Melting behavior of pseudoknots containing adjacent GC and AT rich domains

Background: Z-DNA is very important in transcription when RNA polymerase transcribes and Z-DNA is formed on the 5’ end following the polymerase. Pseudoknots have also been found to play important roles in the biology of RNA, such as the expression of genes and in the overall tertiary structure of RNA molecules. Methods: A combination of UV and circular dichroism (CD) spectroscopies and differential scanning calorimetry (DSC) were used to investigate the UV/CD spectral characteristics and the unfolding thermodynamics of two DNA pseudoknots with the following sequences: d(CGCGCGT5GAAATTCCGCGCGT5 GAATTTC) (CG-PsK) and d(GCGCGCT5CAAATT-GGCGCGCT5CAATTTG) (GC-PsK), where “T5” are loops of five thymines., along with, two control self-complementary duplexes, d(CG)3 (Du-CG) and d(GC)3 (Du-GC). Results: The UV/DSC melts showed sequential biphasic (CG-PsK) and triphasic (GC-PsK) transitions, with TMs independent of the strand concentration. Their DSC unfolding took place in two independent parts, the unfolding of the AT rich stem followed by the GC rich stem. CG-PsK’s thermodynamic profiles under both salt conditions were similar, while the stability and the folding enthalpy were lower in this salt range for GC-PsK. The CD spectrum showed the d(CG)3 portion of the stem of CG-PsK flips into a left handed helix in high salt conditions that could be affecting the loop/stem interplay, yielding a higher folding enthalpy relative to GC-PsK. Conclusions: Each pseudoknot formed intramolecularly with sequential transitions corresponding to the associated AT and GC rich melting domains. The placement of d(CG) 3 in CG-PsK yielded a CD spectra with a partial transition of the full stem to a left-handed helix. The melting behavior of CG-PsK remained unchanged with the addition of salt, while GC-PsK was greatly affected due to a more constrained right stem of the pseudoknot. The main reasons for these differences was the melting behavior of the AT rich motif, which is explained in terms of a loop/stem interplay within GC-PsK, hydration differences of their dA•dT base pairs, and/or the conversion of CG-PsK to a partial left handed helix. This conversion could be helping to stabilize this pseudoknot allowing the right stem to be less constrained at 5 M NaCl.


Introduction
Z-DNA is a rare left-handed double-helix which requires alternating GC rich sequences [1,2]. It is more elongated and narrow than both B and A-DNA and has a zigzag backbone due to the alternate stacking of bases in anti and syn-conformations. Due to the electrostatic repulsion of the phosphates, Z-DNA is more favorable at high salt concentrations by reducing this effect [3,4]. However, these high salt conditions are not present in vivo, but cytosine methylation, spermine, and spermidine can also help to stabilize the Z-conformation [5,6]. It has been found that Z-DNA is mainly present when DNA is undergoing negative supercoiling and also during transcription [7,8]. For Biochemical Compounds ISSN 2052-9341 | Volume 5 | Article 4 CrossMark ← Click for updates example, when the RNA polymerase binds to the DNA the Z-conformation is formed behind the polymerase due to the negative torsional constrain. This is because the polymerase is not rotating while it's transcribing [9,10].
Z-DNA has been found in both prokaryotic and eukaryotic systems. In prokaryotes, B-DNA is transformed to Z-DNA to avoid undesired methylation [11]. Also in E. coli, Z-DNA can form which is regulated by transcription and enhanced by inactivating topoisomerase through mutations [12,13]. Z-DNA in eukaryotes is highly immunogenic, both polyclonal and monoclonal antibodies can bind to this conformation [14,15]. In murine cells it was shown that three transcriptiondependent Z-DNA-forming segments were identified in the 5' region of a gene with two of them near promoters [16,17]. Studies on Z-DNA have shown that the majority forms behind a moving RNA polymerase and is stabilized by the negative supercoiling generated by DNA transcription [18,19].
Pseudoknots are also very important in biological systems. Their functional roles include, but are not limited to, the catalytic cores of various ribozymes, self-splicing introns, riboswitches, and ribosomal frameshifting [20][21][22][23][24]. Pseudoknots are perhaps the foundation of the large number of structures that RNA molecules undertake due to its intramolecular nature. It has also been found that RNA with rich CG regions is able to adopt the Z-conformation [25,26]. It could be possible that pseudoknots with CG rich stems can flip into the Z-conformation aiding in the control of gene expression. Riboswitches are often found near ribosomal binding sites and formation of pseudoknots can stop the ribosome from binding by blocking access to the RBS in the mRNA [27,28], so there could be a similar mechanism as seen with transcription. It is of interest to see whether or not the inclusion of a d(CG) 3 segment can form a left-handed conformation imbedded in a DNA pseudoknot and how this affects the overall melting behavior of the pseudoknot.
In this work, we have designed DNA pseudoknots containing adjacent GC and AT rich domains along with two control duplexes, one being d(CG) 3 and the other d(GC) 3 to mimic the GC rich domains of each pseudoknot. We used temperaturedependent unfolding techniques (UV, CD and DSC), to test the conformational rearrangements within the pseudoknots and measured the associated energetics that accompanies their melting behavior.

