Pressure Dependence and Volumetric Properties of Short DNA

Hairpins

by

Amir Reza Amiri

A thesis submitted in conformity with the requirements for the degree of

MASTERS OF SCIENCE

Graduate Department of Pharmaceutical Sciences

Leslie Dan Faculty of Pharmacy

University of Toronto

©Copyright by Amir Reza Amiri, 2010

ii

ABSTRACT

Pressure Dependence and Volumetric Properties of Short DNA Hairpins

Amir Reza Amiri, MSc, 2010

Department of Pharmaceutical Sciences

Leslie Dan Faculty of Pharmacy

University of Toronto

Previous studies of short DNA hairpins have revealed that loop and stem sequences can

significantly affect the thermodynamic stability of short DNA hairpins. Nevertheless, there has

not been sufficient investigation into the pressure-temperature stability of DNA hairpins, and the

current thermodynamic knowledge of DNA hairpins’ stability is limited to the temperature

domain. In this work, we report the effect of hydrostatic pressure on the helix-coil transition

temperature (TM) for eleven short DNA hairpins at different salt concentrations by performing

UV-monitored melting. The studied hairpins form by intramolecular folding of 16-base self-

complementary DNA oligodeoxyribonucleotides. Model dependent (van’t Hoff) transition

parameters such as ΔHvH and transition volume (ΔV) were estimated from analysis of optical

melting transitions. Experiments revealed the ΔV for denaturation of these molecules range

from -2.35 to +6.74 cm3mol

-1. The difference in the volume change for this transition is related

to differences in the hydration of these molecules.

iii

ACKNOWLEDGMENTS

I would like to express my humble gratitude to the following people:

Dr. Robert Macgregor, Jr., my thesis supervisor, for his great patience, support, approachability,

and valuable advice.

Dr. Tigran V. Chalikian and Dr. Heiko Heerklotz, the members of supervisory committee, for

their time and helpful insights into my research project.

My undergraduate student Zhang Guan Nan for assisting me with sample preparation and optical

melting experiments during my first year as a graduate student.

My lab mates including those from the Chalikian and Heerkoltz laboratories.

Mr. Andras Nagy for fixing the pressure pump instrument.

Mr. Joe Melcher, Nova Biotech, in El Cajon California for his kind patience both through email

and phone for helping me with UV-Spectrophotometer instrument related difficulties.

My family and friends for their support.

iv

TABLE OF CONTENTS

ABSTRACT .................................................................................................................................... ii

ACKNOWLEDGMENTS ............................................................................................................. iii

LIST OF TABLES ......................................................................................................................... vi

LIST OF FIGURES ..................................................................................................................... viii

CHAPTER I. INTRODUCTION .................................................................................................... 1

Background ................................................................................................................................. 1

DNA Hairpins ............................................................................................................................. 2

Hydration and the Stability of DNA under Pressure ................................................................... 3

CHAPTER II. MATERIALS AND METHODS ............................................................................ 8

DNA Oligonucleotides ................................................................................................................ 8

DNA Melting/Denaturation Experiments ................................................................................. 10

Optical Melting Experiments under Hydrostatic Pressure ........................................................ 11

CHAPTER III. RESULTS ............................................................................................................ 13

I. Heterogeneous Loop Hairpins ............................................................................................... 13

Melting Curve Analysis ............................................................................................................ 15

II. Homogenous Loop Hairpins ................................................................................................. 24

Salt Dependence of Melting Temperatures for both Homogenous and Heterogeneous Loop

Hairpins ..................................................................................................................................... 33

Contribution of Hairpin Loops on Transition Volume.............................................................. 37

v

CHAPTER IV. DISCUSSION, CONCLUSIONS & FUTURE PERSPECTIVES ...................... 41

Discussion ..................................................................................................................................... 41

Pressure Denaturation of DNA Hairpin Samples ...................................................................... 47

Conclusion ................................................................................................................................. 48

Future Perspectives ................................................................................................................... 49

REFERENCES ............................................................................................................................. 51

APPENDIX ................................................................................................................................... 59

Effect of Pressure on the Thermally-Induced Denaturation of the Human Telomeric Sequence 59

vi

LIST OF TABLES

Table 1 Atmospheric pressure transition temperatures from optical melting experiments for six

heterogeneous loop hairpin samples (the error in temperature is approximately 0.1 ºC). ............ 18

Table 2 Values for the change in TM as functions of pressure for the six studied heterogeneous

loop hairpins.................................................................................................................................. 19

Table 3 HvH at different salt concentrations evaluated from optical melting experiments for

heterogeneous loop hairpins. ........................................................................................................ 20

Table 4 Volume change of the hairpin-coil transition of six heterogeneous loop hairpins as a

function of sodium ion concentration. .......................................................................................... 22

Table 5 Expansivity values for six heterogeneous loop hairpins. ................................................. 24

Table 6 Atmospheric pressure transition temperatures from optical melting experiments for five

homogenous loop hairpin samples (the error in temperature is approximately 0.1 ºC)................ 27

Table 7 Values for the change in TM as a function of pressure for the five studied heterogeneous

loop hairpins.................................................................................................................................. 28

Table 8 HvH at different salt concentrations evaluated from optical melting experiments for

homogenous loop hairpins. ........................................................................................................... 29

Table 9 Volume change of the hairpin-coil transition of five homogenous loop hairpins as a

function of sodium ion concentration. .......................................................................................... 31

Table 10 Expansivity values for five homogenous loop hairpins. ................................................ 33

Table 11 The salt dependence of the transition temperature (TM/log [Na+]). ........................... 35

Table 12 Release of Na+ from DNA hairpins upon melting (Δn) at atmospheric pressure. ......... 35

Table 13 Release of Na+ from DNA hairpins upon melting (Δn) at 50 MPa. .............................. 36

Table 14 Release of Na+ from DNA hairpins upon melting (Δn) at 100 MPa. ............................ 36

vii

Table 15 Release of Na+ from DNA hairpins upon melting (Δn) at 200 MPa. ............................ 36

Table 16 ΔΔV for both the heterogeneous and the homogenous sequence loop hairpins. ........... 37

Table 17 The difference in transition volumes (ΔV) between all homogenous loop hairpins

studied. .......................................................................................................................................... 39

Table 18 The difference in transition volumes (ΔV) between all six heterogeneous loop hairpins

studied. .......................................................................................................................................... 40

viii

LIST OF FIGURES

Figure 1 Phase diagram for the helix to coil transition of double stranded nucleic acids .............. 7

Figure 2 Sequences of the 11 DNA hairpins of this study .............................................................. 9

Figure 3 A photograph of the temperature regulated iso-hyperbaric spectrophotometer ............. 12

Figure 4 A heat-induced helix-coil transition of AT (TA2T) AT hairpin sample in an aqueous

solution containing 10 mM Na+ at 10 MPa .................................................................................. 14

Figure 5 A normalized heat-induced helix-coil transition of AT (TA2T) AT hairpin sample in an

aqueous solution containing 10 mM Na+ at 200 MPa. ................................................................. 16

Figure 6 Helix-coil transition temperature (TM) of AT(TA2T)AT as a function of pressure at four

Na+ concentrations: 100 mM (▼), 50 mM (▲), 20 mM (), and 10 mM (■). ........................... 17

Figure 7 Molar volume change of the heat-induced denaturation (V) as a function of salt

concentration ................................................................................................................................. 21

Figure 8 Molar volume change of the heat-induced denaturation (V) as a function of

temperature, (■) ATTA2TAT and () AATA2TTT ..................................................................... 23

Figure 9 A heat-induced helix-coil transition of ATG4AT hairpin sample in an aqueous solution

containing 10 mM Na+ at 50 MPa.. .............................................................................................. 25

Figure 10 Helix-coil transition temperature (TM) of ATG4AT as a function of pressure at various

Na+ concentrations: 100 mM (▼), 50 mM (▲), 20 mM (), and 10 mM (■). ........................... 26

Figure 11 Molar volume change of the heat-induced denaturation (V) as a function of salt

concentration for ATG4AT ........................................................................................................... 30

Figure 12 Molar volume change of the heat-induced denaturation (V) as a function of TM, (■)

AAG4TT and () ATG4AT .......................................................................................................... 32

ix

Figure 13 Hairpin-coil transition temperature (TM) as a function of log [Na+] at atmospheric

pressure: ATTC2AT (▼), AATG2TT (▲), ATT4AT (), AAC4TT (■) ...................................... 34

1

CHAPTER I. INTRODUCTION

Background

Deoxyribonucleic acid (DNA) was discovered by the Swiss physician and biologist Johannes

Friedrich Miescher in 1868 only four years after publication of Mendel’s work, the first

quantitative studies of inheritance. Miescher managed to isolate, extract, and purify a white

substance from human pus cells obtained from disposed surgical bandages. He soon found out

that this substance contained nitrogen and phosphorous but not sulphur; moreover, the proportion

of nitrogen and phosphorous was different from any other known cell constituents at the time,

convincing Miescher that he had discovered a new biological substance (James 1970). This new

white purified substance appeared to be associated with the cell nucleus and Miescher decided to

name it nuclein. What Miescher discovered was in fact nucleoprotein, and it was not until 1889

that protein-free nucleic acids were obtained by Richard Altman who, due to their slight acidic

nature, named this substance nucleic acid (Blackburn 2006).

