Unfolding and Targeting Thermodynamics of a DNA Intramolecular

Subscriber access provided by Universiteit Utrecht

Article

Unfolding and Targeting Thermodynamics of a DNA Intramolecular Complex With Joined Triplex-Duplex Domains Sarah E. Johnson, Calliste Reiling-Steffensmeier, Hui-Ting Lee, and Luis A. Marky J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b10379 • Publication Date (Web): 21 Dec 2017 Downloaded from http://pubs.acs.org on December 27, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry B is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Unfolding and Targeting Thermodynamics of a DNA Intramolecular Complex with Joined Triplex-Duplex Domains Sarah E. Johnson, Calliste Reiling-Steffensmeier, Hui-Ting Lee, and Luis A. Marky* Department of Pharmaceutical Sciences, University of Nebraska Medical Center, 986025 Nebraska Medical Center, Omaha, Nebraska 68198-6025

*Corresponding author: [email protected]

1

ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Our laboratory is interested in developing methods that can be used for the control of gene expression. In this work, we are investigating the reaction of an intramolecular complex containing a triplex-duplex junction with partially complementary strands. We used a combination of isothermal titration (ITC), differential scanning calorimetry (DSC) and spectroscopy techniques to determine standard thermodynamic profiles for these targeting reactions. Specifically, we have designed single strands to target one loop (CTTTC) or two loops (CTTTC and GCAA) of this complex. Both reactions yielded exothermic enthalpies of -66.3 and -82.8 kcal/mol by ITC, in excellent agreement with the reaction enthalpies of -72.7 and -88.7 kcal/mol, respectively, obtained from DSC Hess cycles. The favorable heat contributions result from the formation of base-pair stacks involving mainly the unpaired bases of the loops. This shows each complementary strand is able to invade and disrupt the secondary structure. The simultaneous targeting of two loops yielded a more favorable reaction free energy, by approximately -8 kcal/mol, which corresponds to the formation of roughly four base-pair stacks involving the unpaired bases of the 5’-GCAA loop. The main conclusion is that the targeting of loops with a large number of unpaired bases results in a more favorable reaction free energy.

2


Page 2 of 29

Page 3 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Introduction The conformational manifold of DNA and RNA secondary structures is well known including: triplexes, G-quadruplexes, i-motifs, stem-loop motifs, three- and four-way junctions, and pseudoknots,1–9 which have been found to be involved in a variety of biological functions.2,10–20 These particular structures have been linked to cancer and other human diseases, as well as, cellular life span.8,15,21–27 Due to the high selectivity/specificity of nucleic acids, oligonucleotides can be used for the control of gene expression because of their ability to discriminate targets differing by a single base.10,11,22 In the successful targeting of nucleic acids, it is important to know the structure of the targeting motif. This, however, is usually not the case, but if the sequence of the RNA transcript is known its secondary structure can be predicted.28 Single strands can then be designed to interact with the loops of the target molecule; the unpaired bases of the loops drive the reaction forward. The single strand is able to invade and disrupt the secondary structure of the target molecule forming a larger number of base-pair stacks in the duplex products. There are several approaches for the use of oligonucleotides as modulators of gene expression, including the antisense, antigene, and small interfering RNA strategies.22 In the antisense strategy, an oligonucleotide binds to messenger RNA, forming a DNA/RNA hybrid duplex that inhibits translation by blocking the assembly of the translation machinery, inducing an RNase H mediated cleavage of their mRNA target.10 In the antigene strategy, an oligonucleotide binds to the major groove of a DNA duplex, forming a triple helix29 that inhibits transcription, by competing with the binding of proteins that activate the transcriptional machinery.11,30 There are advantages and disadvantages in these two strategies. In the direct targeting of a gene, the antigene strategy offers some advantages over the antisense strategy.

3



First of all, there are only two copies of a particular gene whereas there is a large continuous supply of the mRNA gene transcript. Moreover, blocking the transcription of the gene itself prevents repopulation of the mRNA pool, allowing a more efficient and lasting inhibition of gene expression.31,32 The main disadvantage is that the oligonucleotide needs to cross the nuclear membrane and access its DNA target within the densely packed chromatin structure.23 One disadvantage of the use of oligonucleotides for targeting purposes is that the oligonucleotide needs to cross lipid membranes. For instance, hydrophilic oligonucleotide duplexes do not cross lipid membranes.33 Another disadvantage is the fast degradative action of endonucleases. These disadvantages can be circumvented by using single strands and by chemically modification of its phosphate or sugar groups. The presence of unpaired nucleobases renders the oligonucleotide slightly more hydrophobic, enabling them to cross the cellular membranes or to interact better with polycationic micelles. These polycations can be used as delivery vectors, protecting the oligonucleotide from the action of nucleases. From a thermodynamic point of view, successful control of gene expression depends on the effective binding of a DNA single strand, with specificity and tight affinity, to its full or partially complementary target. This is provided by using a long sequence of 15-20 bases in length.10 Strong specificity is conferred by hydrogen bonding in the formation of Watson-Crick and/or Hoogsteen base-pairs. While, high affinity is provided by the large negative free energy upon formation of a large number of additional base-pair or base-triplet stacks in the duplex or triplex products, respectively. Thereby, if these stacking contributions are large enough the targeting strands will effectively compete with the proteins involved in transcription or translation. Our laboratory is using DNA oligonucleotides to mimic the secondary structures of RNA molecules and their targeting with complementary strands to create a library of thermodynamic