Materials
All oligonucleotides were synthesized by 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 designation are shown in Figure 1. The concentrations of the oligomer solutions were determined at 260 nm and 90°C or 100°C using an Aviv Spectrophotometer Model 14DS UV-Vis and the molar extinction coefficients: 326.5 mM -1 cm -1 (CG-PsK), 320.6 mM -1 cm -1 (GC-PsK), 60.0 mM -1 cm -1 (Du-CG), 59.8 mM -1 cm -1 (Du-GC). These values were obtained by extrapolation of the tabulated values for dimers and monomeric bases [29] at 25°C to 90°C or 100°C using procedures reported previously [30,31]. Inorganic salts from Sigma were reagent grade, and used without further purification. Measurements were made in appropriate buffer solutions: 10 mM sodium phosphate (NaPi), 1 M NaCl or 5 M NaCl at pH 7.0. All oligonucleotide solutions were prepared by dissolving the dry and desalted ODNs in buffer.

Temperature-Dependent UV Spectroscopy (UV)
Absorbance versus temperature profiles were measured at 260, 268, and 275 nm with a thermoelectrically controlled Aviv Spectrophotometer Model 14DS UV-Vis (Lakewood, NJ). The temperature was scanned at a heating rate of approximately 0.6 °C/min, and shape analysis of the melting curves yielded transition temperatures, T M s [30]. The transition molecularity for the unfolding of a particular complex was obtained by monitoring T M as a function of the strand concentration. Intramolecular complexes show a T M -independence on strand concentration, while the T M of intermolecular complexes does depend on strand concentration [31].

Differential scanning calorimetry (DSC)
The total heat required for the unfolding of each oligonucleotide, in appropriate buffer conditions, was measured with a VP-DSC differential scanning calorimeter from Malvern (Northampton, MA). T M s and standard thermodynamic profiles are obtained from these thermograms, ΔC p as a function of temperature, using the following relationships [30,31]: ΔH cal = ∫ΔC p (T)dT; ΔS cal = ∫ΔC p (T )/TdT, and the Gibbs equation, ΔG°( T) =ΔH cal -TΔS cal ; where ΔC p is the anomalous heat capacity of the ODN solution during the unfolding process, ΔH cal and ΔS cal are the unfolding enthalpy and entropy, respectively, assumed to be temperature-independent. ΔG°( T) is the free energy at temperature T, 20°C.

Circular dichroism (CD)
All CD spectra were obtained with an Aviv Circular Dichroism Model 202SF spectrometer (Lakewood, NJ) equipped with a peltier temperature control system. These CD spectra were taken from 320 nm to 220 nm in 1 nm increments with an averaging time of 3 seconds using 0.1 cm free-strained quartz (Suprasil) cuvettes. The reported spectra correspond to the average of at least two scans. The particular conformation of each pseudoknot or duplex (10 mM NaPi with 1 M or 5 M NaCl) was determined from inspection of their CD spectra at 2 or 25°C, where the oligos were completely folded. in 1 M salt: 61.4°C, 51 kcal/mol (Du-CG) and 62.8°C, 53 kcal/ mol (Du-GC). However, the increase in the salt to 5 M yielded decreases in both T M and ΔH vH : 53.6°C, 44 kcal/mol (Du-CG) and 52.1°C, 44 kcal/mol (Du-CG). The higher thermal stability of the pseudoknots was due to the fact that these molecules fold/unfold intramolecularly while the folding/unfolding transitions of the control duplexes were bimolecular.

