Effect of Loop Length and Sequence on the Stability of DNA

Article pubs.acs.org/JPCB

Effect of Loop Length and Sequence on the Stability of DNA Pyrimidine Triplexes with TAT Base Triplets Hui-Ting Lee,† Carolyn E. Carr, Irine Khutsishvili,‡ 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: We report the thermodynamic contributions of loop length and loop sequence to the overall stability of DNA intramolecular pyrimidine triplexes. Two sets of triplexes were designed: in the first set, the C5 loop closing the triplex stem was replaced with 5′‑CTnC loops (n = 1−5), whereas in the second set, both the duplex and triplex loops were replaced with a 5′‑GCAA or 5′‑AACG tetraloop. For the triplexes with a 5 ‑ ′ CTnC loop, the triplex with five bases in the loop has the highest stability relative to the control. A loop length lower than five compromises the strength of the base-pair stacks without decreasing the thermal stability, leading to a decreased enthalpy, whereas an increase in the loop length leads to a decreased enthalpy and a higher entropic penalty. The incorporation of the GCAA loop yielded more stable triplexes, whereas the incorporation of AACG in the triplex loop yielded a less stable triplex due to an unfavorable enthalpy term. Thus, addition of the GCAA tetraloop can cause an increase in the thermodynamics of the triplex without affecting the sequence or melting behavior and may result in an additional layer of genetic regulation.

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INTRODUCTION Triplexes were originally hypothesized to be involved in gene regulation, a function that has been confirmed in vivo.1−4 Their role in the regulation of genes is supported by the fact that many of the sequences of triplex sites occur only once, providing proteins and other biomolecules specific sites to target.5,6 Triplexes have also been implicated in the response of eukaryotic cells against viruses,7,8 post-transcriptional processing,9,10 chromatin organization,11,12 and replication pausing.13−15 In addition, triplexes can induce mutagenesis and DNA repair because of the inherent instability present in these structures.16−18 Although this instability can be a useful regulatory mechanism, it has also been implicated in cancer.19,20 Triplexes consist of three strands, with the first two forming canonical Watson−Crick base pairs with each other and a third complementary strand binding through Hoogsteen or reverseHoogsteen hydrogen bonding. Intramolecular DNA triplexes can be classified into two types: a “pyrimidine” triplex, in which the third strand is pyrimidine-rich, or a “purine” triplex, where the third strand is purine-rich. If the third strand is pyrimidinerich, it runs parallel to the first, or Watson, strand and binds to the Watson strand through Hoogsteen base pairing. Pyrimidine triplexes with cytosine in the third strand are stabilized by low pH to facilitate cytosine protonation.21,22 If the third strand is purine-rich, it runs antiparallel to the Watson strand and binds to the Watson strand through reverse-Hoogsteen hydrogen bonds. Purine triplexes are stabilized by divalent cations instead of low pH because the N3 position of cytosine does not have to be protonated. The triplexes shown in Scheme 1 are examples © XXXX American Chemical Society

of pyrimidine-rich triplexes, with the third Hoogsteen strand inserting into the major groove and interacting with the Watson strand through Hoogsteen hydrogen bonds. Because there are no cytosines, the stability of these triplexes is independent of pH and instead are stabilized by salt.23,24 The effects of the loop sequence and loop length on the stability of straight hairpin loops has been reported by several research groups.25−27 Earlier reports have shown that an ideal loop length consists of four bases and that there is a sequence preference for the base pair flanking the loop, with a CG closing base pair conveying the greatest stability.26,28−30 The loop effects of intramolecular triplexes, which contain two loops, have not been studied and are expected to be more complicated than the case of hairpin loops for two reasons. First, the loop connecting the Watson and Crick strands (duplex loop) can influence the stability of the triplex;31,32 a stronger duplex produces a weaker triplex, whereas a weak duplex yields a stronger triplex.32 This is due to the flexibility of the duplex component of the triplex.31,32 Second, the length and sequence of the loop connecting the Crick strand and Hoogsteen strand (triplex loop) were shown to have a significant effect on the stability and probability of triplex formation.33,34 A combination of these two phenomena, together with solution conditions, affects triplex flexibility and overall stability. A comprehensive study of the effects of loop sequence, loop length, and salt Received: July 31, 2017 Revised: September 5, 2017 Published: September 6, 2017 A