4


Page 4 of 29

Page 5 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


targeting data.34–36 The novelty of this approach is several fold, DNA oligonucleotides are less expensive than RNA oligonucleotides and more stable against hydrolysis. Most importantly, in the targeting of RNA molecules with DNA complementary strands the resulting DNA/RNA thermodynamic data is similar to the DNA/DNA thermodynamic data in terms of nearestneighbor contributions.37–42 However, one slight drawback of this approach is the higher thermodynamic stability of RNA.41,43 This must be taken into consideration when the target is an RNA molecule. Previously, our laboratory has investigated a variety of targeting reactions including: hairpins, G-quadruplexes, triplexes, pseudoknots and three- and four-way junctions.34–36,44 In these targeting reactions, a single loop was targeted to produce a duplex product. We found that when increasing the loop size in these specific structures, a more stable duplex was formed due to a larger number of base pairs formed in the duplex product. These previous structures studied are basic intramolecular secondary structures found in RNA, confirming DNA can be used to mimic the conformational manifold of RNA. In this work, we mimic the targeting of a potential RNA secondary structure, an intramolecular complex containing a triplex and a duplex motif (shown in Scheme 1), which has a higher folding free energy due to the additional contribution of the complementarity of the third strand. We investigated two targeting reactions using DNA partially complementary strands of varying length. The first reaction involves the targeting of the triplex portion including its loop and the second reaction targets both loops of the triplex and duplex motifs. We would like to answer the following questions: can the complex containing a triplex and duplex motif be targeted in the same way as previously studied structures? How does the length of the complementary strand affect the thermodynamics of targeting this complex? What is the effect of

5



targeting multiple loops? Consequently, what are the thermodynamic contributions favoring the formation of these product duplexes? To this end, we have used a combination of isothermal titration (ITC) and differential scanning calorimetric (DSC) techniques to determine the reaction thermodynamics. The results show that each single strand was able to disrupt this complex, yielding favorable free energy contributions, resulting from exothermic enthalpies. This is due to the formation of additional base-pair stacks in the duplex product, which involve the unpaired bases of the loops present in this DNA triplex-duplex complex.

Scheme 1. Designation and Putative Structures of the triplex-hairpin complex and its component control molecules. Materials and Methods Materials. Oligonucleotides were synthesized and HPLC purified by Integrated DNA Technologies, and desalted by column chromatography using G-10 Sephadex exclusion chromatography. Sequences and their designations for this work are shown in Table 1. Oligonucleotide solution concentrations were determined at 260 nm and 90 ºC using an AVIV 14DS spectrophotometer (Lakewood, NJ) and the molar extinction coefficients shown in the last column of Table 1. The molar extinction coefficients were obtained by extrapolation of the tabulated values for dimers and monomeric bases,37,45 from 25 oC to 90 oC using procedures previously reported.37,39 Extinction coefficients for duplexes (not shown) were calculated by averaging the molar extinction coefficients of the complementary strands. Inorganic salts were reagent grade from Sigma and used without further purification. Measurements were made in 10

6


Page 6 of 29

Page 7 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1: Sequences, designation, and molar extinction coefficients of oligonucleotides 5’ to 3’ Oligonucleotide Sequence

Designation

ε260 ×10-5 (M-1cm-1)

d(A7GTA2CGCA2GT2ACT7CT3CT7) d(A7T7CT3CT7) d(GTA2CGCA2GT2AC) d(A7GA3GA7) d(A7GA3GA7GTA2CT2GCG)

Complex 7TAT-Trip GTAAC-Hp Tri-CS TriHP-CS

3.86 2.80 1.44 2.65 3.54

mM sodium phosphate (label as NaPi throughout the text) with 100 mM NaCl, resulting in a total Na+ concentration of 116 mM, at pH 7.0. All oligonucleotide solutions were prepared by dissolving the dry, desalted ODNs in buffer, heating the solution to 90 oC for 5 min, and cooling to room temperature over 25 min. Isothermal Titration Calorimetry (ITC). The heat for the reaction of Complex with a partially complementary single strand was measured directly by ITC using the iTC200 from Malvern (Northampton, MA). Four to five 2 µL injections of Complex into a 0.2 mL reaction cell containing the targeting oligonucleotide were performed using total strand concentrations of ~ 150 µM of the targeting oligonucleotide and Complex. All experiments were carried out at 5 °C using a mixing rate of 1000 rpm. The solution of single strands was placed in the reaction cell to reduce potential base-base stacking interactions due to its high purine content. These injections were designed mainly to measure the heats of each reaction, which corresponds to the formation of the duplex product. Reaction heats were measured by integration of the area of the injection curve, corrected for the dilution heat of the titrant, and normalized by the moles of titrant added. The average of all injections yielded the reaction enthalpy, ∆HITC.46

7



Temperature-Dependent UV Spectroscopy. Absorbance versus temperature profiles (UV melting curves) were measured at 260 nm with a thermoelectrically controlled AVIV Spectrophotometer Model 14DS UV-Vis. The temperature was scanned at a heating rate of approximately 0.6

o

C/min, and shape analysis of the melting curves yielded transition

temperatures, TMs38. The transition molecularity for the unfolding of a particular complex was obtained by monitoring the TM as a function of strand concentration. Intramolecular complexes have TMs independent of strand concentration, while the TMs of intermolecular complexes do depend on strand concentration.38 Differential Scanning Calorimetry (DSC). The total heat required for the unfolding of each reactant and product of these targeting reactions was measured with a VP-DSC from Malvern. We use buffered solutions containing oligonucleotide with concentrations ranging from 78 to 230 µM in total strands, as indicated in the figure legends. TMs and standard unfolding thermodynamic profiles were obtained from DSC experiments using the following relationships:38,39 ∆Hcal = ∫∆Cp(T)dT; ∆Scal = ∫∆Cp(T)/TdT, and the Gibbs equation, ∆G°(T) = ∆Hcal-T∆Scal, where ∆Cp is the anomalous heat capacity of the oligonucleotide solution during the unfolding process, ∆Hcal is the unfolding enthalpy, ∆Scal is the unfolding entropy, and ∆G°(T) is the unfolding free energy extrapolated to a common temperature of 5 °C, assuming that both ∆Hcal and ∆Scal terms are independent of temperature i.e., the unfolding of a nucleic acid is accompanied by a zeroth heat capacity effect.39,47 We obtain this unfolding data for each reactant and product and the coupling of this data corresponds to the reactions performed in the ITC titrations yielded the Hess cycle profiles, ∆HHC and ∆G°HC, as discussed in a later section. Circular Dichroism (CD) Spectroscopy. An AVIV circular dichroism spectrometer, Model 202SF equipped with a peltier temperature control system (Lakewood, NJ) was used to obtain

8


Page 8 of 29

Page 9 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


the spectra of Complex, the control molecules, partially complementary single strands and duplex products. All CD experiments consisted of using an approximately 1 OD sample solution in a 10 mm quartz cell; the spectrum was recorded from 200 nm to 320 nm at 1 nm intervals. Scans were taken at 5 ºC to ensure the molecules were in a 100% helical state. Final spectra were the result of averaging two scans, subtracting a buffer spectrum, and normalizing by concentration.

