Download Structural Energetics of a RNA-DNA Hybrid

Wesleyan University
The Honors College
Structural Energetics of a RNA-DNA Hybrid
Containing a Tract of rA-dT Base Pairs
by
Katherine Leon
Class of 2010
A thesis submitted to the
faculty of Wesleyan University
in partial fulfillment of the requirements for the
Degree of Bachelor of Arts
with Departmental Honors in Chemistry
Middletown, Connecticut
April, 2010
Acknowledgements
I would first like to acknowledge my advisor and mentor, Professor Irina
Russu. Thank you for welcoming me into the Russu lab, for supporting and guiding
me through both this research and my academic career at Wesleyan, and for being an
excellent teacher.
I would also like to thank my labmates: Xiaoli Weng, Jie Zhang, Ryan Coffey,
and especially Yuegao (Golden) Huang for his guidance and patience in the past two
years and for his help in writing this thesis.
I would like to thank the staff and faculty in the Chemistry Department for
being so helpful with anything and everything.
I would like to thank my friends for being so supportive and encouraging
throughout this writing process. I truly appreciate all of you very much. Thank you to
my housemate, Gloria, for the good humor and good company.
Finally, I would like to thank my family, especially my parents, for always
believing in me and supporting me in everything I have done and wish to do.
i
Table of Contents
Acknowledgements ...................................................................................................... i
Table of Contents ........................................................................................................ ii
Abstract....................................................................................................................... iii
List of Figures............................................................................................................. iv
List of Tables .............................................................................................................. vi
Chapter 1. Introduction ............................................................................................ 1
1.1. Nucleic Acids and Intrinsic Transcription Termination ................................... 2
1.2. Imino Proton Exchange................................................................................... 11
1.3. Previous and Current Studies .......................................................................... 14
Chapter 2. Materials and Methods ........................................................................ 17
2.1. Nucleic Acid Synthesis ................................................................................... 18
2.2. Nucleic Acid Purification ............................................................................... 24
2.3. Sample Preparation ......................................................................................... 25
2.4. NMR Spectroscopy ......................................................................................... 26
2.4.1. 1D Proton Experiments ............................................................................ 26
2.4.2. Transfer of Magnetization........................................................................ 29
2.4.3. Fast Heteronuclear Single Quantum Coherence (fHSQC) Experiments . 32
2.4.4. Nuclear Overhauser Effect Spectroscopy (NOESY) Experiments .......... 34
2.5. pH Calculation ................................................................................................ 35
2.6. Model for Imino Proton Exchange in Nucleic Acids...................................... 38
Chapter 3. Results .................................................................................................... 41
3.1. Assignments of Imino Proton Resonances ..................................................... 42
3.2. Imino Proton Exchange................................................................................... 51
Chapter 4. Discussion .............................................................................................. 59
4.1. Analysis of Base-Pair Energetics in the rA-dT Hybrid .................................. 60
4.2. Comparison of the rA-dT Hybrid with Two Homologous GC-rich RNA-DNA
Hybrids.................................................................................................................... 63
4.3. Comparison of rA-dT Hybrid with Its Homologous rU-dA Hybrid and dA-dT
DNA Duplex ........................................................................................................... 67
4.4. Dependence of Base-Pair Openings on Temperature ..................................... 71
Conclusions and Future Work................................................................................. 74
References .................................................................................................................. 76
ii
Abstract
Base sequence has a great effect on the physical and functional properties of
nucleic acid structures. For example, intrinsic (rho-independent) termination of
transcription in prokaryotes relies solely on properties induced by the base sequence
at the termination site. The tR2 terminator site of phage  contains an adenine-rich
tract, which yields a uracil-rich sequence at the 3’ end of the RNA transcript. The
associated dA-dT DNA template duplex and the rU-dA RNA-DNA hybrid have
previously been studied in our laboratory. This thesis focuses on the corresponding
rA-dT RNA-DNA hybrid. The stability and opening dynamics of the base pairs are
determined through the rate of exchange of imino protons using Nuclear Magnetic
Resonance (NMR) spectroscopy.