For years very little work was done on DNA and hence its functions remained undiscovered. It

was not until the late 1920’s that a Russian-American biochemist named P.A. Levene studied the

chemistry of nucleic acids. He characterized the different forms of nucleic acid, and found that

DNA contained adenine, guanine, thymine, cytosine, deoxyribose (a five carbon sugar) and a

phosphate group. Levene first proposed that DNA was made up of equal amounts of adenine,

guanine, thymine, and cytosine which led to the mistaken belief that DNA was a simple

repeating polymer with no sequence variation providing structural support to proteins in

2

chromosomes. As a result, the organization of DNA was believed to be incapable of carying

genetic information. In 1944, Oswald T. Avery, a Canadian-born U.S. physician and medical

researcher, and fellow workers from Rockefeller Institute in New York City would show that

hereditary information is contained in DNA and not in protein (Avery, Macleod et al. 1944). In

1950, Erwin Chargaff, an Austrian scientist at Columbia University, showed that the ratio of

adenine to thymine and cytosine to guanine was close to unity and that differences exist in DNA

nucleotide composition from one organism to another (Chargaff 1950). Despite this fascinating

discovery many still persistently believed that protein was responsible for the hereditary

information until 1952 when Hershey and Chase showed that it is not protein but DNA that is

responsible for carrying hereditary information (Hershey and Chase 1952). It is documented that

Chargaff met Watson and Crick in 1952, and, despite personality conflicts explained his findings

to them. Chargaff’s findings would later help Watson and Crick to deduce the double helical

structure of DNA; Watson and Crick’s proposal and other studies began to lead to a better

understanding of molecular basis of inheritance.

DNA Hairpins

The presence of inverted repeats in DNA or RNA sequences, usually known as palindromes, can

lead to the formation of a snap-back region or hairpin (Sinden 1994). Hairpins are common

secondary structural elements in RNA and DNA. RNA hairpins have functions in initiating

folding and forming tertiary structures and protein binding sites (Williams and Hall 2000;

Proctor, Schaak et al. 2002); DNA hairpins are involved in regulating replication and

transcription (Varani 1995; Willwand, Mumtsidu et al. 1998). In practical applications, hairpin

3

loops have been used as attractive candidates for the design of antisense therapeutics (Poddevin,

Meguenni et al. 1994; Yamakawa, Abe et al. 1998; Park, Miyano-Kurosaki et al. 2000).

Therefore, due to the natural occurrence of hairpin sequences and the therapeutics applications of

DNA hairpins, it is imperative to better understand the sequence dependent stability and the

associated melting thermodynamics of DNA hairpins.

The stability of hairpins and the thermodynamics and kinetics of hairpin-coil transitions have

been the subject of intense investigation (Elson, Scheffler et al. 1970; Haasnoot, Hilbers et al.

1986; Vallone, Paner et al. 1999). Previous thermodynamic studies of DNA hairpins have

revealed that the stability of DNA hairpins depends on both the sequence composition of the

loop and the closing base pair (Senior, Jones et al. 1988; Paner, Amaratunga et al. 1990; Vallone,

Paner et al. 1999; Nakano, Moody et al. 2002). There has been very little investigation into the

pressure-temperature stability of DNA hairpins, and the majority of helix-coil transitions of

DNA hairpins have been studied as a function of temperature at constant atmospheric pressure.

Consequently, the current thermodynamic knowledge of DNA hairpins stability is essentially

limited to the temperature domain and the pressure properties remain unknown.

Hydration and the Stability of DNA under Pressure

The role that water plays in the properties of nucleic acids is crucial to the higher-order structures

they form and requires attention. The conformational stability of DNA, for instance, depends on

hydration. Lee and co-workers have reported that DNA loses its secondary structure below a

certain relative humidity (Lee, Debenedetti et al. 2004). Hydration can be defined as the binding

of water molecules to a solute, in our case DNA, DNA + nH2O ↔ [DNA.H2O] n. The water

4

interacting with nucleic acids can be divided into three categories as previously described by

Chalikian and Macgregor (Chalikian and Macgregor 2007). The first category consists of

ordered water molecules that are stabilized by electrostatic forces or hydrogen bonds. The second

category consists of unordered H2O molecules in forming the first hydration layer that are

distinct from bulk solvent due to their proximity to solute and the differential nature of solvent-

solvent and DNA-solvent interactions. Finally, the third group involves water molecules from

more distant solvation layers that are still affected by the DNA via intermediate water molecules.

These layers of hydration stabilize the DNA helix. For a more detailed description of the

interaction between water and nucleic acids, and to see how these water molecules are detected,

please refer to the review article by Chalikian and Macgregor (Chalikian and Macgregor 2007).

In addition to hydration, DNA stability is determined by base-pair stacking energies, hydrogen

bonding and electrostatic interactions. Base-pair stacking interactions are hydrophobic in nature

and they can be pictured as a stack of coins (aromatic bases in the case of DNA) where the

position of a single coin is stabilized in the stack by the coins above and below it (Sinden 1994).

The electrostatic interactions arise due to the presence of phosphate groups in DNA structures

which repel each other. In general, cations neutralize the phosphate-phosphate repulsion leading

to the stabilization of the helix. This interaction is referred to as counterion condensation

(Manning 1969; Manning 1978).

The effect of hydrostatic pressure on the thermal stability of DNA hairpins remains largely

unexplored; however, it offers an alternative method for the investigation of the role of solvation

on DNA stability. We are interested in the role of water in thermodynamics of DNA hairpin

5

helix-coil transition; to study this we investigate the effect of hydrostatic pressure on thermally

induced helix-coil transition. The most direct methods to determine the role of hydration involve

measurements of volumetric parameters such as volume, compressibility, expansivity, etc. These

parameters can be either measured directly or by studying the temperature and pressure

dependence of the stability of a system. In general, perturbation of the hairpin-coil transition with

pressure yields the molar volume change of the equilibrium. The molar volume change (ΔV) can

be defined as the difference between the molar volume of the products and that of the reactants.

The molar volume can be either positive or negative. In water, negative volume changes are

generally attributed to the formation of stronger interactions with the solvent or, in other words,

more extensive hydration. On the other hand, a positive ΔV indicates that the molar volume of

the coil form is larger than the molar volume of the helix or hairpin form.

The goal of this project is to measure the effect of pressure on the thermal stability of DNA

hairpins in order to investigate if DNA hairpins agree with the pressure-temperature phase

diagram for polymeric nucleic acids published previously (Dubins, Lee et al. 2001). This phase

diagram developed for DNA polymers showed that the thermodynamic stability of a nucleic acid

duplex depends on temperature, pressure and duplex transition temperature in a highly complex

and non-linear fashion. Furthermore, this phase diagram predicted polymeric duplexes to be

destabilized by pressure (negative ΔV) if the TM is <~50ºC and stabilized by pressure (positive

ΔV) if the TM is > ~50ºC (Figure 1).

6

Despite the fact this phase diagram effectively predicts the thermodynamic stability of polymeric

duplexes, it has not yet been thoroughly tested to see if these predictions are applicable to short

oligomeric nuclei acid duplexes or DNA hairpins. For instance, a study by Macgregor on “Chain

Length and Oligonucleotide Stability at High Pressure” showed that oligonucleotides are in fact

stabilized by pressure even though the transition temperatures in some cases were much lower

than 50 ºC (Macgregor 1996). Oligomeric nucleic acid duplexes and polymers exhibit many

differences in thermodynamic properties. One example is that the salt dependence of transition

temperatures (TM) of polymeric duplexes are linear throughout a broad range of monovalent ion

concentration while oligomeric duplexs exhibit non-linear behavior that tend to plateau at ~ 1 M

monovalent ion concentration (Schildkraut 1965; Frank-Kamenetskii 1971; Chalikian and

Macgregor 2007). Additionally, oligomeric nucleic acid duplexes have much shorter sequences

than polymeric duplexes and their stabilities are affected due to the greater relative proportion of

terminal bases (end-fraying). Previous structural, thermodynamic, and kinetic studies of

oligonucleotides demonstrated that the terminal base pairs of oligonucleotides exhibit structures

significantly different from base pairs distant from the ends (Williams, Longfellow et al. 1989;

Braunlin and Bloomfield 1991; Olmsted, Anderson et al. 1991; Stein, Bond et al. 1995;

Macgregor 1996). Thus, the deviation may be caused by the greater significance of tail

interactions to oligomeric duplex behavior. As described, factors such as end-effects are

generally ignored in DNA polymers due to their relatively small contribution to the total stability

and are not included in the theory underlying the phase diagram presented by Dubins (Dubins,

Lee et al. 2001).

7

0 50 1000

2000

4000

6000

8000

10000

12000

20 °C

30 °C

40 °C

60 °C

80 °C

100 °C

PM ,

ba

r

T, °C

Figure 1 Phase diagram for the helix to coil transition of double stranded nucleic acids. The

denaturation pressure, PM, is plotted as a function of temperature, TM, at atmospheric pressure at

20 oC, 30

oC, 40

oC, 60

oC, 80

oC, 100

oC. The various temperatures are represented by different

color lines on the diagram (Dubins, Lee et al. 2001).