Scheme 2. A) Designation and Structures of the reactant and products of the reactions investigated. B) Hess cycle diagram of first reaction

9



Results and Discussion The overall experimental approach is as follows.

First is to determine the unfolding

thermodynamics for Complex along with its control component molecules, 7TAT-Trip and GTAAC-Hp, shown in Scheme 1. Based on its unfolding behavior, we have designed two single strands, which were partially complementary to Complex. A cartoon of these targeting reactions is shown in Scheme 2A. Each reaction was designed to yield a favorable free energy contribution in enthalpy driven reactions due to the additional formation of base-pair stacks involving the targeting of the unpaired bases of the loops of Complex. Then, standard thermodynamic profiles are determined for each targeting reaction. Initially, we used ITC titrations to measure directly the reaction heats. Next, we used DSC to determine unfolding thermodynamic profiles for the reactants and products of each reaction, which are used to set up thermodynamic Hess cycles (Scheme 2B). This experimental approach indirectly determines thermodynamic profiles for each targeting reaction from the unfolding profiles of the reactants and products. To determine the free energy of the ITC experiments, ∆G°ITC, we use the following relationship: ∆G°ITC = ∆G°HC (∆HITC/∆HHC) for each targeting reaction.34–36,48–50 This equation was previously derived empirically and corrects for single-stranded base stacking contributions of the ITC experiments.49,50 Unfolding Thermodynamics of Complex, 7TAT-Trip and GTAAC-Hp. The UV melts (data not shown) of Complex show biphasic transitions, while 7TAT-Trip and GTAAC-Hp show monophasic transitions. Figure 1A shows their TMs as a function of total strand concentration for each molecule. All TMs remain constant over a tenfold range of total strand concentration, indicating the intramolecular nature of each transition. Figure 1B shows the DSC unfolding of Complex and its component molecules and the results are shown in Table 2. The unfolding of

10


Page 10 of 29

Page 11 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Complex takes place through a biphasic transition with TMs of 27.2 and 69.5°C, and unfolding enthalpies of 25.9 and 86.9 kcal/mol, respectively. While, the unfolding of 7TAT-Trip takes place with a TM of 34.2 °C and enthalpy of 83.0 kcal/mol and GTAAC-Hp has a TM of 66.6 °C and enthalpy of 38.4 kcal/mol (Table 2), yielding a combined enthalpy of 121.4 kcal/mol.

Figure 1. TM dependences on Strand Concentration, DSC Thermograms and CD Spectra for Complex and Control Molecules. All experiments were carried out in100 mM NaCl, 10 mM NaPi buffer at pH 7.0. A) Dependence of TM on strand concentration: Complex, 1st Transition (●) and 2nd Transition (O); 7TAT-Trip (); GTAAC-Hp () B) DSC thermograms performed at the indicated strand concentration (in total strands): Complex, 0.078 mM ( ); 7TAT-Trip, 0.065 mM ( ) and GTAAC-Hp, 0.045 mM ( ). C) Left panel: CD spectra of Complex ( ), ), and GTAAC-Hp ( 7TAT-Trip ( ). Right panel: the spectra of Complex ( ) is overlaid with the additive spectra of 7TAT-Trip and GTAAC-Hp ( ).

11



Page 12 of 29

Table 2. Unfolding Thermodynamic Profiles of Complex and its Component Molecules. Transition TM ∆Hcal ∆Go(5) T∆Scal o ( C) (kcal/mol) (kcal/mol) (kcal/mol) Complex 1st 27.4 25.9 1.9 24.0 nd 2 69.5 86.9 16.4 70.5 Total 112.8 18.3 94.5

34.2

7TAT-Trip 83.0

7.9

75.1

66.6

GTAAC-Hp 38.4

7.0

31.4

Total 121.4 14.9 106.5 Experiments were performed in 100 mM NaCl, 10 mM NaPi, pH 7.0. Experimental errors are TM (± 0.5 ºC), ∆Hcal (± 5%), T∆Scal ( ± 5%) and ∆Gº(5) (± 7%). This observed enthalpy is in excellent agreement with the total unfolding enthalpy of Complex (112.8 kcal/mol), indicating Complex is able to retain both triplex and duplex domains. Actually, the substitution of the 5 cytosine loop of 7TAT-Trip with GTAAC-Hp to form Complex (see Scheme 1) yielded a net enthalpy difference of 8.6 kcal, which can be attributed to a compensation of the loss of base stacking of the C5 loop of 7TAT-Trip (two cytosines stacked on the stem of this triplex) with the formation of an additional AG/TC base-pair stack at the triplexduplex junction. Furthermore, this substitution affects the overall melting of Complex because the length of the duplex stem is increased i.e., higher number of base-pair stacks, yielding a sequential biphasic melting behavior: the first transition corresponds to the unfolding of the third strand follow by the unfolding of the duplex stem. The CD spectra for all molecules are shown on the left panel of Figure 1C. All molecule show two main bands with wavelength minimums around 250 nm and wavelength maximums at 275 nm; both bands have similar overall areas, indicating all three oligonucleotide adopt the “B” conformation.51 Complex shows a higher intensity for the minimum peak centered around 250