The rates of base-pair opening in the rA-dT RNA-DNA hybrid reveal the
following: (i) in general, G-C base pairs are more stable than A-T base pairs, (ii) the
stabilities of A-T base pairs are dependent of their location in the base sequence, and
(iii) the A-U base pair plays a role in destabilizing neighboring base pairs.
Comparison with previous results from our laboratory indicates that the base pairs in
the A-T tract in the rA-dT RNA-DNA hybrid are more stable than those in the A-U
tract in the rU-dA hybrid but less stable than those in the A-T tract in the dA-dT
DNA-DNA duplex.
iii
List of Figures
Figure 1.1.1. Structure of a DNA (RNA) chain .......................................................... 6
Figure 1.1.2. Structures of canonical Watson-Crick base pairs .................................. 7
Figure 1.1.3. 3D structures of A-DNA, B-DNA, and Z-DNA .................................... 8
Figure 1.1.4. Flowchart of the transfer of genetic information in cells ...................... 9
Figure 1.1.5.
Base sequence of DNA template and RNA transcript in the tR2
terminator site of phage  ........................................................................................... 10
Figure 1.3.1. Base sequences of (a) two homologous GC-rich DNA-RNA hybrids,
(b) dA-rU DNA-RNA hybrid and dA-dT DNA-DNA duplex, and (c) dT-rA DNARNA hybrid investigated in the present work............................................................. 16
Figure 2.1.1. Base sequences of synthesized RNA and DNA strand........................ 21
Figure 2.1.2. Structures of protected nucleosides used in synthesis of DNA strands
..................................................................................................................................... 22
Figure 2.1.3. Structures of protected nucleosides used in synthesis of RNA strands
..................................................................................................................................... 23
Figure 2.4.1. Pulse sequences used in the NMR experiments .................................. 28
Figure 2.4.2.
An illustration of NMR magnetization transfer experiments:
dependence of the normalized intensity of (rA-dT)5 imino proton resonance on the
exchange delay at 10 C and 36 mM ammonia concentration ................................... 31
Figure 2.4.3. 1D fHSQC pulse sequences used for 15N-labeled samples ................. 33
Figure 2.5.1. Selected region of the NMR spectrum of the RNA-DNA hybrid
investigated showing the two triplet resonances of TEOA ......................................... 37
iv
Figure 3.1.1. (a) Base sequence and (b) imino proton resonances of rA-dT hybrid
investigated at 10 C. .................................................................................................. 45
Figure 3.1.2. Temperature dependent 1D 1H NMR spectra of rA-dT hybrid ........... 46
Figure 3.1.3. 15N/14N-editing of NMR resonances at 10 C of imino protons for a
sample of rA-dT hybrid in which thymines in positions 6 and 7 are labeled with 15N
..................................................................................................................................... 47
Figure 3.1.4. 15N/14N-editing of NMR resonances at 10 C of imino protons for a
sample of rA-dT hybrid in which uracil in position 10 and thymines in positions 6 and
7 are labeled with 15N ................................................................................................. 48
Figure 3.1.5. Expanded regions of the 1H-1H NOESY spectrum at 10 C of the rAdT hybrid ..................................................................................................................... 49
Figure 3.2.1. Imino proton resonances of rA-dT hybrid at various ammonia
concentrations and at 10 C ........................................................................................ 53
Figure 3.2.2. Dependence of imino proton exchange rates on ammonia base
concentration in the rA-dT hybrid at 10 C ................................................................ 54
Figure 3.2.3. Dependence of imino proton exchange rates on ammonia base
concentration in the rA-dT hybrid at 1 C .................................................................. 55
Figure 4.2.1. Base sequences of GC-rich RNA-DNA hybrids ................................. 65