8

CHAPTER II. MATERIALS AND METHODS

DNA Oligonucleotides

All oligonucleotides were synthesized and cartridge purified by ACGT, Inc. (Toronto,

Canada). Self-complementary sequences of 16-base DNA oligomer strands in their folded

hairpin configuration are shown in Figure 2. For simplicity the short-hand notation AX-Y4 (X,

Y= A, T) will be used for referring to each hairpin loop. Oligonucleotide concentrations were

determined using calculated extinction coefficients (Cantor, Warshaw et al. 1970; Vallone, Paner

et al. 1999). All DNA samples were dissolved in 20 mM sodium cacodylate and 0.01 mM

Na2EDTA (pH 6.42), at sodium ion concentrations ranging from 100 to 10 mM1. The sodium ion

concentration was regulated by altering the NaCl concentration. Previous work suggests that for

these DNAs, with the exception of molecules with A4 loops nucleated by AA/TT or AT bp

doublets, melting temperatures are independent of strand concentration from 0.5 to 63.5 µM

(Vallone, Paner et al. 1999). In our experiments, the concentration of DNA samples was

approximately 10 µM (strand). Prior to melting, samples were heated to 100 C for 20 min and

then placed on ice for 30 min.

1 Note: There is an additional 0.02 mM sodium in all of the solutions that comes from the

addition of 0.01 mM Na2EDTA. Thus, 10 mM is actually 10.02 mM, 20 mM is 20.02 mM, etc.

9

Figure 2 Sequences of the 11 DNA hairpins of this study. As illustrated, all studied hairpin

structures have a six-base pair duplex-forming stem linked by a four-base loop. The first four

base pairs in the stem are the same for every molecule.

10

DNA Melting/Denaturation Experiments

The helical structure of DNA is remarkably stable. This stability is derived from two

chemical forces, hydrogen bonding and base stacking interactions. Besides, the helix is solvated

with water molecules which form a shell of hydration around the DNA. To melt or denature the

DNA, these stabilizing forces must be overcome (Sinden 1994).

DNA undergoes a helix-coil transition when heated (Marmur and Doty 1959; Inman and

Baldwin 1962), upon incubation at pH > 12 or pH < 2 due to ionization of the bases (Sinden

1994), in the presence of high concentrations of certain solvents (Herskovits 1962; Sinanoglu

and Abdulnur 1964), and when exposed to high pressure depending on the intrinsic stability of

the DNA (Dubins, Lee et al. 2001). DNA denaturation can be measured in different ways. One

method involves measurement of a characteristic increase in the absorbance as a function of the

perturbant (i.e. temperature, pH, cosolvent concentration, and pressure) called hyperchromicity,

which results from the unstacking of the bases (Tinoco 1960). A plot of absorbance versus a

perturbant such as temperature displays a cooperative sigmoidal shape curve known as the

melting curve. Melting curves are indicators of DNA stability. For an account on the history of

DNA denaturation see a review by Thomas (Thomas 1993). The early DNA melting experiments

exhibited the importance of the G-C content (Marmur and Doty 1959; Marmur and Doty 1962),

and salt concentration (Schildkraut 1965) on the stability of DNA. DNA samples with a greater

G-C content are more stable and melt at higher temperature than those with a lower G-C content,

while increasing the cation concentration stabilizes the DNA by shielding the repulsion between

the negative phosphates, an effect known as counterion condensation (Manning 1969; Manning

1978). It is worth mentioning that the number of counterions condensed per phosphate remains

constant over broad salt concentrations (Manning 1978).

11

Optical Melting Experiments under Hydrostatic Pressure

The temperature regulated iso-hyperbaric spectrophotometer was employed to obtain

the heat-induced melting curves; this instrument has been described previously (Wu and

Macgregor 1993). Briefly, the sample solution (~300 µL) was contained in a cylindrical quartz

cuvette (path length 0.5 cm) positioned in the optical path of a pressure cell equipped with quartz

windows. The high pressure cell was filled with silicon oil, as the pressure transmitting medium.

Pressure up to 200 MPa (~0.1 MPa is equivalent to atmospheric pressure) was generated using

an automated high pressure pump (Porous Materials Incorporated, Ithaca, NY). The temperature

was regulated using a Haake model DC5-k20 circulating bath (Thermo Scientific, Waltham,

MA). A thermocouple connected to an Omega DP80 digital thermometer (Stamford, CT) is

inserted into the pressure-cell in order to measure the temperature. The temperature, pressure and

absorption of the sample were all recorded by the software controlling the experiment. Sample

temperature was increased linearly at a heating rate of 0.9C/min and the helix-coil transition

was monitored by measuring the change in absorption at 268 nm. A photograph of the equipment

described above is shown in Figure 3. Please note that the temperature bath and the computer are

not shown in this Figure.

12

Figure 3 A photograph of the temperature regulated iso-hyperbaric spectrophotometer. For a

more detailed schematic diagram please refer to Wu and Macgregor (Wu and Macgregor 1993).

13

CHAPTER III. RESULTS

I. Heterogeneous Loop Hairpins

Figure 4 shows a melting curve of AT (TA2T) AT (a heterogeneous loop hairpin) sample in an

aqueous solution containing 10 mM Na+ at 10 MPa. The mid-point of the transition is known as

the melting temperature (TM) and is an indicator of the stability of the hairpin. The TM, in this

case, corresponds to a temperature at which half of the DNA samples are in the hairpin state,

whereas the other half are in the single-stranded (coil) state. The transition is highly cooperative

as seen from the shape of the curve. The TM of the transition is 42.1 ºC. As discussed below, the

following thermodynamic parameters are extracted from the heat-induced melting curves; the

melting temperature (TM), the model dependent van’t Hoff enthalpy change of the helix-coil

transition (ΔHvH), and the transition volume of the heat-induced helix-coil transition (ΔV).

14

10 20 30 40 50 60 70 801.55

1.60

1.65

1.70

1.75

1.80

O

D (

26

8 n

m)

Temperature (°C)

Figure 4 A heat-induced helix-coil transition of AT (TA2T) AT hairpin sample in an aqueous

solution containing 10 mM Na+ at 10 MPa. The melting temperature of the transition

corresponds to 42.1 ºC. The rate of heating was 0.9 ºC/min. The hairpin concentration was

approximately 10 µM (strand).

15

Melting Curve Analysis

The fraction of DNA in the coil form at a temperature T, , was calculated using the following

equation:

[ ( ) ( )]

[ ( ) ( )]

OD T L T

H T L T

Eq.1

Where OD (T) is the optical density at temperature T, and L (T) and H (T) are equations for the

lines describing the low-temperature and high-temperature baselines respectively as a function of

temperature. The studied samples are assumed to be in hairpin (helix) form if = 0, while at

= 1 the samples are assumed to be entirely in the single stranded (coil) form. The hairpin-coil

transition temperature (TM) is the temperature at which = 0.5. For more detailed and thorough

explanation of melting curve analysis please refer to the review article by Mergney (Mergny and

Lacroix 2003). Figure 5 illustrates a normalized heat-induced helix-coil transition curve of AT

(TA2T) AT hairpin sample at 10 mM Na+ at 200 MPa.

The van’t Hoff enthalpy (ΔHvH) for each transition was calculated from (/T) Max and the

melting temperature, TM. Hence, enthalpy changes were calculated assuming a van’t Hoff two-

state melting transition as described in Marky and Breslauer (Marky and Breslauer 1987).

2(2 2 ) ( )M

vH T MaxH n RTT

Eq.2

16

Where n is a constant equal to the molecularity (n = 1 in this case), R is the gas constant, and

(/T) Max is the maximum slope of the versus temperature curve at TM (Marky and Breslauer

1987).

10 20 30 40 50 60 70 80

0.0

0.2

0.4

0.6

0.8

1.0

Fra

cti

on

of

den

atu

red

ba

se-p

air

s)

Temperature (°C)

Figure 5 A normalized heat-induced helix-coil transition of AT (TA2T) AT hairpin sample in an

aqueous solution containing 10 mM Na+ at 200 MPa. For this sample, the melting temperature of

the transition corresponds to 41.9 ºC. Heat-induced helix-coil transition curves of all studied 11

hairpin samples generally exhibit the same broad transition.

17

Figure 6 illustrates the pressure dependence of the TM for the AT (TA2T) AT hairpin sample at

four different salt concentrations. The sign and magnitude of TM/P are indicative of the effect

of pressure on the stability of short DNA hairpins. Positive values of TM/P specify that

increasing pressure stabilizes the hairpin form, while negative values of TM/P show that

increasing pressure destabilizes the hairpin state of the oligos. TM values along with TM/P

values for the six heterogeneous sequence loop hairpins are summarized in Tables 1 and 2,

respectively.

0 50 100 150 20041

42

43

44

45

46

47

Tem

per

atu

re (C

)

Pressure (MPa)

Figure 6 Helix-coil transition temperature (TM) of AT(TA2T)AT as a function of pressure at

four Na+ concentrations: 100 mM (▼), 50 mM (▲), 20 mM (), and 10 mM (■). The solid lines

are least-squares fits to the data.

18

Table 1 Atmospheric pressure transition temperatures from optical melting experiments for six

heterogeneous loop hairpin samples (the error in temperature is approximately 0.1 ºC)2.

