (Figure 1C). This implies the GG/TA base-pair stack is strong enough to form in PreQ1 and there is also a potential interaction from the tail dA•dT. The next molecule, PreQ1-ACCC, which prevents pseudoknot formation, had very similar melting profiles (∆Hcal = -41.5 to -71.6 kcal/mol) to PreQ1, Table 1. This enthalpic similarity indicates the modified 3’ tail has no effect on the structure of PreQ1. Next, we characterized the molecules that would inhibit structures involving loop interactions shown in Figures 1D and 1F, named from now on Compact-Hairpin and Kissing-Complex, respectively. PreQ1-TT-Loop had similar melting profiles as PreQ1-T5, with one transition and enthalpies ranging from -41.3 to -43.1 kcal/mol (Table 1). This indicates, only the hairpin’s stem forms, i.e., no kissing interactions are occurring in PreQ1-TT-Loop. Finally, PreQ1-C-Loop also had lower enthalpies compared to PreQ1 (ΔHcal = -28.4 to -38.2 kcal/mol), indicating internal loop interactions are eliminated with the cytosine substitutions. However, it’s important to note these enthalpies are lower than PreQ1-T5 due to its less constrained loop that behaves like a random coil.

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+

3.5

PreQ1

16 mM Na

PreQ1 T5 Loop

3.0

PreQ1 ACCC PreQ1 TT Loop

2.5

PreQ1 C Loop

2.0 1.5 1.0 0.5 0.0 +

3.5

116 mM Na

3.0

∆ Cp (kcal/mol)

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Biochemistry

2.5 2.0 1.5 1.0 0.5 0.0 3.5

+

216 mM Na

3.0 2.5 2.0 1.5 1.0 0.5 0.0 20

40

60

80

o

T ( C)

Figure 4. DSC Unfolding of PreQ1 and Modified Molecules. Unfolding experiments were carried out using concentrations ranging from 41 to 72 µM. The increase in salt concentration increased the thermal stability of each molecule, respectively. DSC fits are provided in the supplementary information. All experiments were carried out in 10 mM Phosphate buffer at pH 7.0 with the NaCl concentrations indicated in the figure.

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Table 1. Folding Thermodynamic Profiles for DNA PreQ1and Modified Molecules. [Na+]

TM (°°C)

∆H (kcal/mol)

16 mM

14.8 19.4 26.2

116 mM

22.3 28.7 38.9

216 mM

24.4 31.6 42.9

-9.0 -15.1 -20.6 -44.7 -5.6 -16.7 -46.8 -69.1 -4.7 -14.6 -52.2 -71.5

16 mM 116 mM 216 mM

37.5 53.3 57.2

16 mM

14.2 17.9 23.6

116 mM

23.7 30.9 40.0

216 mM

25.5 35.3 44.6

16 mM 116 mM 216 mM

19.2 38.2 43.9

16 mM

19.6 26.2

116 mM

31.5 40.9

216 mM

35.3 47.3

T∆S (kcal/mol)

∆G°°(5) (kcal/mol)

∆nion (per mol)

-8.7 -14.4 -19.1 -42.2 -5.3 -15.4 -41.7 -62.4 -4.4 -13.3 -45.9 -63.6

-0.3 -0.7 -1.5 -2.5 -0.3 -1.3 -5.1 -6.7 -0.3 -1.3 -6.3 -7.9

-0.11 -0.60 -2.02 -2.73

-40.2 -37.9 -41.0

-4.7 -6.6 -7.7

-1.88

PreQ1-ACCC -9.5 -9.2 -12.6 -12.0 -19.4 -18.2 -41.5 -39.4 -8.3 -7.8 -23.2 -21.2 -32.9 -29.2 -64.4 -58.2 -3.8 -3.5 -19.4 -17.5 -48.4 -42.4 -71.6 -63.4

-0.3 -0.6 -1.2 -2.1 -0.5 -2.0 -3.7 -6.2 -0.3 -1.9 -6.0 -8.2

-0.11 -0.63 -1.97 -2.71

PreQ1-TT-Loop -41.3 -39.3 -42.1 -37.6 -43.1 -37.8

-2.0 -4.5 -5.3

-2.36

PreQ1-C-Loop -9.9 -9.4 -18.5 -17.2 -28.4 -26.6 -10.4 -9.5 -23.1 -20.5 -33.5 -30.0 -12.5 -11.3 -25.7 -22.3 -38.2 -33.6