DOI: 10.1021/acs.jpcb.7b07591 J. Phys. Chem. B XXXX, XXX, XXX−XXX

Article

The Journal of Physical Chemistry B

Scheme 1. Cartoon of the Sequences of Triplexes and Control Hairpins, Drawn According to Their Hypothesized Structures

sequence of d(A7C5T7C5T7).55 To investigate the effects of the loop length, the C5 triplex loop was replaced with 5′‑CTnC (n = 1−5) loops. To investigate loop sequence effects, both the duplex and triplex loops of 7TAT were replaced with 5′‑GCAA or 5′‑AACG, as shown in Scheme 1. We used a combination of temperature-dependent UV spectroscopy and differential scanning calorimetry (DSC) to determine standard thermodynamic profiles for the unfolding of all of the triplexes under optimized solution conditions. The data show that a triplex loop of five nucleotides has the most favorable free energy of formation. When replacing either the C5 triplex or duplex loop with a 5′‑GCAA loop, the triplex is stabilized; however, replacement of the C5 triplex loop with a 5′‑AACG loop destabilizes the triplex. Replacement of the C5 duplex loop with a 5′‑AACG loop generates a bimolecular triplex due to the base complementarity of all four nucleotides of this loop, and its stability cannot be compared with the other triplexes.

concentration on triplex stability will provide us with fundamental physical factors that can predict their stable formation. Because the probability of triplex formation is influenced by the duplex and triplex loops, one way for DNA to influence the thermal, and possibly the thermodynamic, stability of the triplex without changing the sequence is the application of stabilizing tetraloops. Although loops of four bases were previously shown to impart the highest stability in hairpins, some sequences have significant thermal stability compared with others, and these have been classified as stabilizing tetraloops.35−38 There are three main classes of tetraloops; GNRA, UNCG, and CUYG, where N is any nucleotide, R is a purine, and Y is a pyrimidine. The GNRA class of tetraloops is the most common due to high sequence variability, leading to a higher rate of specific interand intramolecular contacts between the loop and other stems/ loops.35,39 The exceptional stability is due primarily to a noncanonical GA base pair; van der Waals and π−π interactions between the last three nucleotides due to the second and third nucleotides stacking onto the fourth nucleotide, adenine; and 2′ OH hydrogen bonds within the loop.27,40−46 Replacing the duplex and triplex loops with a GNRA tetraloop should alter the stability of the triplexes. Our current understanding of DNA and RNA structures and stability has been enhanced by thermodynamic investigations of the helix → coil transitions of model complexes. In particular, intramolecular hairpin loops are favorable for these thermodynamic studies because they form stable structures with a lower entropy cost47−50 and the resulting higher TM’s allow a better temperature range for investigation. The structure and overall physical properties of stem−loop motifs have been reported earlier,49,51−53 as have the effects of loop size on hairpin stability.28,29,48,54 In this work, we have investigated loop contributions to the stable formation of DNA intramolecular triplexes. We used a well-studied intramolecular triplex, 7TAT, with a 5′ to 3′

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MATERIALS AND METHODS Materials. All oligonucleotides were synthesized in the Eppley Institute Molecular Biology Core facility at the University of Nebraska Medical Center, reverse-phase highperformance liquid chromatography-purified, desalted on a G10 Sephadex column, and lyophilized to dryness before our experiments. The concentrations were determined at 260 nm and 90 °C using the molar extinction coefficients calculated from tabulated values at 25 °C.56,57 The sequences and designations are shown in Scheme 1, and the extinction coefficients (in mM−1cm−1) of all molecules are as follows: 273, 7TAT; 262, CTC-7TAT; 285, CT2C-7TAT; 280, CT3C-7TAT; 284, CT4C-7TAT; 290, CT5C-7TAT; 282, AACG-7TAT; 281, GCAA-7TAT; 285, 7TAT-AACG; 284, 7TAT-GCAA; 183, 7AT Hp; 190, 7AT-GCAA Hp; 190, 7AT-AACF Hp. All experiments were performed in buffer solutions containing 10 mM sodium phosphate at pH 7, adjusted to the desired salt concentration B


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ΔHvH/ΔHcal ratio allows us to inspect whether the unfolding takes place through two-state transitions or through the formation of intermediates. If the ΔHvH/ΔHcal ratio is in the range of 0.9−1.1, the transition takes place in an all-or-none fashion.58 Differential Thermodynamic Binding of Counterions. UV melting curves were carried out as a function of salt concentration to measure the differential binding of counterions, ΔnNa+, between the folded and unfolded states. The ΔnNa+ linking number was determined experimentally with the following relationships59