Na+

(mM)

Loop

Sequence

Nucleation

Stack

TA2T TG2T TC2T

AT/AT 10

20

50

100

42.1

43.2

44.6

46.1

42.8

44.0

45.5

46.8

44.9

46.1

48.7

51.3

AA/TT 10

20

50

100

40.2

41.5

43.3

44.7

37.9

41.1

43.5

45.1

44.0

45.4

47.9

49.9

2 Every value was measured at least twice for all analyzed samples.

TM (ºC)

19

Table 2 Values for the change in TM as functions of pressure for the six studied heterogeneous

loop hairpins.

Na+

(mM)

Loop

Sequence

Nucleation

Stack

TA2T TG2T TC2T

AT/AT 10

20

50

100

-1.09 ± 0.08

0.44 ± 0.19

2.08 ± 0.04

3.67 ± 0.32

-3.09 ± 0.29

-0.55 ± 0.10

3.44 ± 0.45

6.42 ± 0.37

3.75 ± 0.59

4.72 ± 0.07

6.39 ± 0.35

7.95 ± 0.20

AA/TT 10

20

50

100

-5.28 ± 0.06

-3.11 ± 0.14

-0.52 ± 0.09

2.03 ± 0.26

-5.68 ± 0.21

-2.09 ± 0.04

2.09 ± 0.04

5.29 ± 0.06

1.54 ± 0.14

2.36 ± 0.15

3.51 ± 0.35

4.75 ± 0.28

The molar volume change of the heat-induced helix-coil transition (V) was calculated from the

slopes of the data such as the ones shown in Figure 3 using the Clapeyron equation:

MM

T VT

P H

Eq.3

Where H is the calorimetric enthalpy change of the hairpin-coil transition.

1000 × ∂TM/P (ºC/MPa)

(ºC/MPa)

20

It should be noted that for all V calculations the calorimetric enthalpies reported previously by

Benight’s group were used (Vallone, Paner et al. 1999). Table 3 presents the salt dependence of

the van’t Hoff (two-state model) enthalpy (HvH). It is worth mentioning that HvH values are

slightly dependent on pressure (i.e. HvH value decrease for all samples at all salt concentrations

with the increase in pressure).

Table 3 HvH at different salt concentrations evaluated from optical melting experiments for

heterogeneous loop hairpins.

Na+

(mM)

Loop

Sequence

Nucleation

Stack

TA2T TG2T TC2T

AT/AT 10

20

50

100

110

115

116

119

114

113

114

112

131

137

142

142

AA/TT 10

20

50

100

112

106

107

106

98

94

106

106

133

136

140

148

HvH (kJ mol-1

) ± 6%

21

Figure 7 illustrates the molar volume change of the heat-induced transition (V) as a function of

sodium chloride concentration for the AT(TA2T)AT hairpin sample. The value of V varied

linearly with the log [Na+] from -0.44 ± 0.04 cm

3 mol

-1 in 10 mM Na

+ to 1.46 ± 0.14 cm

3 mol

-1 in

100 mM Na+. Table 4 lists the V values for all six studied heterogeneous loop hairpins at

various sodium chloride concentrations.

1.0 1.2 1.4 1.6 1.8 2.0

-0.5

0.0

0.5

1.0

1.5

V

(cm

3/m

ol)

log[Na+

]

Figure 7 Molar volume change of the heat-induced denaturation (V) as a function of salt

concentration. The line is a least-squares fit of the data; the slope of the line is 1.87 ± 0.06 cm3

mol-1

.

22

Table 4 Volume change of the hairpin-coil transition of six heterogeneous loop hairpins as a

function of sodium ion concentration.

Na+

(mM)

Loop

Sequence

Nucleation

Stack

TA2T TG2T TC2T

AT/AT

ΔV/Δlog [Na+]

10

20

50

100

-0.44 ± 0.04

0.18 ± 0.08

0.83 ± 0.04

1.46 ± 0.32

1.87 ± 0.06

-1.41 ± 0.14

-0.25 ± 0.05

1.55 ± 0.21

2.89 ± 0.20

4.33 ± 0.10

1.81 ± 0.29

2.27 ± 0.10

3.05 ± 0.21

3.76 ± 0.18

1.95 ± 0.12

AA/TT

ΔV/Δlog [Na+]

10

20

50

100

-1.96 ± 0.08

-1.15 ± 0.07

-0.19 ± 0.04

0.74 ± 0.10

2.67 ± 0.09

-2.35 ± 0.13

-0.86 ± 0.04

0.85 ± 0.04

2.14 ± 0.09

4.47 ± 0.11

0.78 ± 0.08

1.18 ± 0.09

1.75 ± 0.19

2.35 ± 0.17

1.55 ± 0.10

The effect of TM on the volume change of the transition for two heterogeneous loop hairpins is

presented in Figure 8. From these data we calculate that the V = 0 cm3

mol-1

at 42.9 ºC for

ATTA2TAT. For AATA2TTT V = 0 cm3

mol-1

at 43.5 ºC. The expansivity values (ΔE) for the

six heterogeneous sequence loops are summarized in Table 5.

ΔV (cm3

mol-1

)

23

40 41 42 43 44 45 46

-2.0

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

V

(cm

3m

ol-1

)

Temperature (°C)

Figure 8 Molar volume change of the heat-induced denaturation (V) as a function of

temperature, (■) ATTA2TAT and () AATA2TTT. The solid lines are least-squares fits to the

data. The slope of the line, the ΔE of the transition is equal to 0.48 cm3 K

-1 mol

-1 and

0.58 cm3

K-1

mol-1

for ATTA2TAT and AATA2TTT hairpin samples, respectively.

24

Table 5 Expansivity values for six heterogeneous loop hairpins.

Loop

Sequence

Nucleation

Stack

TA2T TG2T TC2T

AT/AT 0.48 ± 0.02 1.10 ± 0.04 0.30 ± 0.01

AA/TT 0.58 ± 0.02 0.62 ± 0.05 0.26 ± 0.01

II. Homogenous Loop Hairpins

Figure 9 illustrates the heat-induced helix-coil transition curve of ATG4AT (a homogenous loop

hairpin) at 50 MPa at 10 mM Na+. It should be mentioned that for all eleven hairpins studied the

transitions are highly cooperative and that while the transition temperatures increase or decrease

with pressure, the general shape of the curves are generally not altered.

Figure 10 presents the pressure dependence of the TM for ATG4AT hairpin sample at four salt

concentrations. Once again, the effect of pressure on the conformational stability of DNA

hairpins is reflected by the sign and magnitude of TM/P. Positive values of TM/P specify that

pressure stabilizes the native state (hairpin form), while negative values of TM/P show that

pressure destabilizes the hairpin state of the DNA samples. The TM values along with the TM/P

values for the five homogenous loop hairpins are summarized in Tables 6 and 7, respectively.

ΔE (cm3

mol-1

K-1

)

25

10 20 30 40 50 60 70 801.60

1.65

1.70

1.75

1.80

1.85

OD

(2

68

nm

)

Temperature (C)

Figure 9 A heat-induced helix-coil transition of ATG4AT hairpin sample in an aqueous solution

containing 10 mM Na+ at 50 MPa. The melting temperature of the transition corresponds to 43.9

ºC. The rate of heating was 0.9 ºC/min. The hairpin concentration was approximately 10 µM

(strand).

26

0 50 100 150 200

43

44

45

46

47

48

49

50

Tem

pera

ture (

°C)

Pressure (MPa)

Figure 10 Helix-coil transition temperature (TM) of ATG4AT as a function of pressure at various

Na+ concentrations: 100 mM (▼), 50 mM (▲), 20 mM (), and 10 mM (■). The solid lines are

least-squares fits to the data.

27

Table 6 Atmospheric pressure transition temperatures from optical melting experiments for five

homogenous loop hairpin samples (the error in temperature is approximately 0.1 ºC).

Na+

(mM)

Loop

Sequence

Nucleation

Stack

C4 G4 T4

AT/AT 10

20

50

100

42.4

43.7

46.1

49.3

44.0

45.1

46.5

47.7

43.9

45.8

48.2

51.0

AA/TT 10

20

50

100

42.9

44.1

45.6

47.2

38.4

40.0

42.0

43.7

TM (ºC)

28

Table 7 Values for the change in TM as a function of pressure for the five studied heterogeneous

loop hairpins.

Na+ (mM) Loop

Sequence

Nucleation

Stack

C4 G4 T4

AT/AT 10

20

50

100

5.28 ± 0.07

7.08 ± 0.20

9.72 ± 0.19

11.9 ± 0.5

-3.20 ± 0.11

0.54 ± 0.11

5.21 ± 0.28

8.50 ± 0.22

2.63 ± 0.07

4.43 ± 0.17

7.60 ± 0.26

10.6 ± 0.1

AA/TT 10

20

50

100

2.63 ± 0.08

3.19 ± 0.08

4.18 ± 0.08

4.72 ± 0.07

-2.07 ± 0.06

0.56 ± 0.16

3.63 ± 0.18

6.81 ± 0.08

Equation 2 was once again employed to calculate the model dependent van’t Hoff enthalpies for

these five homogenous loop hairpins. The results are summarized in Table 8. A similar trend was

observed for these samples in terms of the slight dependence of HvH values on pressure (i.e.

HvH values slightly decrease for all samples at all salt concentrations with the increase in

pressure).