-0.5 -1.3 -1.8 -0.9 -2.6 -3.5 -1.2 -3.4 -4.6

-0.41 -1.36 -1.77

PreQ1

PreQ1-T5 -44.9 -44.5 -48.7

The reported values in the table were calculated from the DSC melting curves. They show an increase in the TM, enthalpy and overall stability of each molecule as a function of salt, respectively. Δnion is reported in the last column. All experiments were carried out in 10 mM

Phosphate buffer at pH 7.0 and varying salt conditions. Experimental errors are as follows: TM (± 0.5 °C), ∆H (± 5 %), T∆S (± 5 %), ∆G°(5) (± 7%), Δnion (±15%)

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Biochemistry

To decipher the possible structures forming in PreQ1, we have considered all of the potential structures of the modified molecules as a whole. PreQ1 unfolds with a total enthalpy of 44.7 kcal/mol at 16 mM Na+, which is close to the predicted enthalpy of the hairpin in Figure 1B (ΔH = 47.1 kcal/mol). The DSC melting curve only appears to have two transitions; however, the fit is much better with three. Therefore, we can interpret the melting curve as follows, the first transition is likely due to the melting of the 3’tail, while, the next two fitted transitions is the melting of the hairpin stem including the GG/TA base-pair stack. The total enthalpy of the last two transitions is similar to the enthalpy obtained from PreQ1-T5 confirming the assignment of this transition. The increase in salt concentration to 216 mM Na+ increases the overall enthalpy to 71.5 kcal/mol which is similar to the predicted enthalpy of the Compact-Hairpin (ΔH = 69.9 kcal/mol). Again there are three transitions; however, the overall enthalpy is significantly higher, with the third transition having the greatest affect. One possibility for this enthalpy increase is due to the stabilization of additional interactions by closing the hairpin’s loop.

So based on this

analysis from an intramolecular point of view, PreQ1 is forming a hairpin with 10 base-pairs and an additional base-pair in the 3’ tail. This leaves the intermolecular Kissing-Complex to be examined. The strongest data to support the formation of this complex is by comparing the enthalpies of PreQ1, PreQ1-T5, and PreQ1-TT-Loop, see Table 1. If the kissing complex is not forming, then when making the modification in the loop from GC to TT there should be no effect on the observed enthalpy relative to PreQ1 because the other loop bases have not been modified, .i.e, they are still able to form base-pair interactions. However, PreQ1-TT-Loop has one transition with similar enthalpy to PreQ1-T5, which also doesn’t allow for kissing to take place. So, if kissing hairpins are forming in PreQ1, then the enthalpy of the second transition should be doubled due to the bimolecular

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nature of the hexaduplex formed from the base complementarity of their loops, i.e., the overall concentration for a bimolecular complex is half of a monomolecular complex with the total strand concentration being used for the analysis of the DSC melting curves. Therefore, we are obtaining an average enthalpy of -31.1 kcal/mol for the formation of this duplex, which is in moderate agreement with the predicted nearest neighbor enthalpy of -39.8 kcal/mol. One potential reason for the lower enthalpy than predicted is the external AT base-pairs are not completely forming in the duplex portion of the Kissing-Complex. Overall, there is evidence for both the formation of the Compact-Hairpin and Kissing-Complex and we cannot rule out either structure from these experiments alone.

Table 2. Thermodynamic Profiles for PreQ1 in 1 M NaCl Concentration 30 µM

62 µM

T

M

(°°C) 28.6 38.1 54.0 28.6 40.8 53.9

∆H (kcal/mol)

T∆S (kcal/mol)

∆G°°(5) (kcal/mol)

-3.8 -12.3 -54.5 -70.6 -3.8 -15.8 -54.5 -74.1

-3.5 -11.0 -46.3 -60.8 -3.5 -14.0 -46.4 -63.9

-0.3 -1.3 -8.2 -9.8 -0.3 -1.8 -8.1 -10.2

The values listed in Table 2 correspond to the values obtained from the DSC concentration dependence of PreQ1 in 1 M NaCl. The second transition shows a slight decrease in the TM with a decrease in strand concentration which suggests a bimolecular interaction. All experiments were carried out in 10 mM Phosphate buffer at pH 7.0 in 1M NaCl. Experimental errors are as follows: TM (± 0.5 °C), ∆H (± 5 %), T∆S (± 5 %), ∆G°(5) (± 7%).