with sodium chloride. All chemicals used in this study were reagent grade. Temperature-Dependent UV Spectroscopy. Absorbance versus temperature profiles (UV melting curves) of all oligonucleotides were measured at 260 nm with a thermoelectrically controlled Aviv 14-DS spectrophotometer (Lakewood, NJ). The temperature was scanned at a heating rate of ∼0.6 °C/min. These melting curves allowed the measurement of the transition temperatures, TM, or the midpoint of the order−disorder transition and model-dependent van’t Hoff enthalpies, ΔHvH, from analysis of the shape of the melting curves. We used the relationship ΔHvH = (2n + 2)RTM2(∂α/ ∂T)T=TM that has been described earlier,58 where R is the universal gas constant, α is the fraction of single strands in the helical state, (∂α/∂T) is the slope of the α versus T curve measured around the TM, and n is the molecularity of the transition. TM’s of multiphasic melting curves were obtained from the peak maxima of the corresponding differential melting curves. To determine the molecularity of the transition(s) of each triplex, we carried out UV melting curves as a function of total strand concentration. The TM of a monomolecular transition is independent of strand concentration, whereas the TM of a transition with a molecularity greater than 1 is dependent on the total strand concentration. This dependency can be used to measure the ΔHvH for bimolecular transitions of self-complementary oligonucleotides, using equation 1/TM = [R/ΔHvH] lnCT + ΔS/ΔHvH, as described earlier.58 In this equation, R/ΔHvH is obtained from the slope of the plot of 1/ TM versus ln CT and ΔS/ΔHvH is the intercept of such plot.58 Circular Dichroism (CD) Spectroscopy. We used a thermoelectrically controlled Aviv circular dichroism spectrometer model 202SF (Lakewood, NJ) to measure the CD spectrum of each triplex. The analysis of these spectra yielded the conformation adopted by the helical state of each molecule. Typically, we prepared a triplex solution with an absorbance of 0.9 in 10 mM sodium phosphate buffer with 0.1 M NaCl at pH 7.0. The CD spectrum is measured from 310 to 200 nm every 1 nm at 2 °C, using a quartz cuvette with a path length of 1 cm. At 2 °C, all of the triplexes are in a 100% helical state. The reported spectra correspond to an average of at least three scans. Differential Scanning Calorimetry (DSC). To thermodynamically investigate the helix−coil transition of each molecule, excess heat capacity functions were measured with a MicroCal VP-DSC differential scanning microcalorimeter (Northampton, MA). Two cells, the sample cell containing 0.65 mL of an oligonucleotide solution and the reference cell filled with an equal volume of buffer solution, were heated from 0 to 90 °C at a rate of 0.6 °C/min. Analysis of the resulting thermograms yielded standard thermodynamic profiles (ΔHcal, ΔScal, and ΔG°(T)). Each thermodynamic parameter was obtained from the following relationships, using procedures described previously:58 ΔHcal = ∫ ΔCpa dT and ΔScal = ∫ (ΔCpa/T) dT, where ΔCpa represents the anomalous heat capacity during the unfolding process. The free energy change at any temperature, ΔG°(T), is obtained from the Gibbs equation: ΔG°(T) = ΔHcal − TΔScal. Shape analysis of the experimental DSC curves allows us to determine ΔHvH’s according to the following relationship: ΔHvH = A/[(1/T1) − (1/T2)], where A is a constant equal to 7.0 (monomolecular transition) and 10.14 (bimolecular)58 and T1 and T2 correspond to the lower and upper temperatures, respectively, at the half-height width of the DSC curve. The

ΔnNa + = (ΔHcal /RTM 2)[dTM /d ln(Na +)] = 1.1(ΔHcal /RTM 2)[dTM /d ln[Na +]]

(1)

The first term in parentheses was measured directly from DSC experiments, whereas the second term in brackets was measured from the slope of the TM dependences on salt, and 1.1 is a factor for converting solution activities into concentrations.

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RESULTS AND DISCUSSION Triplex Design. All oligonucleotides were designed to form triplexes with a common triple helical domain consisting of seven TAT base triplets (Scheme 1). To prevent i-motif formation when increasing the length of the cystosine loop, the C5 triplex loop is replaced with a CTnC loop for the CTnC7TAT series of triplexes. The two end-loop cytosines were kept to avoid strand slipping caused by a homothymine loop. The real length of this triplex loop is equal to n + 2, for example, CT3C-7TAT has a five-membered loop. The GNRA tetraloop sequence of GCAA was chosen because this is the most common of the GNRA sequences but still imparts significant thermal stability, whereas the AACG tetraloop sequence is not stabilizing and should behave as a loop with a random sequence, displaying a similar thermodynamic profile to 7TAT. 60−62 However, the sequence of 7TAT-AACG, A7AACGT7, is self-complementary and forms a long (16 bp) duplex, which can form a bimolecular triplex, with the 3′‑C5T7 end looping back around (Scheme 1). Three hairpin controls were studied, with the stems of all three mimicking the Watson−Crick duplex of the 7TAT triplex. Two of the hairpins contained either a GCAA or AACG tetraloop to mimic the loop effect on the duplex. Regardless of the loop length or sequence, all hairpins and triplexes are stabilized by increasing the concentration of salt. Therefore, all experiments were conducted in 10 mM sodium phosphate buffer containing 0.1 M NaCl to stabilize the triplex. All Oligonucleotides except 7TAT-AACG Fold Intramolecularly. Figure S1 shows typical UV melts for each triplex in 10 mM sodium phosphate with 0.1 M NaCl at pH 7. All transitions follow a sigmoidal unfolding behavior typical for helical nucleic acid structures. Almost all of the UV melting curves are monophasic, except for 7TAT-AACG and AACG7TAT, which are biphasic. UV melts over a total strand concentration range of 2−130 μM were obtained to determine the transition molecularities of each molecule; the corresponding TM dependences are shown in Figure 1. The TM’s of all of the molecules, except 7TAT-AACG, are independent of strand concentration, consistent with their monomolecular formation. The TM of the first transition of 7TAT-AACG is independent of strand concentration and thus is related to the unfolding of a monomolecular structure. This transition could correspond to C