12


Page 13 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


nm, indicating larger stacking contributions from the additional stacking of base-triplets. 7TATTrip has an intermediate intensity, while GTAAC-Hp has the lowest intensity. This is due to contributions from exclusively base-triplet stacking (7TAT-Trip) and base-pair stacking (GTAAC-Hp). The peak centered around 275 nm is due to the sugar puckering of these molecules. Furthermore, the small split of the positive band, 260-265 nm, is characteristic of the homopurine/homopyrimidine sequences (A7/T7) used in Complex and 7TAT-Trip. A third and negative band is observed at 210 nm for both Complex and 7TAT-Trip, this is a characteristic feature of the formation of DNA triple helices.52,53 On the right panel of Figure 1C, the CD spectrum of Complex overlays well with the sum spectra of 7TAT-Trip and GTAAC-Hp, confirming that in this particular complex the sum of the parts equals the whole. One difference includes the lower magnitude at 250 nm of Complex, which is indicative of the additional AG/TC stack at the triplex-duplex junction. Another small difference is the higher magnitude at 275 nm of the sum spectra, which may be attributed to contributions of the 5 cytosines loop in 7TAT-Trip. Overall Targeting Approach. Scheme 2A shows a cartoon of the targeting reactions investigated, we used two single strands with different lengths that are partially complementary to Complex. In the first reaction, the single strand (Tri-CS) is targeting the triplex domain on the left side of Complex, including its loop. While in the second reaction, the single strand (TriHP-CS) is targeting both the full triplex and duplex domains, including the duplex loop on the right side of Complex. The effective targeting of Complex will generate two duplex products, Tri-Dup and TriHP-Dup (Scheme 2A). Each reaction is investigated directly using ITC and indirectly by using DSC Hess cycles resulting from the DSC unfolding thermodynamic profiles of the reactants and products of each targeting reaction.

13

The expectation is that we will obtain



exothermic enthalpies in both reactions, which results from the net compensation of an endothermic heat from breaking base-triplet stacks and base-pair stacks of Complex with an exothermic heat from the formation of base-pair stacks of the duplex products. These exothermic heats primarily result from the participation of the unpaired bases of the loops, 5 (triplex loop) and 9 (both loops). Therefore, in these two reactions we are actually determining the heat contributions of the loops to the overall thermodynamics of each targeting reaction. Furthermore, we expected to measure reaction enthalpies of -69.5 and -114.1 kcal/mol, respectively. These values are estimated from Hess cycles using the folding enthalpies of Complex, -112.8 kcal/mol (Table 2) and predicted enthalpies from nearest-neighbors of the duplex products, -182.3 (TriDup) and -226.9 (TriHP-Dup) kcal/mol.41,42,54 Enthalpy Contributions of reaction one: In this reaction, Complex is targeted with a partially complementary strand (Tri-CS) to form a duplex with a hairpin and dangling end (Tri-Dup). In the ITC titration (Figure 2A), Complex was titrated into Tri-CS, yielding a ∆HITC of -66.3 kcal/mol. This enthalpy is in excellent agreement with the predicted enthalpy of -69.5 kcal/mol. Next, we measured indirectly the enthalpy of this reaction by creating a Hess cycle (Scheme 2B). The unfolding of Complex is biphasic while Tri-Dup is triphasic. UV melts as a function of strand concentration (Figure 2B) for the reactants and products showed the TM of Complex is independent of strand concentration, while the TM of the first transition of Tri-Dup, increases with increasing strand concentration, indicating a bimolecular formation. However, the TM of its second transition remains the same (Figure 2B), which corresponds to the unfolding of an intramolecular structure. The DSC unfolding thermodynamic profiles are shown in Table 3. The

14


Page 14 of 29

Page 15 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Reaction One Characterization. A) ITC of Complex (0.149 mM) into Tri-CS (0.149 mM). B) Dependence of TM on strand concentration: Complex 1st Transition (●), 2nd Transition Complex (O) and Tri-Dup 1st Transition (), 2nd Transition (), 3rd Transition () C) DSC thermograms of Complex 0.078 mM ( ), Tri-Dup, 0.23 mM ( ) D) Left panel: CD spectra of Complex ( ) and Tri-CS ( ) ). Right panel: the spectra of Complex and Tri-CS ( overlaid with Tri-Dup ( ). All experiments were performed in 100 mM NaCl, 10 mM NaPi at pH 7.0

15



Table 3. Unfolding Thermodynamic Profiles for Complex and Duplex Products TM ∆Hcal ∆Go(5) T∆Scal o ( C) (kcal/mol) (kcal/mol) (kcal/mol) Complex 1st 27.4 25.9 1.9 24.0 nd 2 69.5 86.9 16.4 70.5 Total 112.8 18.3 94.5 Tri-Dup 1 35.4 73.9 7.3 66.6 2nd 48.9 26.6 3.6 23.0 rd 3 69.4 85.0 16.0 69.0 Total 185.5 26.9 158.6 TriHP-Dup 1st 60.9 152.0 25.4 126.6 nd 2 71.1 49.5 9.5 40.0 Total 201.5 34.9 166.6 Experiments were performed in 10 mM sodium phosphate buffer, 100 mM NaCl, at pH 7.0. Experimental errors are TM (± 0.5 ºC), ∆Hcal (± 5%), T∆Scal (± 5%), ∆Gº(5) (± 7%). st

unfolding of Complex has been discussed in the previous section. The DSC unfolding of a TriCS yielded a broad transition with a folding enthalpy of 3.4 kcal/mol (data not shown), while the unfolding of Tri-Dup, shown in Figure 2C, yielded three transitions with TMs/∆HDSCs of 35.4 °C/73.9 kcal/mol, 48.9 °C/26.6 kcal/mol, and 69.4 °C/85.0 kcal/mol, respectively (Table 3). The sum of these enthalpies (185.5 kcal/mol) is in excellent agreement with the N-N estimate of 182.3 kcal/mol for this duplex.41,42,54 A closer examination of the melting profile of Tri-Dup reveals the first two transitions correspond to the unfolding of the duplex product followed by the melting of the long hairpin of 12 base-pairs, which reform after Tri-CS dissociates. This reaction ∆HHC is obtained by subtracting the unfolding enthalpy of Tri-Dup from the unfolding enthalpy of Complex, the unfolding enthalpy of Tri-CS is not included because it does not have a defined transition. The Hess cycle for this first reaction yielded a ∆HHC of -72.7 kcal/mol for the formation of Tri-Dup (Table 4). This value takes into account the enthalpy contributions for the unfolding of Complex and the formation of the duplex product. This value