Figure 4.3.1. Base sequences of rU-dA RNA-DNA hybrid, dA-dT DNA duplex, and
rA-dT RNA-DNA hybrid ............................................................................................ 69
v
List of Tables
Table 3.1.1. Imino Proton Connectivities of rA-dT Hybrid ...................................... 50
Table 3.1.2. Chemical Shifts of Imino Proton Resonances of rA-dT Hybrid at 10 C
..................................................................................................................................... 50
Table 3.2.1. Base-Pair Opening Parameters in the rA-dT RNA-DNA Hybrid at 10 C
..................................................................................................................................... 56
Table 3.2.2. Base-Pair Opening Parameters in the rA-dT RNA-DNA Hybrid at 1 C
..................................................................................................................................... 57
Table 3.2.3. Estimation of Enthalpy and Entropy Changes for Base-Pair Opening in
the rA-dT RNA-DNA Hybrid at 1 C and 10 C ........................................................ 58
Table 4.2.1. Base-Pair Opening Parameters in the GC-rich RNA-DNA Hybrid I at 10
C ................................................................................................................................ 66
Table 4.2.2. Base-Pair Opening Parameters in the GC-rich RNA-DNA Hybrid II at
10 C ........................................................................................................................... 66
Table 4.3.1. Equilibrium Constants and Free-Energy Changes for Base-Pair Opening
in the rU-dA RNA-DNA Hybrid at 10 C .................................................................. 70
Table 4.3.2. Equilibrium Constants and Free-Energy Changes for Base-Pair Opening
in the dT-dA DNA Duplex at 10 C ........................................................................... 70
Table 4.4.1. Enthalpy changes for base-pair opening in two DNA duplexes ........... 73
Table 4.4.2. Enthalpy changes for base-pair openings in a DNA duplex containing a
TATA box sequence ................................................................................................... 73
vi
Chapter 1. Introduction
1
1.1. Nucleic Acids and Intrinsic Transcription Termination
Nucleic acids are complex macromolecules which store and transmit genetic
information. The two most common types of nucleic acids are deoxyribonucleic acid
(DNA) and ribonucleic acid (RNA). DNA contains the information that is transcribed
in messenger RNA, which then leads to synthesis of proteins as a form of gene
expression.
Both DNA and RNA are made up of nucleotide units linked by
phosphodiester bonds. Each nucleotide unit contains a nucleobase, a five-membered
sugar, and a phosphate group. The sugar in DNA is 2’-deoxyribose, and the sugar in
RNA is ribose. The five nucleobases are adenine (A), cytosine (C), guanine (G),
thymine (T), and uracil (U). The bases C, T, and U are classified as pyrimidines, and
the bases A and G are classified as purines. A, C, and G bases are found in both DNA
and RNA, but T is found mostly in DNA and U is found mostly in RNA. An example
of a fragment of a DNA (or RNA) chain is shown in Figure 1.1.1.
DNA typically exists as a double-stranded helix. The bases on one strand
hydrogen bond to complementary bases on the other strand. In canonical WatsonCrick base pairs, there are two hydrogen bonds between A and T (or U in RNA
helices), and three hydrogen bonds between G and C. The structures of the base pairs
are shown in Figure 1.1.2. DNA is primarily in the right-handed B-DNA form in vivo,
with 10 base pairs per turn of the helix, helical-twist angle of 36, and helical rise of
3.4 Å. Under different environmental conditions, DNA can conform to right-handed
A-DNA (11 base pairs per turn of the helix, helical-twist angle of 33, and helical rise
of 2.9 Å) or left-handed Z-DNA (12 base pairs per turn of the helix, helical-twist
2
angle of 30, and helical rise of 3.7 Å). Structures of the three forms are shown in
Figure 1.1.3. RNA can adopt a greater variety of structures, including hairpin loops,
bulges, internal loops, and single strands. Double-stranded RNA is typically in the Aform.