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Figure 1. TM dependence on strand concentration at 260 nm in 10 mM sodium phosphate with 0.1 M NaCl at pH 7.0. (Top) TM dependence of 7TAT (black), CTC-7TAT (red), CT2C-7TAT (green), CT3C-7TAT (blue), CT4C-7TAT (cyan), and CT5C-7TAT (purple). (Bottom) TM dependence of AACG-7TAT (black), GCAA-7TAT (red), 7TAT-AACG (green), and 7TAT-GCAA (blue). The closed symbols are first transitions and the open circles are second transitions.

Figure 2. CD spectra at 2 °C in 10 mM sodium phosphate with 0.1 M NaCl at pH 7.0. (Top) CD spectra of 7TAT (black), CTC-7TAT (red), CT2C-7TAT (green), CT3C-7TAT (blue), CT4C-7TAT (cyan), and CT5C-7TAT (magenta). (Bottom) CD spectra of GCAA-7TAT (black), AACG-7TAT (red), 7TAT-GCAA (green), and 7TAT-AACG (blue).

the unfolding of the triplex structure because the stability of the bimolecular duplex is strong, which would conversely weaken the stability of the triplex, which is reflected in its TM of 9.3 °C. This transition is partially intramolecular and thus would explain the lack of concentration dependence. The second transition of 7TAT-AACG shows a significant dependence on strand concentration (Figure 1), indicating that this transition corresponds to the unfolding of a bimolecular complex, consistent with the unfolding of a long duplex, as shown in Scheme 1. All Oligonucleotides Form Triplexes and Are in the BConformation. The CD spectra of all triplexes at 2 °C are shown in Figure 2. All spectra consist of a positive band at ∼280 nm with a crossover at 254−259 nm and two negative bands centered at ∼250 and ∼210 nm. The band at ∼280 nm is related to the sugar puckering, whereas the band at ∼250 nm corresponds to the extent of base-pair stacking. Because the band at 250 nm is similar in all cases, this indicates that basepair stacking is relatively unchanged regardless of the loop length and sequence. In all spectra, the 280 nm band has a small hump at ∼260 nm, which is consistent with the homopurine/homopyrimidine nature of their duplex sequence.63 Each complex exhibits a negative band at 210 nm, which has been observed in previous reports,55,64 and is considered a characteristic feature of triplex formation.65 The magnitudes of the bands at 280 and 250 nm indicate that all triplexes exhibit the typical CD spectra of a nucleic acid helix in the “B” conformation and that binding of the third strand does not impose major distortions on the geometry of the duplex. The peak at ∼210 nm for 7TAT-GCAA and GCAA-7TAT has increased in intensity compared to all of the other

oligonucleotides, indicating an increase in triplex character, which is expected to occur with an increase in stability caused by the GCAA tetraloop. The GCAA loop in the 7TAT-GCAA triplex forms an additional base-pair stack (GA) at the 3′-end of the duplex;62 this may reduce the effect caused by strain from the random coil loop, leading to a stronger base-pair stack and contributing to a more homopurine−homopyrimidine duplexlike spectrum, and explains the presence of a peak at ∼260 nm. Unfolding of Triplexes as a Function of Loop Length. DSC curves of the CTnC triplexes are shown in Figure 3 (top), and the resulting thermodynamic profiles are shown in Table 1. The DSC curve of control hairpin 7AT Hp is shown in Figure 3 (bottom, black). The unfolding of all triplexes undergoes highly reproducible monophasic transitions. The TM’s from the DSC thermograms were obtained at a much higher concentration than from UV melts, but the values were the same, consistent with their intramolecular formation. Because all of the triplexes display only one unfolding transition, this indicates that the triplex and duplex structures are unfolding at the same time. The thermal stability of the CTnC triplexes follows the following order: 7TAT = CTC7TAT = CT2C-7TAT = CT3C-7TAT > CT4C-7TAT > CT5C7TAT, whereas the enthalpy follows the following order: 7TAT = CT4C-7TAT = CT3C-7TAT > CT5C-7TAT > CT2C-7TAT > CTC-7TAT, with the 7TAT control triplex underlined for clarification. Taken together, these results indicate that a loop length of five is ideal for the triplex loop to improve the overall thermal stability of the structure without compromising the strength of the base-pair stacks. Triplexes with a loop length D