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62 µM 30 µM

∆Cp (kcal/mol)

2

1

0

20

40

60

80 o

Temperature ( C) 30 µM

PreQ1 1M Chi = 1025 2

o

∆ Cp (kcal/mole/ C)

3

2

1

0 20

40

60

80 o

Temperature ( C) 62 µM

PreQ1

3

1M 2 Chi = 663

o

∆ Cp (kcal/mole/ C)

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Biochemistry

2

1

0 20

40

60

80 o

Temperature ( C)

Figure 5. Concentration Dependence of PreQ1 in 1 M NaCl. DSC melting curves show a broadening with a decrease in strand concentration due to the second transition having a slightly smaller TM. This suggests a bimolecular interaction. All experiments were carried out in 10 mM Phosphate buffer at pH 7.0 in 1 M NaCl.

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With the question of whether or not PreQ1 is truly forming a biomolecular interaction, a concentration dependence was performed at 1 M NaCl. The increase in salt and strand concentration helps to stabilize bimolecular interactions.50 Based on the differential of the UV melting curves, two distinct transitions were observed with TMs of 28 and 54 °C which were concentration independent (Figure S2), however, a possible third transition was observed at higher strand concentration. To determine if there are three transitions, DSC melting experiments were performed at two different strand concentrations (Figure 5). The DSC melting curves at both strand concentrations show three transitions (Figure 5). The DSC of the low concentration had a much broader melting curve, which was due to a shift in the second transition’s TM, Table 2. However, this decrease in TM is small compared to previous studied bimolecular complex.51 This decrease in TM dependence could be due to the ability of this sequence to also form the Compact-Hairpin, which would minimize this effect. Therefore, we propose an equilibrium of these two species to occur in solution. Correlation of the differential binding of ions, ∆nNa+, with Structure Formation. We determine ∆nNa+, according to Eq. 1. The ∆H/RTM2 term is obtained directly from the DSC thermograms while the ∂TM/∂ln [Na+] term is obtained from the slope of TM vs ln [Na+] shown in Figure 6. The resulting ∆nNa+ values, in mol Na+ per mol of molecules, are summarized in the last column of Table 1 next to the highest salt condition. Taking into account the total number of

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phosphates, we obtained ∆nNa+s of 0.078 (PreQ1), 0.067 (PreQ1-T5), 0.077 (PreQ1-ACCC), 0.067 (PreQ1-TT-Loop) and 0.051 mol of Na+/mol of phosphate (PreQ1-C-Loop). These values are small but consistent with counterion uptake by short DNA duplexes of 6 to 8 base-pairs.

2,52

In

general, the ∆nNa+ values increase when there are additional loop interactions (PreQ1 and PreQ1-ACCC) relative to the molecules with loop modifications (PreQ1-T5, PreQ1-TT-Loop and PreQ1-C-Loop). However, the ion binding for PreQ1-C-Loop is lower than PreQ1-T5 and PreQ1-TT-Loop due perhaps to partial protonation of the cytosine rich loop resulting in some screening effects.52–54 This lower uptake probably reflects the decrease in the negative charge density due to the partial protonation.55 A similar affect was seen with hairpins, triplexes and i-motifs. There was a smaller uptake on ions in the molecules containing the GC+ base-pairs and C+GC base-triplets compared to their unprotonated counterparts.18,20 Also, with a loop of 12 bases, with only base stacking occurring, it could be approaching random coil like behavior, having a lower uptake of counterions50 compared to PreQ1-T5 and PreQ1-TT-Loop, which have more structured loops. For a better assessment of the differential binding of counterions, we considered just the phosphates associated with the helical stems, including the two loop phosphates adjacent to each stem; the other loop phosphates behave like random coil phosphates. Since we suggest an equilibrium between the Compact-Hairpin and Kissing-Complex, both structures will be considered in this analysis. PreQ1 and PreQ1-ACCC would have a slightly different number of helical phosphates for the formation of the Compact-Hairpin, 25, and Kissing-Complex, 28. To better understand what is occurring, we assign the number of helical phosphates involved in each transition of these molecules. PreQ1 unfolds in three transitions, the last transition correspond to the unfolding of a hairpin with 7 base-pairs in the stem, which has 14 helical phosphates. The