The Clapeyron equation (Equation 3) was used to calculate the transition volumes for the

homogenous loop hairpins. Figure 11 illustrates the molar volume change of the heat-induced

1000 × ∂TM/P (ºC/MPa)

(ºC/MPa)

29

transition (V) as a function of sodium chloride concentration for the ATG4AT hairpin sample.

The value of V varied linearly with the log [Na+] from -1.72 ± 0.09 cm

3 mol

-1 in 10 mM Na

+ to

4.52 ± 0.22 cm3 mol

-1 in 100 mM Na

+. Table 9 lists the V values for all five studied

homogenous loop hairpins at various sodium chloride concentrations.

Table 8 HvH at different salt concentrations evaluated from optical melting experiments for

homogenous loop hairpins.

Na+

(mM)

Loop

Sequence

Nucleation

Stack

C4 G4 T4

AT/AT 10

20

50

100

138

143

141

135

107

110

112

100

129

132

135

134

AA/TT 10

20

50

100

144

148

149

150

87.5

84.9

87.0

84.1

HvH (kJ mol-1

) ± 6%

30

1.0 1.2 1.4 1.6 1.8 2.0

-2

-1

0

1

2

3

4

5

V

(cm

3/m

ol)

log[Na+

]

Figure 11 Molar volume change of the heat-induced denaturation (V) as a function of salt

concentration for ATG4AT. The line is a least-squares fit of the data; the slope of the line is 6.25

± 0.13 cm3 mol

-1.

31

Table 9 Volume change of the hairpin-coil transition of five homogenous loop hairpins as a

function of sodium ion concentration.

Na+

(mM)

Loop

Sequence

Nucleation Stack C4 G4 T4

AT/AT

ΔV/Δlog [Na+]

10

20

50

100

3.07 ± 0.13

4.10 ± 0.20

5.58 ± 0.25

6.74 ± 0.38

3.69 ± 0.06

-1.72 ± 0.09

0.29 ± 0.06

2.79 ± 0.19

4.52 ± 0.22

6.25 ± 0.13

1.38 ± 0.07

2.30 ± 0.13

3.92 ± 0.20

5.40 ± 0.22

4.03 ± 0.25

AA/TT

ΔV/Δlog [Na+]

10

20

50

100

1.38 ± 0.07

1.67 ± 0.08

2.18 ± 0.10

2.45 ± 0.10

1.09 ± 0.05

-0.83 ± 0.04

0.22 ± 0.06

1.44 ± 0.09

2.68 ± 0.11

3.45 ± 0.14

The effect of TM on the volume change of the transition for two homogenous loop hairpins are

depicted in Figure 12. From these data we calculate that the V = 0 cm3

mol-1

at 45.0 ºC for

ΔV (cm3

mol-1

)

32

ATG4AT. For AAG4TT V = 0 cm3

mol-1

at 39.7 ºC. The expansivity values (ΔE) for the five

homogenous loops are summarized below in Table 10.

38 39 40 41 42 43 44 45 46 47 48

-2

-1

0

1

2

3

4

5

V

(cm

3/m

ol)

Temperature (°C)

Figure 12 Molar volume change of the heat-induced denaturation (V) as a function of TM, (■)

AAG4TT and () ATG4AT. The solid lines are least-squares fits to the data. The slope of the

line, the ΔE of the transition is equal to 0.65 cm3 K

-1 mol

-1 and 1.72 cm

3 K

-1 mol

-1 for AAG4TT

and ATG4AT hairpin samples, respectively.

33

Table 10 Expansivity values for five homogenous loop hairpins.

Loop

Sequence

Nucleation

Stack

C4 G4 T4

AT/AT 0.53 ± 0.06 1.72 ± 0.06 0.58 ± 0.02

AA/TT 0.26 ± 0.02 0.65 ± 0.01

Salt Dependence of Melting Temperatures for both Homogenous and

Heterogeneous Loop Hairpins

For all hairpins, increasing the Na+ concentration from 10 to 100 mM resulted in the shift of the

melting curves to higher temperatures. The increase in salt concentration shifted the hairpin-coil

transition towards the conformation with higher charge density parameter. Plots of TM versus

log [Na+] for four of the eleven hairpin samples are shown in Figure 13. According to equation 4

(Eq. 4), the salt dependence of the transition temperature (TM/log [Na+]) can be used to

estimate the number of sodium cations released, Δn, when each sequence melts (Gruenwedel

1975; Record, Anderson et al. 1978; Rayan and Macgregor 2005).

Eq.4

Where R is the gas constant (0.008314 kJ K−1

mol−1

) and Δn is the number of sodium cations

released.

ΔE (cm3

mol-1

K-1

)

34

The salt dependence of the transition temperature (TM/log [Na+]) and, thus, the calculated

number of sodium cations released, Δn, for the eleven hairpin samples are summarized in Tables

11 and 12, respectively.

1.0 1.2 1.4 1.6 1.8 2.0

39

42

45

48

51

TM

(°C

)

log [Na+

]

Figure 13 Hairpin-coil transition temperature (TM) as a function of log [Na+] at atmospheric

pressure: ATTC2AT (▼), AATG2TT (▲), ATT4AT (), AAC4TT (■). The solid lines are least-

squares fits to the data. Slopes of these plots provided evaluations of the counterion release, n.

35

Table 11 The salt dependence of the transition temperature (TM/log [Na+]).

∂Tm/∂log [Na+]

(ºC)

Loop Sequence

Nucleation stack T4 G4 C4 TA2T TG2T TC2T

AT/AT 6.98 ± 0.47 3.62 ± 0.05 6.76 ± 0.90 3.91 ± 0.23 3.94 ± 0.11 6.48 ± 0.67

AA/TT 5.28 ± 0.11 4.22 ± 0.22 4.58 ± 0.03 7.06 ± 0.78 5.99 ± 0.31

Table 12 Release of Na+ from DNA hairpins upon melting (Δn) at atmospheric pressure.

Δn

Loop Sequence

Nucleation stack T4 G4 C4 TA2T TG2T TC2T

AT/AT 0.630 0.329 0.680 0.268 0.304 0.537

AA/TT 0.367 0.377 0.292 0.513 0.515

The number of sodium cations released, Δn, for the all hairpin samples at 50 MPa, 100 MPa, and

200 MPa are summarized in Tables 13 and 14, and 15, respectively. These Tables show that the

number of sodium cations released during the hairpin-coil transition increases with pressure for

all eleven hairpin samples. It is worth mentioning that similar pressure dependence of Δn was

reported by Wu and Macgregor (Wu and Macgregor 1993) and Rayan and Macgregor (Rayan

and Macgregor 2005) for DNA polymers.

36

Table 13 Release of Na+ from DNA hairpins upon melting (Δn) at 50 MPa.

Δn

Loop Sequence

Nucleation stack T4 G4 C4 TA2T TG2T TC2T

AT/AT 0.666 0.384 0.713 0.285 0.347 0.541

AA/TT 0.402 0.389 0.315 0.558 0.526

Table 14 Release of Na+ from DNA hairpins upon melting (Δn) at 100 MPa.

Δn

Loop Sequence

Nucleation stack T4 G4 C4 TA2T TG2T TC2T

AT/AT 0.702 0.440 0.737 0.297 0.388 0.572

AA/TT 0.425 0.396 0.341 0.604 0.534

Table 15 Release of Na+ from DNA hairpins upon melting (Δn) at 200 MPa.

Δn

Loop Sequence

Nucleation stack T4 G4 C4 TA2T TG2T TC2T

AT/AT 0.779 0.556 0.824 0.339 0.467 0.609

AA/TT 0.495 0.415 0.387 0.684 0.572

37

Contribution of Hairpin Loops on Transition Volume

It is reasonable to propose that the transition volumes of hairpins arise from two factors, one

being the stem and one the loop. Our goal in this part is to attempt to extract more information

from the transition volume values reported in Tables 4 and 9.

Table 16 illustrates the ΔΔV for both the heterogeneous and the homogenous loop hairpins. This

is done for every pair of hairpin samples that has the same composition. First we will hold the

loop-nucleating base pairs constant examining the difference between nucleating base pair

sequences for each of the loop sequences. In other words, assuming the loops are constant, we

can isolate changes resulting from nucleating base pairs to see the effect of nucleation stack on

volumetric properties. Therefore, by ignoring the interactions between the loop and the

nucleating base pair (i.e. the interpretation is dependent on our assumption of independence), it

can be assumed that the reported values in Table 16 correspond to the contribution of the

nucleating base pairs to transition volumes of the hairpin samples.

Table 16 ΔΔV for both the heterogeneous and the homogenous sequence loop hairpins.

ΔΔV (cm3

mol-1

)

Loop Sequence

Nucleation stack Na+ G4 C4 TA2T TG2T TC2T

(AT/AT)-(AA/TT)

10 -0.89 1.69 1.52 0.94 1.03

20 0.07 2.43 1.33 0.61 1.09

50 1.35 3.40 1.02 0.70 1.30

100 1.84 4.29 0.72 0.75 1.41

38

Tables 17 and 18 illustrate the difference in transition volumes between different homogenous

and heterogeneous loop hairpins, respectively. In this case, assuming we consider the same

nucleating base pairs, we can isolate changes resulting from loop sequences and, hence, see the

effect of loop sequences on volumetric properties. Since the stem and the nucleation stacks are

identical for the samples summarized in Tables 17 and 18, it can be claimed that the differences

are due to the loops. In fact, the difference in transition volumes at various Na+ concentrations

between poly [d(A-T)] and poly(dA).poly(dT) (Wu and Macgregor 1993), in addition to a

nearest neighbor analysis study of double stranded DNA, i.e. AA TT and AT TA in this case,

carried out by Dubins and Macgregor (Dubins and Macgregor 2004) at 25 mM Na+ show that the

differences in transitions volumes are close to zero and negligible. This further suggests that the

differences in transition volumes for hairpin samples are mainly due to the loops.