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but reduce the ΔHvH value because the shape of the curve would appear to be due to one cooperative transition. Unfolding of Triplexes as a Function of Loop Sequence. DSC curves of the GCAA/AACG triplexes are shown in Figure 3 (middle), and the resulting thermodynamic profiles are shown in Table 1. DSC curves of control hairpins 7AT-GCAA Hp and 7AT-AACG Hp are shown in Figure 3 (bottom). The unfolding of all triplexes undergoes highly reproducible monophasic or biphasic transitions. The TM’s from the DSC thermograms were obtained at a much higher concentration than from UV melts, but the values were the same, consistent with their intramolecular formation. Both GCAA-7TAT and 7TAT-GCAA display very similar melting profiles with almost identical TM’s. The TM’s of both are higher than those of 7TAT or any of the triplexes with varying loop length, indicating that the GCAA tetraloop has a stabilizing effect on the unfolding of the triplex. For GCAA7TAT, there is a decrease in the enthalpy relative to 7TAT (−84.8 vs −91.3 kcal/mol), which is expected based on the results seen for CT2C-7TAT (−81.7 kcal/mol), indicating that the reduction in enthalpy is likely an effect of loop length and not sequence. The tetraloop does impart thermal stability to the triplex, indicating that the stabilizing structure of the tetraloop is present despite the decrease in enthalpy caused by strain from the short loop. There is no reduction in enthalpy for 7TAT-GCAA compared to that for 7TAT most likely because a loop of four for a duplex is optimal;25,26,48 the noncanonical GA base pair from the tetraloop should improve the stability of the duplex and conversely weaken the triplex. However, only one transition is visible, indicating that weakening of the triplex does not change the thermal stability. There is no change in enthalpy, although it could be that the increased stability of the duplex yields a higher enthalpy that is then offset by weakening of the triplex. Both triplexes have a ΔHvH/ΔHcal ratio less than 1, indicating that, like the triplexes with varying loop length, both the duplex and triplex are unfolding at the same time. Thus, a GCAA tetraloop imparts stability but does affect the unfolding mechanism of the molecule or its enthalpy. AACG-7TAT and 7TAT-AACG are the only triplexes in the study that have biphasic melting profiles, although due to different phenomena. AACG-7TAT has two transitions at 23.6 and 39.2 °C, which are both monomolecular. The enthalpy of each transition is approximately the same and together (ΔHcal = −83.4 kcal/mol) equals the enthalpy of GCAA-7TAT and CT2C-7TAT, indicating that once again the loop length, but not the loop sequence, affects the enthalpy. The first transition at a lower temperature and the reduced enthalpy of the transition is most likely the unfolding of the triplex and indicates that the AACG tetraloop is destabilizing and thus the unfolding of the triplex occurs at a lower temperature than the unfolding of the duplex. The transition at 39.2 °C occurs at the same temperature as GCAA-7TAT, which is most likely attributed to the unfolding of the duplex. This result indicates that the cooperative unfolding of the triplex and duplex seen in the other molecules causes the TM of the duplex to change, decreasing it to match the unfolding of the triplex. The ΔHvH/ ΔHcal ratio of the first transition is less than 1, indicating intermediate states or perhaps the unfolding of both sides of the triplex. The ΔHvH/ΔHcal ratio of the second transition is 1.2, indicating a near-two-state transition, likely the unfolding of just the duplex.

Figure 3. DSC thermograms in 10 mM sodium phosphate with 0.1 M NaCl at pH 7.0. (Top) Thermograms of 7TAT (black), CTC-7TAT (red), CT2C-7TAT (green), CT3C-7TAT (blue), CT4C-7TAT (cyan), and CT5C-7TAT (magenta). (Middle) Thermograms of AACG-7TAT (black), GCAA-7TAT (red), 7TAT-GCAA (green), and 7TAT-AACG (blue). (Bottom) Thermograms of 7AT Hp (black), 7AT-GCAA Hp (red), and 7AT-AACG Hp (green).

higher than five have the weakest thermal stability, which does not compromise the enthalpy, whereas triplexes with a loop length shorter than five have high thermal stability, but this stability compromises the base-pair stacks, leading to a decrease in enthalpy. Table 1 also shows van’t Hoff enthalpies determined from the shape of the DSC curves, which are similar to the ones obtained from UV melting curves within experimental error. To determine the ΔHvH/ΔHcal ratios, the optical and calorimetric ΔHvH’s for the triplexes were averaged. A ΔHvH/ΔHcal of 0.9− 1.1 indicates that the molecule unfolds in a two-state, all-ornothing, transition. A ratio greater than 1 indicates that more than one equilibrium66,67 is involved, whereas a ratio of less than 1 indicates the presence of intermediate states. In all cases for the triplexes with varying loop lengths, the ΔHvH/ΔHcal ratios are in the range of 0.4−0.6, which indicates that these triplexes unfold in non-two-state transitions.58 This is likely due to the unfolding of both the triplex and duplex occurring at the same time, which would show an increase in the ΔHcal value E