16


Page 16 of 29

Page 17 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


is in excellent agreement with the enthalpy (-66.3 kcal/mol) obtained directly from ITC. The overall result confirms the net exothermic enthalpy of this targeting reaction corresponds to a complete override of the endothermic heat contributions from the disruption of base-triplet and base-pair stacking of Complex by the exothermic heat contributions from the formation of a larger number of base-pair stacks in the duplex product.35,49,55 This demonstrates that we can mimic the targeting of RNA due to DNA’s ability to form similar structures. This particular single strand is able to disrupt the triplex portion of this complex due to the additional base-pair and base-pair stacks involving mainly the unpaired bases of the loops. Table 4. Reaction Thermodynamic Profiles from ITC and Hess Cycles. ITC

DSC (Hess Cycle) Duplex Product ∆HITC ∆G°ITC ∆HHC T∆SHC ∆G°HC (kcal/mol) (kcal/mol) (kcal/mol) (kcal/mol) (kcal/mol) Tri-Dup -66.3 -7.8 -72.7 -64.1 -8.6 TriHP-Dup -82.8 -15.6 -88.7 -72.1 -16.7 All parameters measured in 10 mM sodium phosphate buffer, 100 mM NaCl, at pH 7.0. Experimental errors as follows: ∆HITC (±5%), ∆G°ITC (±7%), ∆HHC (±10%), T∆SHC (±10%), ∆G°HC (±14%). The CD spectra of Complex and Tri-CS are shown on the left panel of Figure 2D. The spectrum of Tri-CS is similar to a right-handed helix due to its sequence rich in adenine,56 basebase stacking is higher for these sequences relative to a single strand rich in pyrimidine bases. Complex has a lower ellipticity at 250 nm, higher stacking contributions, due to its base-pair and base-triplet stacks. The right panel of Figure 2D shows the overlay CD spectra of Tri-Dup spectrum and the sum spectra of Complex plus Tri-CS, at 250 nm the magnitude of the ellipticity at 250 nm is larger for Tri-Dup, confirming the enthalpy results from higher stacking contributions that are formed in the duplex product.

17



Enthalpy contributions of reaction two. In this reaction, Complex is targeted with TriHP-CS to form a longer duplex with a dangling end (TriHP-Dup). Figure 3A shows the ITC titration in which Complex was titrated with TriHP-CS, the average heat of the five injections yielded a ∆HITC of -82.8 kcal/mol. This enthalpy is much lower than the predicted enthalpy of -114.1 kcal/mol, this difference will be discussed below. Next, we determine melting profiles for each reactant and product and create a Hess cycle for this second reaction (Scheme 2B). The UV melts of both Complex and TriHP-Dup are biphasic, while no defined transition was observed for the TriHP-CS single strand (data not shown). The TM-dependences on strand concentration for the transitions of TriHP-Dup are shown in Figure 3B. The TM of the first transition increases, while the TM of the second transition remains the same with the increase in strand concentration, indicating the first transition is due to the dissociation of TriHP-CS and the second transition is due to the unfolding of Complex, which may be reformed after the complementary strand is removed. The DSC unfolding of Complex has been discussed earlier, while the DSC unfolding of a TriHP-CS yielded a broad transition with an unfolding enthalpy of 6.8 kcal/mol (data not shown). The unfolding of TriHP-Dup is shown in Figure 2C and the overall thermodynamic profiles are reported in Table 3. The unfolding of TriHP-Dup yielded two transitions with TMs/∆HDSCs of 60.9 °C/152.0 kcal/mol and 71.1 °C/49.5 kcal/mol, respectively. The total enthalpy of these transitions is 201.5 kcal/mol, which is in fair agreement with a N-N estimate of 226.9 kcal/mol.41,42,54 A closer examination of the unfolding of TriHP-Dup reveals the first transition is due to the dissociation of TriHP-CS, which correlates with the concentration dependence observed from the UV melting experiments. The second transition is intramolecular with a TM of 71.1 °C, which reflects the melting temperature of the long 12 base-pair hairpin.

18


Page 18 of 29

Page 19 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Reaction Two Characterization. A) ITC of Complex (0.149 mM) into TriHP-CS (0.149 mM), B) TM dependence on strand concentration: Complex, 1st Transition (●), 2nd Transition (O); TriHP-Dup 1st Transition (), 2nd Transition (), C) DSC thermograms of Complex, 0.078 mM ( ) and TriHP-Duplex, 0.18 mM ( ), E) Left panel: CD spectra of Complex ( ) and TriHP-CS ( ). Right panel: summation spectra of Complex and TriHP-CS ( ) overlaid with TriHP-Dup ( ). All experiments were performed in 100 mM NaCl, 10 mM NaPi at pH 7.0.

19



However, its enthalpy is lower than the experimental enthalpy of the second transition of Complex (86.9 kcal/mol), suggesting only 57% of the long hairpin is reforming after TriHP-CS dissociates due to the higher TM of this duplex. This could be one explanation for the enthalpy difference of 25.4 kcal/mol in the unfolding of TriHP-Dup. If 100% of this hairpin were to reform, there would be an experimental enthalpy of 238.9 kcal/mol for this duplex, which would be in better agreement with the predicted enthalpy. The ∆HHC of the second reaction is obtained by subtracting the unfolding enthalpy of TriHpDup from the unfolding enthalpy of Complex, the unfolding enthalpy of TriHp-CS is not included because it does not have a defined transition. This exercise yielded a ∆HHC of -88.7 kcal/mol for the formation of TriHP-Dup (Table 4). This value is in excellent agreement with the enthalpy of -82.8 kcal/mol obtained directly from ITC. From these results, we confirm this reaction takes place with a greater net exothermic enthalpy of 16.3 kcal/mol compared to reaction one, a higher number of base-pairs are formed in this reaction product.35,49,55 This greater exothermic enthalpy confirms that targeting a larger number of unpaired bases increases the probability of the reaction to take place, i.e., a larger favorable free energy term. This result should be considered in the designing of complementary single strands by targeting a larger of unpaired bases, which normally exists in the loops of RNA secondary structures.The CD spectra of Complex and TriHP-CS are shown on the left panel of Figure 3D. The spectrum of Complex was discussed previously, however, the spectrum of TriHP-CS is similar to the previous single strand (Tri-CS) and shows some base-base stacking contributions due to its sequence rich in adenines.56 The right panel of Figure 3D shows the overlayed CD spectra of TriHP-Dup spectrum and the sum spectra of Complex and TriHP-CS; at 250 nm the magnitude of the ellipticity is larger for TriHP-Dup, confirming the ITC and DSC results of higher stacking