The properties of nucleic acids play important roles in the effectiveness of
gene expression. In the transfer of genetic information, a DNA strand is replicated to
form a complementary RNA strand, and protein is synthesized from this geneencoded RNA strand. A diagram of this process is shown in Figure 1.1.4. In this
study, we are focused on the transcription step in the transfer of genetic information.
The formation of the RNA transcript is divided into two phases: (i) activation and
transcript initiation and (ii) transcript elongation and termination. RNA polymerase
binds to the promoter site on the DNA duplex and unwinds the two DNA strands.
This activates RNA synthesis, in which nucleotides are added to the 3’-end of the
growing RNA chain. The DNA template transiently forms a short hybrid complex
(about 12 base pairs) with the nascent RNA transcript that is being synthesized. This
DNA-RNA hybrid, in which a DNA strand binds to a RNA strand, is a type of nucleic
acid structure that exists in the cell during transcription. The hybrid, which was found
to be similar to A-form RNA (O’Brien and MacEwan, 1970; Pardi et al., 1981), is of
interest because of its role in transcription termination.
In prokaryotes, the release of the nascent RNA transcript from the DNA
template is controlled by either rho-dependent termination or intrinsic termination.
Rho-dependent termination requires the rho protein to bind to the RNA transcript in
order to signal for release. Intrinsic termination, on the other hand, is rho-independent
3
and relies on the properties induced by the base sequence at the termination site. The
tR2 terminator site of phage  contains a guanine/cytosine-rich inverted repeat motif,
which forms a hairpin loop in the transcribed RNA, and an adenine-rich tract on the
DNA template, which yields a uracil-rich sequence at the 3’-end of the RNA
transcript. Transcription termination occurs in this tract of uracil residues. An
illustration of this site is shown in Figure 1.1.5.
A thermodynamic model of intrinsic termination (Yager and von Hippel,
1991) postulates that the free energy of the formation of an elongation complex at a
particular template position can be defined as:
G complex  G DNA DNA  G DNA RNA  G NA polymerase
(1)
where G complex is the net free energy that stabilizes the elongation complex against
dissociation, G DNA DNA is the unfavorable free energy of opening the DNA-DNA
base pairs, G  DNA RNA is the favorable free energy of forming the DNA-RNA base
pairs in the hybrid, and G NA poylmerase is the net free energy of nucleic acidpolymerase interaction in the complex. The terms in this model help to explain the
thermodynamic control of transcription termination.
The G/C-rich hairpin has been known to have a role in intrinsic transcription
termination, though this role is not clearly defined. It has been proposed that the
hairpin gives rise to a pause at the intrinsic termination site, increasing the probability
of termination (Farnham and Platt, 1981) and that the hairpin destabilizes the DNARNA complex (Yager and von Hippel, 1991). Yager and von Hippel’s proposed
model for intrinsic termination states that the DNA-RNA complex destabilizes as it
4
passes through the intrinsic transcription termination site, lowering the G DNA RNA
contribution to G complex . More recent studies (Wilson and von Hippel, 1995) have
found that both the hairpin and the uracil-rich sequence, not just the hairpin alone, are
required in order to effectively terminate transcription, since intrinsic termination
occurs at positions 7 or 8 of the uracil-rich tract. If destabilization of the complex
were not dependent on this tract, transcription would occur more upstream of these
positions.
The rU-dA hybrid corresponding to the DNA-RNA hybrid at the transcription
termination site has been found to be relatively unstable (Martin and Tinoco, 1980).