39

Table 17 The difference in transition volumes (ΔV) between all homogenous loop hairpins

studied.

AT(C4) - AT(G4)

Na+

(mM)

ΔΔV

(cm3 mol

-1)

AT(T4) - AT(G4)

Na+

(mM)

ΔΔV

(cm3 mol

-1)

10 4.79 10 3.10

20 3.81 20 2.01

50 2.79 50 1.13

100 2.22 100 0.88

AT(C4) - AT(T4)

Na+

(mM)

ΔΔV

(cm3 mol

-1)

AA(C4) - AA(G4)

Na+

(mM)

ΔΔV

(cm3 mol

-1)

10 1.69 10 2.21

20 1.80 20 1.45

50 1.66 50 0.74

100 1.34 100 -0.23

40

Table 18 The difference in transition volumes (ΔV) between all six heterogeneous loop hairpins

studied.

AT(TC2T) - AT(TG2T)

Na+

(mM)

ΔΔV

(cm3 mol

-1)

AA(TC2T) - AA(TG2T)

Na+

(mM)

ΔΔV

(cm3 mol

-1)

10 3.22 10 3.13

20 2.52 20 2.04

50 1.50 50 0.90

100 0.87 100 0.21

AT(TC2T) - AT(TA2T)

Na+

(mM)

ΔΔV

(cm3 mol

-1)

AA(TC2T) - AA(TA2T)

Na+

(mM)

ΔΔV

(cm3 mol

-1)

10 2.25 10 2.74

20 2.09 20 2.33

50 2.22 50 1.94

100 2.30 100 1.61

AT(TA2T) - AT(TG2T)

Na+

(mM)

ΔΔV

(cm3 mol

-1)

AA(TA2T) - AA(TG2T)

Na+

(mM)

ΔΔV

(cm3 mol

-1)

10 0.97 10 0.39

20 0.43 20 -0.29

50 -0.72 50 -1.04

100 -1.43 100 -1.40

41

CHAPTER IV. DISCUSSION, CONCLUSIONS & FUTURE

PERSPECTIVES

Discussion

In this project, we have studied the effect of hydrostatic pressure on the hairpin-coil transition

temperature (TM) for a number of short DNA hairpins at different salt concentrations by

performing UV-monitored melting. As mentioned previously, the studied hairpins form by

intramolecular folding of 16-base partially self-complementary DNA oligodeoxyribonucleotides.

All 11 hairpins structures studied have a six-base pair duplex-forming stem linked by a four-base

loop. We have reported some important thermodynamic parameters most sensitive to hydration

such as transition volume and expansivity. These measurements enable a more detailed and

complete thermodynamic characterization of these short DNA hairpins, and the thermal

stabilities of these hairpins are no longer restricted to the temperature domain at constant

atmospheric pressure (Vallone, Paner et al. 1999).

The maximum terrestrial pressure is approximately 100 - 110 MPa at the bottom of the Marianas

Trench (Ashcroft 2000). The existence of organisms living under such harsh conditions of

temperature and pressure is indicative of the stability of DNA under such rigorous conditions

(Somero 1992). The major use of elevated hydrostatic pressure in our studies, however, was to

investigate the role of pressure on the conformational stability of DNA hairpins in order to gain

information about the role of water (hydration) in the thermodynamics of the hairpin-coil

transition. One way to study this is to investigate the volume change for the hairpin-coil

transition through investigation of its pressure dependence.

42

Changes in pressure lead to changes in volume. At pressures employed in these experiments, the

molecules and ions constituting the system are essentially incompressible and behave like rigid

spheres (i.e. bond lengths and bond angles remain constant). In accordance with Le Chatelier’s

principle, increasing pressure will force a shift in equilibrium toward the state with the smallest

molar volume. In the absence of significant compressibility3, pressure-induced changes in the

position of an equilibrium are often related to differences in the extent of interaction between the

solvent and solutes. In other words, the system responds by reducing the free volume, and by

favouring interactions between the solvent and the solute that minimize the volume. The molar

volume change (V) for the hairpin-coil transition equals the difference between the partial

molar volume of the single strands (coil form) and the partial molar volume of the helix (hairpin)

form. Figure 7 and Table 4 (heterogeneous loops) and Figure 11 and Table 9 (homogenous

loops) demonstrate that by altering the ionic strength of the solution the volume changes for the

hairpin-coil transition can be either positive or negative. We suspect these volume changes are

due to interactions between the loop and loop-nucleating base pair interactions, since as shown in

Figure 2, the stem is essentially the same for all hairpin samples, except the nucleating base pair.

According to Scaled Particle Theory, a statistical mechanical theory of liquids developed to

interpret the thermodynamic parameters of solutions (Pierotti 1965; Stillinger 1973; Pierotti

1976), the volume change that results from formation of a complex, V, can be decomposed into

3 Compressibility changes are below the level of detection of our system. All our data are linear

within accuracy and range of our experiments.

43

a sum of three components (Chalikian, Totrov et al. 1996; Chalikian, Volker et al. 1999;

Chalikian 2003):

Eq.5

Where VT is the thermal volume change, VI is the hydration (interaction) volume change and,

VM is the intrinsic volume change of the solute. The thermal volume, VT, is the volume of the

void space surrounding the solvent accessible surface of the solute molecules and according to

Chalikian and colleagues is proportional to the solvent accessible surface area (SA) of the solute,

i.e. DNA (Chalikian, Volker et al. 1999). This volume arises from the thermal motion of the

solute and solvent molecules. The hydration volume change, VI, is the volume change

generated from exchange between relatively high-density water in the hydration shell of solutes

and lower density bulk water. The intrinsic volume, VM, is the geometric volume of the solute

molecules; the change in intrinsic volume, VM, is negligible for macromolecules such as DNA

that are tightly packed and have no significant internal voids (Chalikian, Totrov et al. 1996;

Chalikian 2003). Equation 4 is, therefore, further simplified to:

Eq.6

Each term can then be rewritten in terms of the difference between the volume of the helix

(hairpin in this case) and coil states. Hence, Equation 6 becomes:

Eq.7

The subscripts C and H refer to the coil and hairpin states, respectively.

It is logical to assume that the thermal volume change, VT, is positive for the helix-coil

transition since the coil form should in theory have a greater solvent accessible surface area than

44

the hairpin form and hence . Assuming VT is positive, then the hydration volume

change, VI , can be either negative or positive at low temperatures (i.e. temperatures where the

samples are still in hairpin form, 10 - 35 ºC for most studied samples in this experiment). We

believe there can be two cases in terms of explaining the sign of VI at low temperatures.

1) VI is negative for some hairpin samples such as ATG4, AAG4, ATTA2T, AATA2T,

ATTG2T, and AATG2T at low [Na+] and hence low temperatures. In other words, the

interactions with water are stronger for the coil form than for the helix form at lower

temperatures (i.e. ).

2) VI stays positive at the studied [Na+] and hence temperatures for a number of hairpin

samples such as ATT4, ATC4, AAC4, ATTC2T, and AATC2T.

We once again suspect that this sign and magnitude dependency of VI depends strongly on the

interactions within the loop and loop-nucleating base pair interactions.

The molar volume change of the transition becomes more positive with temperature for all

studied samples. The measured expansivities (ΔE) are, therefore, positive as shown in Tables 5

and 10. The origin of this positive change (∂V/∂T > 0) accompanying strand separation is due

to a balance between the volume changes arising from electrostatic (VI) and hydrophobic (VT)

contributions. The electrostatic component arises from the cation release upon transformation to

the coil form whereas the hydrophobic (base-pair stacking) component is due to greater solvent

exposure (i.e. greater solvent accessible surface area) to the bases. Moreover, as mentioned, it is

reasonable to assume that the change in thermal volume would be positive and that the

45

magnitude of VT would increase with temperature (∂VT /∂T >0) since with the increase in

temperature the coil form will become more and more random (entropic force) resulting in a

larger solvent-accessible surface area. Similar temperature dependence is expected for the

hydration volume change, VI. The value of ∂VI /∂T should also be positive and this is mainly

due to interactions such as hydrogen bonding and electrostriction which become less and less

favorable with the increase in temperature. Experimentally, the measured volume change of the

hairpin-coil transition increases with temperature. While both the hydration and thermal

components exhibit similar behaviour with the increase in temperature, we cannot predict which

one predominates.

With increasing sodium chloride concentration, the molar volume changes become increasingly

positive, i.e. ΔV changes linearly with log [Na+], as illustrated in Tables 4 and 9. This change is

attributed to a decrease in the number of cations released upon melting as reflected in the

decreasing magnitude of TM/log [Na+] with increasing salt concentration (Record, Anderson et

al. 1978). Each ion coordinates several water molecules, and even water molecules involved in

outer-sphere hydration of ions have higher densities than bulk water (Spiro, Revesz et al. 1968).