20


Page 20 of 29

Page 21 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


contributions formed in this duplex product. Moreover, the magnitude of its ellipticity is greater than that of the duplex product (Tri-Dup) of the first reaction. This is indicative of higher basepair stacking contributions, consistent with its higher folding enthalpy. Standard thermodynamic profiles for the targeting reactions. To determine the free energy of each targeting reaction, ∆G°ITC, we use the relationship: ∆G°ITC = ∆G°HC (∆HITC/∆HHC), where ∆G°HC is calculated from the DSC data in a similar manner as the ∆HHC terms discussed previously. The use of this equation allows us to correct the ∆G°HC term for base-base stacking contributions of the single strands. For instance, in the formation of a duplex from the mixing of two complementary strands, the overall heat obtained from ITC is always lower than the heat obtained from the DSC unfolding, |∆HITC| < |∆HDSC|. The overall thermodynamic data for these two reactions at 5 °C are shown in Table 4. In the first reaction the triplex portion of Complex is targeted, which includes the 5 unpaired bases of the triplex loop (d(CTTTC)). We obtained a ∆G°HC = -8.6 kcal/mol and ∆G°ITC = -7.8 kcal/mol, which are in good agreement (Table 4). For reaction two, the triplex and duplex portions of Complex are targeted, which include the 9 unpaired bases of both loops (d(CTTTC) and d(GCAA)). This yielded a ∆G°HC = -16.7 kcal/mol and ∆G°ITC = -15.6 kcal/mol. We obtained a ΔΔG°

ITC

of -7.8 kcal/mol between these two

targeting reactions. This differential term corresponds to the formation of four additional basepair stacks, which is exactly the difference in the number of unpaired bases (9-5 = 4) between reaction two and reaction one . A similar exercise but using reaction ITC enthalpies instead, yielded a ∆∆HITC of -16.5 kcal/mol (Table 4). This differential term corresponds to the formation of at least two additional base-pair stacks. A similar calculation for ∆(T∆SITC) and using the ITC parameters of Table 4, yielded a ∆(T∆SITC) term of -8.7 kcal/mol. This differential entropy term

21



corresponds to differences in the putative ordering of the strands in their respective duplex state and the uptake of counterions and water molecules. In summary, both reactions yielded favorable free energy contributions, i.e., both targeting reactions occur spontaneously. The higher the number of unpaired bases targeted results in higher favorable free energy terms. Overall, these favorable free energy terms resulted from large compensations of a favorable enthalpy and unfavorable entropy contributions. The enthalpy contributions correspond mainly to the formation of base-pair stacks. While, unfavorable entropy contributions include the bimolecular association of two strands and the putative immobilization of water molecules and counterions

Conclusions We have investigated two reactions using DNA single strands as reagents to target an intramolecular DNA complex. Specifically, we have used a combination of ITC, DSC and spectroscopy techniques to determine standard thermodynamic profiles for the reaction of a triplex-duplex complex with different complementary strands. The enthalpies are measured directly from ITC titrations and compared to those obtained indirectly from Hess cycles using DSC unfolding data. Both reactions investigated yielded favorable free energy terms, i.e., each single strand is able to invade and disrupt the corresponding intramolecular DNA structure with all techniques used, resulting from the typical compensation of favorable enthalpy-unfavorable entropy contributions. These exothermic heat contributions are due primarily to the formation of additional base-pair stacks in the duplex products. Overall, we have determined an increase in favorable free energy by approximately 2 kcal/mol for each additional base-pair stack formed in the second reaction, relative to the first reaction.

22


Page 22 of 29

Page 23 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The application of this approach to control gene expression can yield a more favorable reaction if loops of larger sizes or multiple loops are targeted, especially in the targeting of RNA structures. In this type of targeting, if the structure of the RNA is known then DNA single strands can be designed to target the largest number of unpaired bases of its secondary structure. This will increase the favorable free energy term, allowing the single strand to compete with the binding of potential proteins. Longer oligonucleotides will also help to disrupt the compact nature of RNA by driving the reaction forward to form a more stable duplex product. Similarly, the simultaneous targeting of several loops of the secondary/tertiary structure of mRNA with several oligonucleotides will also yield favorable free energy terms. Acknowledgments Grant MCB-1122029 from the National Science Foundation supported this work.

23



References 1.

Rich, A. DNA Comes in Many Forms. Gene 1993, 135 , 99–109.

2.

Juliano, R.L.; Astriab-Fisher, A.; Falke, D. Macromolecular Therapeutics: Emerging Strategies for Drug Discovery in the Postgenome Era. Mol. Interv. 2001, 1 , 40–53.

3.

Kaushik, M.; Suehl, N.; Marky, L.A. Calorimetric Unfolding of the Bimolecular and IMotif Complexes of the Human Telomere Complementary Strand, d(C3TA2)4. Biophys. Chem. 2007, 126 , 154–164.

4.

Gehring, K.; Leroy, J.L.; Guéron, M. A Tetrameric DNA Structure with Protonated Cytosine.cytosine Base Pairs. Nature 1993, 363 , 561–565.

5.

Isambert, H.; Siggia, E.D. Modeling RNA Folding Paths with Pseudoknots: Application to Hepatitis Delta Virus Ribozyme. Proc. Natl. Acad. Sci. USA 2000, 97 , 6515–6520.

6.

Pleij, C.W.A. RNA Pseudoknots. Curr. Opin. Struct. Biol. 1994, 4 , 337–344.

7.

Baker, E.S.; Dupuis, N.F.; Bowers, M.T. DNA Hairpin, Pseudoknot, and Cruciform Stability in a Solvent-Free Environment. J. Phys. Chem. B 2009, 113 , 1722–1727.