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The Journal of Physical Chemistry B Table 1. Thermodynamic Profiles for the Folding of Triplexes and Control Hairpinsa TM (°C)

ΔHcal (kcal/mol)

TΔS (kcal/mol)

ΔG°(5) (kcal/mol)

34.0

−91.3

−82.7

−8.6

ΔHVH (kcal/mol)

ΔnNa+ (mol Na+/mol)

ΔnNa+ (mol Na+/hPi)

−51

−3.5

−0.15

−31

−3.0

−0.13

−43

−3.4

−0.15

−47

−3.4

−0.15

−38

−4.1

−0.18

−31

−3.9

−0.17

−45

−3.5

−0.15

−29 −47

−1.4 −1.7

−0.20 −0.11

−37

−3.7

−0.16

−36 −107

−3.4 −6.0

−0.34 −0.18

−35

−1.3

−0.90

−38

−1.3

−0.90

−107

−5.4

−0.17

7TAT CTC-7TAT 34.1

−77.7

−70.3

−7.4 CT2C-7TAT

33.7

−81.7

−74.1

−7.6 CT3C-7TAT

34.0

−87.2

−79.0

−8.2 CT4C-7TAT

32.1

−90.2

−82.2

−8.0 CT5C-7TAT

30.0

−82.7

−75.9

−6.8 GCAA-7TAT

39.2

−84.8

−75.5

−9.3 AACG-7TAT

23.6 39.2

−44.9 −38.5

−42.1 −34.3

−2.8 −4.2

40.5

−93.1

−82.6

−10.5

7TAT-GCAA 7TAT-AACG 9.3 51.9

−42.2 −99.6

−41.6 −85.2

37.1

−42.1

−37.7

47.9

−47.6

−41.2

54.2

−146.6

−124.6

−0.6 −14.4 7AT Hp −4.4 7AT-GCAA Hp −6.4 7AT-AACG Hp −22.0

All experiments are performed in 10 mM sodium phosphate buffer with 0.1 M NaCl at pH 7. Experimental errors are as follows: TM (±0.5 °C), ΔHcal (±5%), TΔS (±5%), ΔG°(5) (±7%), ΔHvH (±15%), and ΔnNa+ (±12%).

a

The unfolding of 7TAT-AACG is also biphasic, but the first transition occurs at 9.3 °C, significantly lower than any transition in the other triplexes, and the second transition has a dependency on TM, indicating a bimolecular unfolding event. The enthalpy of the transition at 9.3 °C is similar to the enthalpy for the first transition in AACG-7TAT and likely corresponds to the unfolding of the end triplexes. The transition at 51.9 °C is most likely the unfolding of the long (16 bp) duplex, as shown in Scheme 1. This duplex forms because the AACG loop can base-pair with itself, forming a long duplex with no mismatches or bulges, whereas the GCAA loop cannot, which favors the formation of a triplex. The bimolecular duplex portion of the triplex unfolds with an enthalpy of 99.6 kcal/mol, whereas its control hairpin, which also forms a duplex (7AT-AACG Hp), unfolds monophasically with an enthalpy of 146.6 kcal/mol. The duplex stem of these molecules is predicted to unfold with an enthalpy of 127.4 kcal/ mol using nearest-neighbors parameters,68 which is in good agreement with the unfolding of 7AT-AACG Hp. However, this enthalpy is 27.8 kcal/mol more than the enthalpy of the second transition of 7TAT-AACG and indicates that the base-pair stacking contributions are much weaker in the triplex. This molecule forms a weaker triplex due to the disruption of two unpaired adenines at the end of the stem, which is consistent with previous findings that bimolecular triplexes are less compact than their intramolecular counterparts.31 Differential Binding of Counterions. Equation 1 was used to calculate the differential binding of counterions. The slope of the TM dependence on salt concentration was obtained

from UV melting curves as a function of [Na+] (Figure S2), whereas the average ΔHcal/RTM2 term is obtained from the DSC melts at several salt concentrations. The folding of each oligonucleotide is accompanied by an uptake of ions due to the shift of the helix−coil equilibrium toward the conformation with a higher charge density parameter.69 The ΔnNa+ values are presented in Table 1, and their magnitude indicates the strength of ion binding by the triplexes. The ion uptake for the triplexes with varying loop length follows the order CT4C7TAT = CT5C-7TAT > 7TAT = CT2C-7TAT = CT3C-7TAT > CTC-7TAT, indicating that a larger loop length facilitates a larger uptake of ions. The values shown in Table 1 can be normalized per helical phosphate by considering the total number of helical phosphates, including each of the two loop phosphates adjacent to the stem. These values are shown in the last column of Table 1 and yield an average of −0.16 mol Na+/ mol helical phosphates; this value is similar to the values of −0.17 mol Na+/mol phosphate for DNA polymers.70−72 These high values are expected due to the high charge density inherent in a compact triplex structure (Figure 4), consistent with the need of high salt for their formation. Triplexes GCAA-7TAT, 7TAT-GCAA, and AACG-7TAT have total ΔnNa+ consistent with what was obtained for the control triplex, 7TAT. This is true even for AACG-7TAT, where the unfolding of the triplex and duplex has been uncoupled, indicating that the values obtained for the other triplexes are simply addition of the ion uptake from the folding of duplex and then the triplex. The 7TAT-AACG triplex has a total ΔnNa+ value of −9.4 mol Na+ per mol oligonucleotide; when F