Optical melting experiments were done on oligomers of dT-rA DNA-RNA hybrid,
dA-rU DNA-RNA hybrid, dA-dT DNA duplex, and rA-rU RNA duplex. The melting
temperatures of the oligomers reveal the stability of the structures from most unstable
to most stable: dA-rU DNA-RNA hybrid, rA-rU RNA duplex, dT-rA DNA-RNA
hybrid, and dA-dT DNA duplex. Circular dichroism experiments show that the dAdT duplex is in the B-form, the rA-rU RNA duplex is in the A-form, and both dT-rA
and dA-rU DNA-RNA hybrids are similar to the A-form. However, the hybrid
structures are unstable because the DNA strands are unstable in the A-form. These
data show the relative stabilities of the whole structures of double-stranded nucleic
acids. To understand how each base pair contributes to the instabilities of the
structures, our laboratory has employed imino proton exchange.
5
Figure 1.1.1. Structure of a DNA (RNA) chain.
6
Figure 1.1.2. Structures of canonical Watson-Crick base pairs. The imino protons are
circled.
7
Figure 1.1.3. 3D structures of A-DNA, B-DNA, and Z-DNA. (Stryer, 1995)
8
Figure 1.1.4. Flowchart of the transfer of genetic information in cells.
9
Figure 1.1.5.
Base sequence of DNA template and RNA transcript in the tR2
terminator site of phage . The GC-rich invert repeat motif is shown in bold and the
AU-tract is shown in red.
10
1.2. Imino Proton Exchange
Unlike the crystallographic representations of their stable native states,
proteins and nucleic acids do not have rigid macromolecular structures. They are
flexible and experience movements ranging from sub-angstrom atomic vibrations to
unfolding of the whole molecule (Englander and Kallenbach, 1984). This property
allows for better interaction with the surfaces of other molecules (Monod, Wyman &
Changeux, 1965; Koshland, Nemethy & Filmer, 1966). In this study, the flexibilities
of nucleic acids allow us to analyze their stabilities through proton exchange.
Proton exchange has been an effective method of studying proteins and
nucleic acids (Gueron and Leroy, 1995; Russu, 2004). The properties of a specific
proton reveal information about the structure at that specific site. Proton exchange
rates in biomolecules span more than 10 orders of magnitude, from less than 10-8 s-1
to about 102 s-1 or faster (Russu, 2004). Nuclear Magnetic Resonance (NMR)
spectroscopy can detect exchange rates in two ranges: hydrogen-deuterium (H-D)
experiments can observe exchange rates slower than 10-2-10-3 s-1, and transfer of
magnetization from water experiments can observe exchange rates faster than about
0.1-0.5 s-1, up to about 100 s-1. Exchange rates from 100 s-1 to 1000 s-1 can be
observed indirectly by linewidth measurements. In ranges other than those mentioned
above, experimental conditions such as pH and temperature can be modified to slow
down or speed up exchange rates into the observable ranges. The exchange of imino
protons of guanine, thymine, and uracil in nucleic acids (Figure 1.1.2.) is of interest in
this study. Imino protons are chosen for two reasons: (i) there is an imino proton
11
present in every canonical base-pair and (ii) the resonances of these protons are
generally well-resolved and separated from the other resonances in the NMR
spectrum.
Since nucleic acids are flexible, the nucleobases can flip “open” out of the
native “closed” conformation. In the “open” state, imino protons exchange with
solvent protons. The model of exchange of protons between these states is:
k op


ex , open
( H ) closed  ( H ) open 
( H ) exchanged
k
k cl
(2)
where (H) is the imino proton, k op is the rate of the opening reaction, k cl is the rate of
the closing reaction, and k ex ,open is the rate of exchange from the open state.
Proton acceptors have been found to catalyze the exchange of imino protons
(Bendel, 1987; Leroy et al., 1988). The exchange rates are dependent on the
concentrations of proton acceptor. At infinite catalyst concentration, the observable
exchange rate is equal to the rate of the opening reaction (EX1 regime). The catalysis
is illustrated below with a guanine nucleobase as:
where H* is a hydrogen atom from solution. In the middle step, the imino proton
forms a hydrogen bond with the proton acceptor B.