Temperature-induced UV melting of Pseudoknots
Based on both the shape of the pseudoknot UV melts and the melting data of their last transitions, the main observations are: GC-PsK was more stable in 1 M salt while their stability was similar in 5 M salt. A similar trend was observed with the control duplexes i.e., Du-GC was more stable in 1 M whereas similar stability for both duplexes was obtained in 5 M. The overall observation was consistent with their number of base pair stacks, three CG/GC & two GC/CG (CG-PsK) and two CG/GC & three GC/CG (GC-PsK). On the other hand, it is difficult to discuss the melting behavior of the AT rich motif of these pseudoknots, which differ mainly by two base pair stacks i.e., two CT/GA in CG-PsK are replaced for two CA/TG in GC-PsK, which may interact differently with the adjacent loop of 5 thymines. However, the DSC results of a later section will show some light on the melting behavior of this AT rich motif of the pseudoknots.

Circular Dichroism (CD)
The CD spectra obtained in both 1 M and 5 M NaCl for all four molecules are shown in Figure 3. The CD spectra of CG-PsK, GC-PsK, Du-CG, and Du-GC in 1 M NaCl showed two bands with similar intensities and areas, and with minimums around 250 nm and maximums at 275 nm, which are characteristic of a nucleic acid in the "B" conformation [32]. In 5 M salt, the positive band of the CG-PsK spectrum ( Figure 3A) showed a slight shift to 272 nm; however, an additional band with a minimum at 293 nm was observed. The GC-PSK spectrum remained the same. The CD spectra of the duplexes showed a trend similar to the pseudoknots in 5 M salt; however, the spectra of Du-CG was completely flipped with a minimum at 294 nm and a maximum at 265 nm, indicating a complete left-handed helix was formed [33]. The CD spectrum of Du-GC remained the same i.e., Du-GC remained in the B-conformation. The additional band in the CG-PsK spectrum at 5 M was therefore due to a partial flip into a left-handed helix, which was confirmed by the addition of the spectra of Du-GC and Du-CG at 5 M salt that produced a similar dip in the spectra at 294 nm (data not shown). This exercise was simulating the right-handed stem (GAAATTC/GAATTTC) and left-handed stems (CGCGCG/GCGCGC) of CG-PsK. This observation indicated that at this high salt concentration the 13 base pair stem of CG-PsK is a mixture of both a right-handed (right stem) and a left-handed (left stem) helix.