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The main observation is that triplexes with a five-member triplex loop have the highest thermal and enthalpic stability. When there are three to five bases in the loop, the thermal stability increases while sacrificing the strength of the base-pair stacks, which results in decreasing enthalpy. The thermal stability decreases with loop length greater than five, but this does not correlate with a reduction in enthalpy. This suggests that the magnitude of the enthalpy depends on the number and strength of the base triplet stacks, which is dependent on the loop length (Figure 5). Interestingly, previous research suggested that for each thymine included in the loop of a hairpin, from three to nine bases, the TM decreases by 4.2 °C per thymine.74 For the triplexes studied, changing the loop length from three to five bases yields no effect on the TM, and when increasing the loop length past five, the TM drops by ∼2 °C per base. Thus, the structure of the triplex negates the thermal destabilization caused by increasing the loop length up to five. The resulting free energy terms shown in Figure 5 (bottom) have a slightly different order than that of the enthalpy and entropy trends, as follows: CT5C-7TAT (−6.8 kcal/mol) < CTC-7TAT (−7.4 kcal/mol) < CT2C-7TAT (−7.6 kcal/mol) < CT4C-7TAT (−8.0 kcal/mol) ≤ CT3C-7TAT (−8.2 kcal/mol). The comparison of all of the thermodynamic parameters of Figure 5 shows that the ΔH and TΔS terms reach the maximum when the loops have six bases, whereas the ΔG°(T) term reaches a maximum when the loop has five bases. At six bases, the unfavorable enthalpy is offset by the unfavorable entropy. These dependences suggest that with a loop length less than 5, the number of base triplets in the stem is reduced due to the short length of the loop, that is, the length of the triplex loop is not long enough to go to the other side of the double helix, reducing the number and/or strength of the base triplets formed in the molecule. However, when the loop length is greater than five, the longer triplex loop facilitates better formation of the base triplets but it is less favorable to fold this longer loop due to its higher entropic penalty. Overall, the above ranking indicates that the magnitude of the free energy trend depends on the favorable enthalpy/ unfavorable entropy compensation, which in turn depends on the length of the triplex loop. This statement is true only when considering the effect of the triplex loop because the triplex stability will depend on the duplex stability. Thermodynamic Profiles for the Folding of Triplexes: Contributions of the Loop Sequence. Close inspection of Table 1 and Figure 6 provides the contribution of the loop sequence to the stability of each triplex. Replacement of the triplex loop with a 5′‑GCAA tetraloop stabilizes the whole triplex by ΔTM of 5.2 °C and ΔΔG°(T) of 0.7 kcal/mol relative to the control triplex, 7TAT, whereas replacement of the duplex loop is more stabilizing, with a ΔTM of 6.5 °C and ΔΔG°(T) of 1.9 kcal/mol (Table 1). All three thermodynamic parameters, enthalpy, entropy, and free energy, for the 7TAT-GCAA triplex are greater than those for control triplex 7TAT (Figure 6), which indicates a more stable triplex conformation when compared with that of any of the CTnC-7TAT triplexes. On the other hand, GCAA-7TAT has enthalpy and entropy terms similar to those of 7TAT (Figure 6) but has a greater free energy due to the increased thermal stability. These results show that both GCAA-7TAT and 7TAT-GCAA are more stable than any of the triplexes with a random loop sequence, which is consistent with the earlier reports of the stabilizing effect of a 5′‑ GCAA tetraloop next to an AT base pair in a DNA duplex.54,60,62 This stabilizing effect of a GCAA tetraloop next

Figure 4. Structure of an intramolecular triplex (PDB ID: 1GN7). (Left) Side view and (right) top view. The third strand is colored yellow to highlight its interaction with the double helix.

normalized per helical phosphate, this triplex yields ΔnNa+ values of −0.34 and −0.18. The value of −0.34 for the triplex is unusually high but is so thermally unstable that this peak cannot be seen at salt concentrations lower than 0.1 M NaCl and lacks a concrete baseline even at this salt concentration. The value for the duplex, −0.18, is consistent with the values obtained for the triplexes as well as the values obtained for long polymers. Thermodynamic Profiles for the Folding of Triplexes: Contributions of the Triplex Loop Length. Standard thermodynamic profiles for the formation of each molecule are summarized in Table 1 and Figure 5. The ΔG°(T) and TΔS terms are estimated at 5 °C, where all of the molecules are in the helical state. Inspection of Table 1 indicates that the folding of each molecule is accompanied by a favorable free energy term, which results from the characteristic compensation of favorable enthalpy and unfavorable entropy contributions.73