Consequently, the release of fewer cations upon denaturation causes the electrostatic contribution

to the volume change (VI), and therefore the overall ΔV becomes more positive. Therefore, the

effect of sodium chloride concentration on the molar volume change of transition arises from the

decrease in ion release with increasing ionic strength. The average release of Na+

counterions for

the eleven hairpin samples studied is 0.437 ± 0.143 mol Na+/mol hairpin at atmospheric pressure,

which is indicative of their low charge density. The magnitudes of Δn values for these hairpins

46

samples are greater by a factor of ~3 compared to DNA polymers previously reported by

Macgregor and colleagues (Wu and Macgregor 1993; Rayan and Macgregor 2005).

Vallone and Benight have proposed that hydrophobic interactions of bases within the loop, and

the exclusion of water from tight loops, may be a significant factor in the stability of hairpins

with small loops, i.e. 4-5 bases (Vallone and Benight 1999). In other words, the loop

immobilizes a high degree of structural water due to a larger exposure of bases to solvent (Soto,

Kankia et al. 2001). Bevilacqua and colleagues further suggest that a network of hydrogen bonds

in the loop region, in addition to interactions between the loop and nucleating base pairs

contribute significantly to the stability of hairpins (Moody and Bevilacqua 2003; Moody and

Bevilacqua 2003; Moody and Bevilacqua 2004). It is worth mentioning that loop formation is

unfavourable due to backbone charge-charge repulsions, i.e presence of phosphate groups. The

contribution of these Coulombic repulsions can be modulated by the ions in solutions.

Introduction of salt (Na+ in this case) can increase the loop flexibility by neutralizing the

phosphate charges, consequently causing the loop formation to be less unfavourable (i.e. the

increase in salt concentration leads to the decrease of the free energy cost for loop formation).

Investigations by Tan (Tan and Chen 2008) and Kuznetsov (Kuznetsov, Ren et al. 2008) reveal

that the ion-dependence of loop free energy plays an important role in the overall salt-

dependence of hairpin stability. Furthermore, in their work, Kuzentsov and co-workers suggest

that sodium cations interact specifically with loops, and stabilize them. Once again, an increase

in [Na+] favours loop formation; a higher [Na

+] would reduce repulsive forces discussed above

due to stronger charge neutralization and thus improves loop stability. We suspect this is the

main reason for the larger expansivity values of these hairpin samples compared to those of

47

double stranded nucleic acid polymers (Wu and Macgregor 1993; Chalikian, Volker et al. 1999;

Rayan and Macgregor 2005).

Pressure Denaturation of DNA Hairpin Samples

As described earlier, under certain conditions DNA samples can undergo a pressure-induced

helix-coil transition (Rayan and Macgregor 2005). In theory, pressure denaturation can be

performed on samples which have negative transition volumes (i.e increase in pressure

destabilizes the sample or simply decrease the TM). Compared to DNA polymers, such as the

ones studied by Rayan and Macgregor, hairpin samples analyzed in this study have a very broad

transition. This can be seen by closely looking at the heat-induced transition curves reported in

the results section. All eleven hairpin samples analyzed in this study had a very broad transition

of about 30-40 ºC, i.e. the difference in temperature between coil and hairpin states, compared to

DNA polymers where the transitions were not as broad and were about 12 ºC. Please see Figure

1 in Rayan and Macgregor publication (Rayan and Macgregor 2005). When the helix-coil

transition is sharp such as the one in Rayan’s publication, the equilibrium constant changes much

more drastically with increase or decrease in pressure and hence a pressure-induced helix-coil

transition can be observed. In our case, a number of samples with the largest negative transition

volumes such as the ATTG2TAT hairpin sample was analyzed to see if they can be pressure

denatured. However, only a linear change in the OD as a function of pressure was observed for

these samples. This increase in absorbance (OD) arose from compression of the samples with

pressure. Consequently, we claim that the change in equilibrium constant and hence the free

energy (ΔG) is small and insignificant with the increase in pressure for these hairpin samples,

48

leaving us unable to pressure-denature the hairpin samples with negative transition volumes. It is

worth mentioning that the small change in equilibrium constant, and consequently the free

energy (ΔG) is directly dependent on the magnitude of ΔV which is not that large for most

hairpins samples studied.

Conclusion

The effect of pressure on 11 different 16 base hairpin samples has been reported. The goal of this

project was to see if these hairpin samples behave the same way as the double stranded nucleic

acids and the phase diagram described earlier (Figure 1). We have observed both negative and

positive transition volumes (ΔV) for the studied hairpins. A positive ΔV indicates that the hairpin

(helix) form is stabilized by pressure (i.e. increase in TM with the increase in pressure); whereas,

a negative ΔV specifies that the hairpin form is destabilized by pressure (i.e. decrease in TM with

the increase in pressure). Our results are not in full agreement with the temperature-pressure

phase diagram for DNA polymers. This disagreement, nonetheless, is not unanticipated as the

reported phase diagram depends on a number of thermodynamic parameters such as pressure,

transition volume, expansivity, heat capacity, adiabatic compressibility, and isothermal

compressibility (Dubins, Lee et al. 2001). In general, changes in expansibility accompanying

conformational transitions of nucleic acids are very well related to hydration change, i.e.

interactions with water. Our results reveal that the expansivities for short DNA hairpins are much

greater than those of the double-stranded nucleic acids, a thermodynamic parameter that the

phase diagram is very dependent on and perhaps most sensitive to. Large expansivity values are

most likely attributable to the excess stability of hairpins and hairpin loops in particular, which

49

has been attributed to favorable hydrophobic interactions of the bases within the loop and

between the loop and the stem. Moreover, as mentioned earlier, the greater expansivity values

can also be attributed to the specific interactions of sodium cations (Na+) with the loops, which

enhance the stability of DNA hairpins. Unfortunately, with the techniques described and

employed in our experiments we are not able to measure all thermodynamic parameters involved

in determining the shape and thermodynamics of the phase diagram illustrated in Figure 1, i.e.

heat capacity, adiabatic compressibility, and isothermal compressibility. Consequently, more

thermodynamic knowledge of hairpin samples is required in order to have a more thorough

understanding of the stability of short DNA hairpin as a function of temperature and pressure.

Future Perspectives

I have only studied one of the simplest cases of DNA hairpins and there are many other types of

hairpins such as hairpins with dangling ends (Doktycz, Paner et al. 1990), dumbbell hairpins

(Paner, Amaratunga et al. 1992; Paner, Gallo et al. 1993; Paner, Riccelli et al. 1996) or even

longer DNA hairpins where the current thermodynamic knowledge is restricted to atmospheric

pressure. I imagine the goal is to ultimately come up with a phase diagram similar to the one for

double-stranded nucleic acid polymers; however, more thermodynamic knowledge of different

hairpin samples is required prior to reaching that stage. As mentioned earlier, my results on the

effect of elevated pressure (up to 200 MPa) on the stability of the 11 studied hairpin samples do

not fully agree with the phase diagram (Dubins, Lee et al. 2001); nevertheless, the phase diagram

depends on some thermodynamic parameters and already my results show that the expansivities

for short DNA hairpins are much greater than those of the double-stranded nucleic acids.

50

Up until now, we have been using UV-melting at high pressure (described earlier) to study these

DNA samples. Our lab is equipped with a High Pressure IR Instrument (HPIRI) and one would

imagine that studying these samples under high pressure using an IR instrument would further

enhance our knowledge and understanding of DNA hairpins and their structural behaviours as a

function of pressure. Not only is HPIRI a great spectroscopy technique in terms of studying the

hydration properties of DNA hairpins, it also enables us to go to much higher pressures. It is very

important to study the effects of pressures greater than 200 MPa on the stability of different

DNA hairpins.

51

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59

APPENDIX

Effect of Pressure on the Thermally-Induced Denaturation of the Human

Telomeric Sequence

Introduction

G-quadruplexes exhibit unique structural features and play an important role in a variety

of cellular processes. More recently they have been considered novel targets for drug therapy in

aging and anticancer research (Blackburn 1994; Duquette, Handa et al. 2004; Seenisamy,

Bashyam et al. 2005; Olsen, Gmeiner et al. 2006; Tang and Shafer 2006; Olsen, Lee et al. 2009).

G-rich nucleic acid sequences can fold into four stranded DNA structures that contain stacks of

G-quartets. Tetraplexes can form by the intermolecular association of four DNA molecules,

dimerization of sequences that contain two G-tracts, or by the intermolecular folding of a single

strand containing four blocks of guanines (Simonsson 2001; Davis 2004; Burge, Parkinson et al.

2006; Phan, Kuryavyi et al. 2006).

In general, G-quadruplexes are stabilized by G-tetrads that are separated by non-G loop-

forming regions. These loops, as a result, play a major role in the stability of G-tetraplexes; the

sequence and the length of the loops can stabilize or destabilize a G-tetraplex (Henderson,

Hardin et al. 1987; Williamson and Rybicki 1989; Macaya, Schultze et al. 1993). This

stabilization or destabilization effect has been attributed to molecular interactions such as

hydrogen bonding, base-base stacking interaction within the loops in addition to the stacking of

60

the loops onto the G-quartets (Keniry, Owen et al. 1997; Hazel, Huppert et al. 2004; Olsen,

Gmeiner et al. 2006; Olsen, Lee et al. 2009).