8.

Wu, Y.; Brosh, R.M. G-Quadruplex Nucleic Acids and Human Disease. FEBS J. 2010, 277 , 3470–3488.

9.

Correll, C.C.; Wool, I.G.; Munishkin, A. The Two Faces of the Escherichia Coli 23 S rRNA Sarcin/ricin Domain: The Structure at 1.11 A Resolution. J Mol Biol. 1999, 292 , 275–287.

10.

Crooke, S.T. Molecular Mechanisms of Action of Antisense Drugs. Biochimica et Biophysica Acta - Gene Structure and Expression 1999, 1489 , 31–43.

11.

Hélène, C. Rational Design of Sequence-Specific Oncogene Inhibitors Based on Antisense and Antigene Oligonucleotides. Eur. J. Cancer Clin. Oncol. 1991, 27 , 1466–1471.

12.

Firulli, A.B.; Maibenco, D.C.; Kinniburgh, A.J. Triplex Forming Ability of a c- Myc Promoter Element Predicts Promoter Strength. Arch. Biochem. Biophys. 1994, 310 , 236– 242.

13.

Fox, K.R. Long (dA)n.(dT)n Tracts Can Form Intramolecular Triplexes under Superhelical Stress. Nucleic Acids Res. 1990, 18 , 5387–5391.

14.

Han, H.; Hurley, L.H. G-Quadruplex DNA: A Potential Target for Anti-Cancer Drug Design. Trends in Pharmacological Sciences 2000, 21 , 136–142.

15.

Mills, M.; Lacroix, L.; Arimondo, P.B.; Leroy, J.-L.; François, J.-C.; Klump, H.; Mergny, J.-L. Unusual DNA Conformations: Implications for Telomeres. Curr. Med. Chem. Anticancer. Agents 2002, 2 , 627–644.

24


Page 24 of 29

Page 25 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


16.

Bock, L.C.; Griffin, L.C.; Latham, J.A.; Vermaas, E.H.; Toole, J.J. Selection of SingleStranded DNA Molecules That Bind and Inhibit Human Thrombin. Nature 1992, 355 , 564–566.

17.

Wang, K.Y.; Krawczyk, S.H.; Bischofberger, N.; Swaminathan, S.; Bolton, P.H. The Tertiary Structure of a DNA Aptamer Which Binds to and Inhibits Thrombin Determines Activity. Biochemistry 1993, 32 , 11285–11292.

18.

Rando, R.F.; Ojwang, J.; Elbaggari, A.; Reyes, G.R.; Tinder, R.; McGrath, M.S.; Hogan, M.E. Suppression of Human Immunodeficiency Virus Type 1 Activity in Vitro by Oligonucleotides Which Form Intramolecular Tetrads. J. Biol. Chem. 1995, 270 , 1754– 1760.

19.

Somogyi, P.; Jenner, A.J.; Brierley, I.; Inglis, S.C. Ribosomal Pausing during Translation of an RNA Pseudoknot. Mol. Cell. Biol. 1993, 13 , 6931–6940.

20.

Maizels, N. Dynamic Roles for G4 DNA in the Biology of Eukaryotic Cells. Nat. Struct. Mol. Biol. 2006, 13 , 1055–1059.

21.

Huard, S.; Autexier, C. Targeting Human Telomerase in Cancer Therapy. Curr. Med. Chem. Anticancer. Agents 2002, 2 , 577–587.

22.

Beal, P.A.; Dervan, P.B. Second Structural Motif for Recognition of DNA by Oligonucleotide- Directed Triple-Helix Formation. Science. 1991, 251 , 1360–1363.

23.

Brown, P.M.; Madden, C.A.; Fox, K.R. Triple-Helix Formation at Different Positions on Nucleosomal DNA. Biochemistry 1998, 37 , 16139–16151.

24.

Zahler, A.M.; Williamson, J.R.; Cech, T.R.; Prescott, D.M. Inhibition of Telomerase by G-Quartet DMA Structures. Nature 1991, 350 , 718–720.

25.

Jung, S.; Schlick, T. Candidate RNA Structures for Domain 3 of the Foot-and-MouthDisease Virus Internal Ribosome Entry Site. Nucleic Acids Res. 2013, 41 , 1483–1495.

26.

Brar, G.A.; Amon, A. Emerging Roles for Centromeres in Meiosis I Chromosome Segregation. Nat. Rev. Genet. 2008, 9 , 899–910.

27.

Counter, C.M. The Roles of Telomeres and Telomerase in Cell Life Span. Mutat. Res. Genet. Toxicol. 1996, 366 , 45–63.

28.

M. Zuker; D. H. Mathews; D. H. Turner. Algorithms and Thermodynamics for RNA Secondary Structure Prediction: A Practical Guide. in RNA Biochemistry and Biotechnology (1999) 11–43.

29.

Mahato, R.I.; Cheng, K.; Guntaka, R. V. Modulation of Gene Expression by Antisense and Antigene Oligodeoxynucleotides and Small Interfering RNA. Expert Opin Drug Deliv 2005, 2 , 3–28.

25



30.

Sinden, R.R. Triple-Helical Nucleic Acids. Vitr. Cell. Dev. Biol. - Anim. 1996, 32 , 591– 592.

31.

Maher, L.J.; Wold, B.; Dervan, P.B. Inhibition of DNA Binding Proteins by Oligonucleotide-Directed Triple Helix Formation. Science 1989, 245 , 725–730.

32.

Fox, K.R. Targeting DNA with Triplexes. Curr. Med. Chem. 2000, 7 , 17–37.

33.

Vasquez, K.M.; Dagle, J.M.; Weeks, D.L.; Glazer, P.M. Chromosome Targeting at Short Polypurine Sites by Cationic Triplex-Forming Oligonucleotides. J. Biol. Chem. 2001, 276 , 38536–38541.

34.

Lee, H.T.; Carr, C.; Siebler, H.; Waters, L.; Khutsishvili, I.; Iseka, F.; Domack, B.; Olsen, C.M.; Marky, L.A. A Thermodynamic Approach for the Targeting of Nucleic Acid Structures Using Their Complementary Single Strands. Methods Enzymol. 2011, 492 , 1– 26.