Figure 5. Effect of loop length on the thermodynamic parameters. G


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exception of 7TAT-AACG, which forms a long intermolecular duplex with two weak intramolecular triplexes at each end. All of the CTnC-7TAT triplexes unfold in monophasic transitions, and analysis of their thermodynamic profiles reveals that five bases is the optimal triplex loop length in terms of stability; a smaller or larger loop leads to a less negative free energy term. This result is consistent with previous reports that longer loops require more energy for triplex formation and are less stable.33,34,75 A smaller loop may form a lower number of base triplets due to steric constraints because the loop may be too short and cause weak base-pair stacks near the loops. A longer loop generates stable base-pair stacks but has a high entropic penalty, resulting in a lower free energy term. In addition, this result is not entirely unexpected because previous results indicated that the optimal duplex loop length for DNA is four bases. Our results suggest that the third strand does not have significantly further distance to cross, leading to an optimal loop length of five bases. The inclusion of the 5′‑GCAA or 5′‑AACG sequences in the loops yielded more diverse changes than those in the CTnC loops. All of these triplexes are formed intramolecularly with the exception of 7TAT-AACG, which forms a bimolecular complex. Both GCAA-7TAT and 7TAT-GCAA are more stable than the triplexes containing five-member loops (Table 1), with more favorable free energy (ΔΔG°(5)) terms. This result is in good agreement with previous reports on the stabilizing effect of this loop sequence.54,60,62 On the other hand, the inclusion of the reverse sequence, 5′‑AACG, destabilizes the removal of the third strand, whereas the stability of the duplex remains the same, indicating that whereas some sequences lead to more favorable folding events, others are destabilizing and may hinder triplex formation. These tetraloops may be an additional layer of regulation because a destabilizing loop would increase the rate of mutagenesis and DNA repair, whereas a stabilizing loop would decrease these phenomena. Overall, we have shown the effect of loop length and sequence on triplex stability. A triplex loop with five bases is most favorable and can be further stabilized by replacing the loop with a 5′‑GCAA tetraloop. A future study will address the stabilization of the duplex in conjunction with the triplex loop to assess the relative influence of one loop over the other. In addition, we will address the effects of varying duplex loop length when the triplex structure is being stabilized by a GCAA tetraloop. These data should improve our understanding of triplex formation and help future predictions on how to generate stable nucleic acid triple helical structures.

Figure 6. Effect of loop sequence on the thermodynamic parameters. 7TAT control (black), GCAA-7TAT (white), AACG-7TAT (red), and 7TAT-GCAA (green). 7TAT-AACG omitted due to bimolecular formation.

to an AT closing base pair is expected to be less than what should be obtained with a CG closing base pair.40 The thermal stabilization caused by the GCAA tetraloop seen for the triplexes is minor when compared to the stabilization observed for hairpins; the reason for this is unclear, but this may be due to the stabilization being spread out across the entire triplex structure. Flipping of the loop sequence to 5′‑AACG in the triplex loop destabilized the thermal stability of the triplex relative to the control triplex (7TAT) by reducing the TM for the removal of the third strand by 10.4 °C and ΔΔG°(T) by 1.6 kcal/mol (Table 1 and Figure 6). This effect may be due to a smaller stacking contribution of the bases in the loop and/or the lower stacking of the loop bases adjacent to the end of the helical stem. However, the true reason for this destabilization is unclear because AACG is expected to behave as a random sequence. Overall, the inclusion of the 5′‑GCAA loop on the triplex side is stabilizing, whereas the inclusion of the 5′‑AACG loop is not. Furthermore, all of these loop-substituted triplexes have a similar differential binding of counterions compared to that for the 7TAT triplex. This indicates that these loop substitutions have similar ionic contributions to the overall unfavorable entropy of formation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b07591. Raw UV−vis melting curves and TM dependence on salt (PDF)

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AUTHOR INFORMATION

Corresponding Author

CONCLUSIONS We used a combination of temperature-dependent UV spectroscopy and differential scanning calorimetry to characterize the melting behavior of a set of triplexes with a common helical domain as a function of both their loop length and loop sequence. All triplexes form intramolecular triplexes with the

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

Carolyn E. Carr: 0000-0002-9688-6015 Luis A. Marky: 0000-0001-5572-7707 H


Article

The Journal of Physical Chemistry B Present Addresses

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‡

Institute of Physics, Javakhishvili State University, Tbilisi 0186, Georgia (I.K.). † Department of Biophysics, John Hopkins University, Baltimore, Maryland 21218, United States (H.-T.L.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by Grants MCB-1122029 and MCB0616005 from the National Science Foundation.

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Effect of Loop Length and Sequence on the Stability of DNA

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