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comparison of this hairpin among these molecules indicates an uptake of 0.14 (PreQ1), 0.14 (PreQ1-ACCC) mol of Na+/mol of helical phosphate. These ∆nNa+ values are in good agreement with the 0.13 value of PreQ1-T5, confirming the hairpin assignment. The second transition of PreQ1 and PreQ1-ACCC may correspond to formation of internal loop interactions and/or bimolecular kissing loops. Due to the difference in molecularity (monomolecular vs. bimolecular) the ΔH/RTM2 term needs to be adjusted accordingly i.e., the bimolecular interaction enthalpy should be doubled from what is reported in Table 1. Using the appropriate ΔH/RTM2 values and the total number of helical phosphates for the internal loop interactions, 11, and kissing loops, 14, PreQ1 has an uptake of either 0.055 or 0.086 mol of Na+/mol of helical phosphate, respectively, while PreQ1-ACCC has an uptake of 0.058 or 0.091 mol of Na+/mol of helical phosphate, respectively. This comparison again shows similar uptakes, indicating similar structures formed: Compact-Hairpin and Kissing-Complex, confirming the enthalpy results.

Figure 6. TM dependence on salt concentration. Using the melting temperatures obtained from the DSC experiments we plotted TM as a function of salt concentration. It is observed for each transition there is an increase in the TM with an increase in salt. The slope was used to calculate Δnion. All experiments were done in 10 mM NaPi with increasing salt concentrations ranging from 16 mM to 216 mM at pH 7.0; TM (± 0.5 °C).

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Biochemistry

Standard Folding Thermodynamic Profiles from DSC. Standard thermodynamic profiles for the folding of all molecules are listed in Table 1. The overall stability of the putative structures formed by these oligonucleotides follows a similar trend to the folding enthalpies, i.e., folding of all molecules are enthalpy driven. The favorable folding free energies result from the characteristic favorable enthalpy – unfavorable entropy compensations; the favorable enthalpies have been discuss earlier in terms of the contribution of the formation of base-pair stacks, while the unfavorable entropy terms comes from the ordering of the single strands and the putative uptake of counterions.56 PreQ1 and PreQ1-ACCC have the highest overall stability due to additional loop interactions forming at the higher salt concentrations. Furthermore, the hairpin stability determined from the last transition of these molecules is in good agreement with the stability of both PreQ1-TT-Loop and PreQ1-C-Loop, which formed a hairpin with a similar stem. This transition in PreQ1 has a ΔG°(5) ranging from -1.9 to -6.3 kcal/mol at these salt concentrations. PreQ1-T5 cannot be directly compared due to its shorter loop yielding a higher TM, resulting in the higher stability of this hairpin. The entropy cost in the folding of PreQ1 increases as the salt concentration is increased, due to formation of additional base-pairs and optimization of base-pair stacks, resulting in a higher uptake of counterions.

CONCLUSION We have used a single stranded DNA oligonucleotide with the sequence of the preQ1 aptamer domain, to answer an important question regarding DNA conformational plasticity and its intramolecular/intermolecular folding. We determined the folding/unfolding thermodynamics of the DNA analog and modified molecules by using a combination of temperature-dependent UV spectroscopy and differential scanning techniques. Based on the analysis of the calorimetric data

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at three different salt concentrations, specifically the parsing of the enthalpy and differential binding of counterions for the observed transitions of all molecules, allows us to conclude the DNA analog under physiological conditions is unable to form a pseudoknot-like structure. Instead, it is forming two potential structures, Compact-Hairpin and Kissing-Complex, which could be in equilibrium. Although this ssDNA is able to form similar structures as its RNA counterpart, it is important to note the RNA oligonucleotide will likely have a higher stability.57 We have previously shown RNA duplexes are more stable and hydrated than DNA duplexes.57 In future studies, we intend to compare the thermodynamic profiles between the DNA analog and the RNA aptamer domain to determine similarities and differences for these secondary structures. In summary, we have shown that this thermodynamic approach can be a useful tool in deciphering different and complex nucleic acid structures.

Funding Information. This work was supported by National Science Foundation Grant MCB-1122029.

Supporting Information. 1) DSC fits for each molecule at three different salt concentrations 2) Concentration dependence for PreQ1 at 1 M NaCl, including: differential curves of UV melts at different strand concentrations, as well as, two DSC thermograms at different strand concentration with their corresponding fits

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Structural Insight on the Unbound State of the DNA Analog for the preq1 Riboswitch: A Thermodynamic Approach Calliste Reiling-Steffensmeier and Luis A. Marky

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