Unfolding Thermodynamics Obtained From Differential Scanning Calorimetry (DSC)
The DSC thermograms of each pseudoknot and control duplex are shown in Figure 4 and standard thermodynamic profiles for the folding of each molecule are shown in Table 1 had a ΔT M of 4.3 and 2.7°C in 1 and 5 M NaCl, respectively. This indicated the intermolecular formation of the control duplexes due to T M dependence on strand concentration. Overall, the favorable folding of each pseudoknot, negative ΔG°, took place through the typical favorable enthalpy-unfavorable entropy compensation. Favorable enthalpy contributions correspond to the energy gained by the formation of base pairs and base pair stacking interactions, while unfavorable entropy contributions correspond to the higher ordered state of the helical state of each molecule and the putative uptake of ions and water molecules [34][35][36][37]. In 1 M salt concentration, ΔG°s of -11.1 kcal/mol (CG-PsK) and -12.2 kcal/mol (GC-PsK) were obtained, which were driven by favorable folding enthalpies of -86.8 kcal/mol (CG-PsK) and -78.8 kcal/mol (GC-PsK), respectively. These folding enthalpies were lower than the ones obtained from nearest-neighbor parameters (-105.8 and -105.6 kcal/ mol, respectively) [38] in similar salt concentrations and using the full sequence of the pseudoknot stems. This may be explained in terms of the thymine loops that are somewhat short and constrained producing a reduction on the base pair stacking in the stem, consistent with previous results of pseudoknots with different loop lengths [39]. The increase in salt concentration to 5 M resulted in less favorable ΔG° terms (by 0.4 and 2.7 kcal/mol, respectively), which resulted from a similar enthalpy (CG-PsK) and less favorable enthalpy (CG-PsK), by 10.2 kcal/mol. The unfolding of each pseudoknot took place with distinctive melting profiles. For instance, CG-PsK unfolded in two transitions while GC-PsK unfolded in three transitions; the initial transitions correspond to unfolding of the right stem of each pseudoknot (AT rich domain) and the last transition to the unfolding of their left stem (GC rich domain), which was confirmed by the DSC thermograms of the control duplexes, Du-CG and Du-GC, respectively. One of the goals of this study was to investigate the folding/unfolding thermodynamics of pseudoknots containing a GC rich motif next to an AT rich motif. The GC rich motif (d(CGCGCG) 2 ) of CG-PsK was capable of forming a left handed helix when the salt is increased from 1 M to 5 M, as described in the CD section. Before describing the thermodynamic melting behavior of pseudoknots in terms of the two different motifs, it is best to compare what is happening between the control duplexes, Du-CG and Du-GC, which pertain to the GC rich helical domain on the left of the pseudoknots (Figure 1). In 1 M salt concentration, ΔG°s of -6.7 kcal/mol (Du-CG) and -7.0 kcal/mol (Du-GC) were obtained, which were enthalpic driven, -51.0 kcal/mol for both duplexes. These folding enthalpies were in excellent agreement with nearest-neighbor enthalpies (-51.4 and -50.6 kcal/mol, respectively) [38]. In 5 M salt, the ΔG° terms were less favorable (by 1.4 and 1.9 kcal/mol, respectively), while the enthalpy terms were slightly lowered by 1.9 kcal/mol (Du-CG) and 2.7 kcal/mol (Du-GC). Furthermore, we used these thermodynamic profiles to estimate thermodynamic parameters for the following hypothetical reaction: Du-CG → Du-GC, which assumed that the single strands were similar at high temperatures. Therefore, the thermodynamic profiles for the folding of Du-CG were subtracted from the profiles of Du-GC. This exercise yielded a ΔΔH = -0.3 kcal/mol, with a ΔΔG° of 0.3 kcal/mol in 1 M NaCl and ΔΔH of -1.1 kcal/mol, with a ΔΔG° of -0.2 kcal/mol in 5 M. This showed that there is little to no thermodynamic effect on the conversion of the helix from left handed to right headed. The analysis of the pseudoknot data in 1 M NaCl (Table 1), and using similar hypothetical thermodynamic cycles, indicated the folding of GC-PsK was entropically, (Δ(TΔS)=9.1 kcal/mol) more stable than CG-PsK by ΔΔG°2 0 =-1.1 kcal/mol; while in 5 M, CG-PsK was entropically (Δ(TΔS)=16.8 kcal/mol) more stable than GC-PsK by ΔΔG°2 0 =-1.2 kcal/mol. In the following paragraph, we have analyzed each of the two motifs in the pseudoknots in a similar way.
The thermodynamic comparison of the second and third transition of CG-PsK and GC-PsK, respectively, produced the effect of the presence of the GC rich domain within the pseudoknot. In 1 M NaCl, GC-PsK had a more favorable ΔG° of -0.9 kcal/mol and more favorable ΔH of -1.6 kcal, while in 5 M NaCl, CG-PSK had a more favorable ΔG° of -0.4 kcal/mol and more favorable ΔH of -3.3 kcal/mol. Both sets were very similar to the comparative data of the control duplexes. This indicated that the inclusion of this GC rich motif in the pseudoknot does not affect its overall thermodynamic profiles, even if the d(CGCGCG) 2 undergoes a conformational change to a left handed helix in 5 M NaCl. This observation was consistent with the zeroth energy change for the helical transition of a right to left handed helix [40]. This also showed that once the bottom strand is melted, there was no strain on the left stem. Therefore, the left stem in either pseudoknot was able to unfold similar to the corresponding duplex.
The comparison of the thermodynamic parameters of the first transition of CG-PsK with those of the initial two transitions of GC-PsK yielded the melting behavior of the AT rich domain i.e., the right stem of the pseudoknots. In 1 M NaCl, GC-PsK was more stable, by ΔΔG°=-0.2 kcal/mol and had a less favorable ΔΔH of 9.6 kcal/mol. In 5 M NaCl, GC-PsK was less stable, by a ΔG°=0.8 kcal/mol with a less favorable ΔΔH of 14.7 kcal/mol. However, the GA/CT and TC/AG base pair stacks of CG-PsK and CA/GT and TG/AC stacks of GC-PsK only differed by 0.6 kcal/mol. This indicated that the large difference in the folding enthalpies of these pseudoknots was due to an interplay of the right stem of the pseudoknots with its loop; the stem base pairs may be weakened by the presence of the adjacent loop of 5 thymines that can be partially complementary to four bases on the stem, 3 adenines and 1 guanine. For instance, the differences between the two stems were the bases in the 7th position of the stem, G in GC-PsK and C in CG-PsK, and the 13 th position of the stem, C in GC-PsK and G in CG-PsK; each guanine could be in a wobble dG•dT base pair with the first and last thymine of the loop in GC-PsK and CG-PsK., respectively, creating additional constrain because each loop would now have only four thymines. Most likely a dG•dT base pair would form in GC-PsK and not in CG-PsK because the two bases are adjacent. In addition, three loop thymines could form three dT*dA•dT base triplets, but this is unlikely because the folding enthalpy would be much higher. The main observation was this loop/stem interplay could form loose base pair stacks in the right stem of GC-PsK, yielding a lower folding enthalpy for this pseudoknot in 1 & 5 M NaCl. Furthermore, the lower enthalpy of GC-PsK obtained in 5 M, compared to CG-PsK, may be explained in terms of the lowered hydration of the AT base pairs of this motif (41) and/ or the formation of the left handed helix in the native form of CG-PsK may be lowering the interplay of the loop with the stem; thus, lowering the overall loop/stem constrain.