There has been very little investigation into the pressure-temperature stability of G-

quadruplexes, and the majority of quadruplex-coil transitions of tetraplexes have been studied as

a function of temperature at constant atmospheric pressure. Consequently, the current

thermodynamic knowledge of quadruplexes stability is essentially limited to the temperature

domain and the pressure properties remain unknown. The effect of hydrostatic pressure on the

thermal stability of DNA tetraplexes offers an alternative method for the investigation of the role

of solvation on DNA stability. We are interested in the role of water in thermodynamics of the

human telomeric sequence 5' AGGG(TTAGGG)3 3' (a G-quadruplex) quadruplex-coil transition;

to study this we investigate the effect of hydrostatic pressure on thermally induced quadruplex-

coil transition. The most direct methods to determine the role of hydration involve measurements

of volumetric parameters such as volume, compressibility, expansivity, etc. These parameters

can be either measured directly or by studying the temperature and pressure dependence of the

stability of a system. In general, perturbation of the quadruplex-coil transition with pressure

yields the molar volume change of the equilibrium. The molar volume change (ΔV) equals the

difference between the molar volume of the products and that of the reactants. The molar volume

can be either positive or negative. In water, negative volume changes are generally attributed to

the formation of stronger interactions with the solvent or, in other words, more extensive

hydration. On the other hand, a positive ΔV indicates that the molar volume of the coil form is

larger than the molar volume of the helix or hairpin form. The goal of this project is to measure

the effect of pressure on the thermal stability of the folded quadruplex structure formed by

5' AGGG(TTAGGG)3 3' G-tetraplex.

61

DNA Oligonucleotides

The oligodeoxyribonucleotide, 5' AGGG(TTAGGG)3 3' was synthesized and cartridge

purified by ACGT, Inc. (Toronto, Canada). The DNA sample was then dissolved in water from a

MilliQ filtration system. The sample was then dialyzed thrice at 4 ºC in 10 mM phosphate form

free acid, 1 mM Na2EDTA, and 1 mM NaN3 (titrated to pH 7) with tetrabutylammonium

hydroxide 30-hydrate buffer for a total of at least 36 hours. The Na+ concentrations ranged from

20 mM to 100 mM. The dialysis tubing (1k MWCO Tube-O-Dialyzer) was obtained from G

Biosciences (St. Louis, MO, USA). The sodium ion concentration was regulated by altering

the NaCl concentration. The DNA stock concentrations were determined spectrophotmetrically

from the Beer-Lambert law at 260 nm with molar extinction coefficient of 228500 M-1

cm-1

(Owczarzy, Vallone et al. 1997).

Optical Melting Experiments under Hydrostatic Pressure

The temperature regulated iso-hyperbaric spectrophotometer was employed to obtain the

heat-induced melting curves; this instrument has been described previously (Wu and Macgregor

1993). Briefly, the sample solution (~300 µL) was contained in a cylindrical quartz cuvette (path

length 0.5 cm) positioned in the optical path of a pressure cell equipped with quartz windows.

The high pressure cell was filled with silicon oil, as the pressure transmitting medium. Pressure

up to 100 MPa (~0.1 MPa is equivalent to atmospheric pressure) was generated using an

automated high pressure pump (Porous Materials Incorporated, Ithaca, NY). The temperature

62

was regulated using a Haake model DC5-k20 circulating bath (Thermo Scientific, Waltham,

MA). A thermocouple connected to an Omega DP80 digital thermometer (Stamford, CT) is

inserted into the pressure-cell in order to measure the temperature. The temperature, pressure and

absorption of the sample were all recorded by the software controlling the experiment. Sample

temperature was increased linearly at a heating rate of 0.9C/min and the helix-coil transition

was monitored by measuring the change in absorption at 295 nm. Prior to melting, samples were

heated to 100 ºC and then allowed to cool naturally to room temperature.

Results and Discussion

Figure A1 shows a melting curve of 5' AGGG(TTAGGG)3 3' sample in an aqueous

solution containing 50 mM Na+ at 10 MPa. The mid-point of the transition is known as the

melting temperature (TM) and is an indicator of the stability quadruplex. The TM, in this case,

corresponds to a temperature at which half of the DNA is in the quadruplex conformation,

whereas the other half is in the coil state. The transition is highly cooperative as seen from the

shape of the curve. The TM of the transition is 38.5 ºC. As discussed below, the following

thermodynamic parameters are extracted from the heat-induced melting curves; the melting

temperature (TM), the model dependent van’t Hoff enthalpy change of the helix-coil transition

(ΔHvH), and the transition volume of the heat-induced helix-coil transition (ΔV).

63

10 20 30 40 50 60 70 800.93

0.94

0.95

0.96

0.97

0.98

0.99

1.00

1.01

1.02

OD

(2

95

nm

)

Temperature (C)

Figure A1 A heat-induced quadruplex to coil transition of AGGG (TTAGGG) 3 at 80 MPa in an

aqueous solution containing 50 mM Na+. The melting temperature of the transition corresponds

to 38.5 ºC. The rate of heating was 0.9 ºC/min.

64

A typical relative absorbance versus temperature curve is show in Figure A1. Plots of this

type show the relative decrease in absorbance at 295 nm associated with melting. In this stage of

analysis each OD versus temperature curve was smoothed by passing the data through a low pass

digital filter using the OriginPro 8 program. The derivative plots, d OD/ d T versus temperature

were obtained using the derivative function of the OriginPro 8 first derivative function. From

plots of d OD/ d T versus temperature the transition temperature (TM) was determined as the

temperature at peak height maximum, d OD/ d Tmax.

The van’t Hoff enthalpy (ΔHvH) for each transition was calculated from (OD/T) Max and

the melting temperature, TM as described in Marky and Breslauer (Marky and Breslauer 1987).

2(2 2 ) ( )M

vH T Max

ODH n RT

T

Where n is a constant equal to the molecularity (n = 1 in this case), R is the gas constant, and

(OD/T) Max is the slope of the OD at 295 nm versus temperature curve at TM (Marky and

Breslauer 1987).

Figure 2A illustrates the pressure dependence of the TM for the quadruplex sample at

three different salt concentrations. The sign and magnitude of TM/P are indicative of the effect

of pressure on the stability of short DNA quadruplex. Positive values of TM/P specify that

65

increasing pressure stabilizes the quadruplex form, while negative values of TM/P show that

increasing pressure destabilizes the quadruplex state of the oligos. (NOTE: Every value was

measured only once for all analyzed samples.)

0 20 40 60 80 10026

28

30

32

34

36

38

40

42

44

46

48

50

52

54

56

Tem

per

atu

re (C

)

Pressure (MPa)

Figure 2A Quadruplex-coil transition temperature (TM) of AGGG (TTAGGG) 3 as a function of

pressure at various Na+ concentrations: 100 mM (■), 50 mM (), and 20 mM (▲). The solid

lines are least-squares fits to the data.

66

The molar volume change of the heat-induced quadruplex-coil transition (V) was

calculated from the slopes of the data such as the ones shown in Figure 2A using the Clapeyron

equation:

MM

T VT

P H

Where H is the calorimetric enthalpy change of the hairpin-coil transition.

Figure 3A illustrates the molar volume change of the heat-induced transition (V) as a

function of sodium chloride concentration for the G-quadruplex sample. The value of V varied

linearly with the log [Na+] from -67.8 0.9cm

3 mol

-1 in 20 mM Na

+ to -56.3 1.9 cm

3 mol

-1 in

100 mM Na+.

The effect of TM on the volume change of the transition for G-quadruplex sample is

presented in Figure 4A. From these data we calculate that the V = 0 cm3

mol-1

at 130 ºC for

AGGG (TTAGGG) 3 G-quadruplex sample.

All thermodynamic parameters extracted from the heat-induced quadruplex-coil

transition at three different Na+ concentrations are summarized in Table 1A.

67

1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0 2.1

-68

-66

-64

-62

-60

-58

-56

V (

cm

3/m

ol)

log[Na+]

Figure 3A Molar volume change of the heat-induced denaturation (V) as a function of salt

concentration. The line is a least-square fit of the data; the slope of the line is 16.6 ± 1.9 cm3

mol-1

.

68

38 40 42 44 46 48 50 52 54 56

-68

-66

-64

-62

-60

-58

-56

V

(cm

3m

ol-1

)

Temperature (C)

Figure 4A Molar volume change of the heat-induced denaturation (V) as a function of TM. The

solid line is a least-square fit to the data. The slope of the line, the ΔE of the transition is equal to

0.74 ± 0.13 cm3 K

-1 mol

-1.

69

Table 1A Thermodynamic parameters extracted from the heat-induced quadruplex-coil

transition at three different Na+ concentrations.

Na+ (mM) TM (C) 100 × dTM/dP (C/MPa) V (cm

3/mol) ΔH (kJ/mol)

20

39.4 0.1

-11.6 0.1

-67.8 0.9

183 2

50

47.4 0.1

−11.3 0.1

-60.1 2.2

170 6

100

54.9 0.1

−11.3 0.1

-56.3 1.9

164 5

70

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