35.

Lee, H.T.; Olsen, C.M.; Waters, L.; Sukup, H.; Marky, L.A. Thermodynamic Contributions of the Reactions of DNA Intramolecular Structures with Their Complementary Strands. Biochimie 2008, 90 , 1052–1063.

36.

Reiling, C.; Marky, L.A. Contributions of the Loops on the Stability and Targeting of DNA Pseudoknots. Biochem. Compd. 2014, 2

37.

Borer, P. Optical Properties of Nucleic Acids, Absorption and Circular Dichroism Spectra. in CRC Handbook of Biochemistry and Molecular Biology: Nucleic Acids. (ed. G.D. Fasman) (1975). , 589–595.

38.

Marky, L.A.; Breslauer, K.J. Calculating Thermodynamic Data for Transitions of Any Molecularity from Equilibrium Melting Curves. Biopolymers 1987, 26 , 1601–1620.

39.

Marky, L.A.; Maiti, S.; Olsen, C.; Shikiya, R.; Johnson, S.E.; Kaushik, M.; Khutsishvili, I. Building Blocks of Nucleic Acid Nanostructures: Unfolding Thermodynamics of Intramolecular DNA Complexes. in Biomedical Applications of Nanotechnology (John Wiley & Sons, Inc., 2007). , 191–226.

40.

Sugimoto, N.; Nakano, S.; Katoh, M.; Matsumura, A. Thermodynamic Parameters to Predict Stability of RNA/DNA Hybrid Duplexes. Biochemistry 1995, 34 , 11211–11216.

41.

SantaLucia, J. A Unified View of Polymer, Dumbbell, and Oligonucleotide DNA NearestNeighbor Thermodynamics. Proc. Natl. Acad. Sci. 1998, 95 , 1460–1465.

42.

SantaLucia, J.; Allawi, H.T.; Seneviratne, P.A. Improved Nearest-Neighbor Parameters for Predicting DNA Duplex Stability. Biochemistry 1996, 35 , 3555–3562.

43.

Xia, T.; SantaLucia, J.; Burkard, M.E.; Kierzek, R.; Schroeder, S.J.; Jiao, X.; Cox, C.; Turner, D.H. Thermodynamic Parameters for an Expanded Nearest-Neighbor Model for Formation of RNA Duplexes with Watson - Crick Base Pairs. Biochemistry 1998, 37 ,

26


Page 26 of 29

Page 27 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


14719–14735. 44.

Prislan, I.; Lee, H.T.; Lee, C.; Marky, L.A. The Size of the Internal Loop in DNA Hairpins Influences Their Targeting with Partially Complementary Strands. J. Phys. Chem. B 2015, 119 , 96–104.

45.

Cantor, C.R.; Warshaw, M.M.; Shapiro, H. Oligonucleotide Interactions. III. Circular Dichroism Studies of the Conformation of Deoxyoligonucleolides. Biopolymers 1970, 9 , 1059–1077.

46.

Wiseman, T.; Williston, S.; Brandts, J.F.; Lin, L.N. Rapid Measurement of Binding Constants and Heats of Binding Using a New Titration Calorimeter. Anal. Biochem. 1989, 179 , 131–137.

47.

Olsen, C.M.; Shikiya, R.; Ganugula, R.; Reiling-Steffensmeier, C.; Khutsishvili, I.; Johnson, S.E.; Marky, L.A. Application of Differential Scanning Calorimetry to Measure the Differential Binding of Ions, Water and Protons in the Unfolding of DNA Molecules. Biochim. Biophys. Acta - Gen. Subj. 2016, 1860 , 990–998.

48.

Rentzeperis, D.; Kupke, D.W.; Marky, L.A. Differential Hydration of dA.dT Base Pairs in Parallel-Stranded DNA Relative to Antiparallel DNA. Biochemistry 1994, 33 , 9588–9591.

49.

Zieba, K.; Marky, L.A.; Chu, T.M.; Kupke, D.W. Differential Hydration of dA•dT Base Pairing and dA and dT Bulges in Deoxyoligonucleotides. Biochemistry 1991, 30 , 8018– 8026.

50.

Marky, L.A.; Rentzeperis, D.; Luneva, N.P.; Cosman, M.; Geacintov, N.E.; Kupke, D.W. Differential Hydration Thermodynamics of Stereoisomeric DNA-Benzo[a]pyrene Adducts Derived from Diol Epoxide Enantiomers with Different Tumorigenic Potentials. J. Am. Chem. Soc. 1996, 118 , 3804–3810.

51.

Miyahara, T.; Nakatsuji, H.; Sugiyama, H. Helical Structure and Circular Dichroism Spectra of DNA: A Theoretical Study. J. Phys. Chem. A 2012, 117 , 42–55.

52.

Plum, G.E.; Breslauer, K.J. Calorimetry of Proteins and Nucleic Acids. Curr. Opin. Struct. Biol. 1995, 5 , 682–690.

53.

Soto, A.M.; Marky, L.A. Thermodynamic Contributions for the Incorporation of GTA Triplets within Canonical TAT/TAT and C+GC/C+GC Base-Triplet Stacks of DNA Triplexes. Biochemistry 2002, 41 , 12475–12482.

54.

Breslauer, K.J.; Frank, R.; Blöcker, H.; Marky, L.A. Predicting DNA Duplex Stability from the Base Sequence. Proc. Natl. Acad. Sci. U. S. A. 1986, 83 , 3746–3750.

55.

Marky, L.A.; Kupke, D.W. Enthalpy-Entropy Compensations in Nucleic Acids: Contribution of Electrostriction and Structural Hydration. Methods in Enzymology 2000, 323 , 419–441.

27



56.

Protozanova, E.; Macgregor, R.B. Circular Dichroism of DNA Frayed Wires. Biophys. J. 1998, 75 , 982–989.

28


Page 28 of 29

Page 29 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


TOC Graphic

29


Unfolding and Targeting Thermodynamics of a DNA Intramolecular

Recommend Documents