Conclusions
We have investigated the thermodynamic stability of two pseudoknots. Specifically, we used a combination of UV, CD, and DSC techniques to determine the unfolding thermodynamics of a pair of pseudoknots and their control duplexes. The favorable folding of DNA pseudoknots resulted from the typical favorable enthalpy-unfavorable entropy compensation; enthalpy contributions corresponded to formation of doi: 10.7243/2052-9341-5-4 base pair stacks while the entropy contributions were due to the ordering of the oligonucleotides and uptake of ions and water. This confirmed the flexibility of DNA strands being able to form pseudoknots that can be used to mimic known RNA secondary structures. The UV and DSC unfolding curves showed biphasic (CG-PsK) and biphasic/triphasic (GC-PsK) transitions, respectively, with T M s independent on the strand concentration, i.e., each pseudoknot unfolds intramolecularly. All transitions were sequential and correspond to the associated melting domains. In addition, the placement of the d(CGCGCG) 2 in CG-PsK formed a left-handed helix in 5 M NaCl. Overall, CG-PsK unfolded in two transitions corresponding to the AT and GC rich stem motifs of the pseudoknot. The salt concentration did not significantly affect the overall thermodynamics of this pseudoknot. GC-PsK unfolded in three transitions with the first two corresponding to the AT rich stem and the last transition being the GC rich stem. However, the folding enthalpy of this pseudoknot was affected by the increase in salt concentration from 1 M to 5 M. The differences in the melting behavior of these pseudoknots, especially the AT rich motif, can be explained in terms of the loop/stem interplay within the GC-PsK pseudoknot, hydration differences of their dA•dT base pairs, and/or the conversion of CG-PsK to a partial left handed helix. This conversion could be helping to stabilize this pseudoknot allowing the right stem to be less constrained at 5 M NaCl.