12
The observable exchange rate ( k ex ) is then given as (Gueron and Leroy, 1995;
Russu, 2004):
k ex 
k op  k ex ,open
(3)
k cl  k ex ,open
and the equilibrium constant of the opening reaction ( K op ) is:
K op 
k op
(4)
k cl
Our laboratory has used imino proton exchange to study various nucleic acid
molecules, including TATA-box DNA (Chen and Russu, 2004), CAGA 12-mer DNA
and CG 10-mer DNA (Every and Russu, 2007), GC-rich DNA-RNA hybrids (Huang
et al., 2009), and A-U DNA-RNA hybrid and A-T DNA duplex (Huang et al., 2010).
13
1.3. Previous and Current Studies
Figure 1.1.5. shows the tR2 terminator site of phage . Various nucleic acid
molecules relating to this termination site have been studied in our laboratory,
including the two homologous GC-rich DNA-RNA hybrids (Huang et al. 2009), the
dA-dT DNA duplex and the dA-rU DNA-RNA hybrid (Huang et al., 2010). The base
sequences of these molecules are shown in Figure 1.3.1.
The GC-rich DNA-RNA hybrids are involved in intrinsic transcription
termination by forming a RNA hairpin loop upstream of the termination site. The
exchange rates for the base pairs in the two homologous hybrids show that A-T base
pairs are more stable than A-U base pairs, but both are unstable compared to G-C
base pairs. Base pairs neighboring the A-U base pairs are also found to be
destabilized.
The dA-rU DNA-RNA hybrid previously studied (Huang et al., 2010) is
analogous to the hybrid formed at the termination site. Analysis of the imino proton
exchange at each base pair shows that A-U base pairs in the A-U tract are much more
unstable than an A-U base pair in a mixed sequence. This finding suggests that the
instability of the DNA-RNA hybrid plays a role in intrinsic transcription termination
by allowing for the release of the RNA transcript from the DNA template.
The dA-dT DNA-DNA duplex previously studied by our laboratory is
analogous to the duplex formed by template strands (Figure 1.1.5.). The exchange
rates show that the A-T base pairs are very stable, more so than the A-U base pairs in
the dA-rU DNA-RNA hybrid. Also, the A-T base pairs in the tract of A-T base pairs
14
are more stable than the A-T base pair in a mixed sequence, suggesting that the A-T
tract plays a role in stabilizing base pairs.
The dT-rA DNA-RNA hybrid is being investigated in the present work. The
hybrid has the same base sequence as the dA-rU hybrid except that the RNA and
DNA strands are interchanged and thymines and uracils are interchanged. Exchange
rates of imino protons in each base pair allow us to examine the same base sequence
in different structural contexts.
15
Figure 1.3.1. Base sequences of (a) two homologous GC-rich DNA-RNA hybrids,
(b) dA-rU DNA-RNA hybrid and dA-dT DNA-DNA duplex, and (c) dT-rA DNARNA hybrid investigated in the present work.
16
Chapter 2. Materials and Methods
17
2.1. Nucleic Acid Synthesis
The DNA and RNA strands were synthesized by Yuegao Huang on an
automated DNA synthesizer (Applied Biosystems 381 A) using solid-support
phosphoramidite chemistry. The base sequences of the synthesized strands are shown
in Figure 2.1.1.
For the DNA oligonucleotide synthesis, the first nucleoside was attached to a
solid support by the 3’-hydroxyl end. The 5’-hydroxyl was protected by a
dimethoxytrityl (DMT) group. The nucleoside-cyanoethyl phosphoramidites that
were used in the synthesis were protected at the 5’-hydroxyl ends by DMT groups
and at the 3’-phosphorous moiety by a diisopropylamino and a -cyanoethyl
protecting group. The amino groups on the deoxyribonucleosides were also protected:
the exocyclic amines on adenosine (A) and cytosine (C) were protected by a benzoyl
group (Abz, Cbz) and the exocyclic amines on guanosine were protected by an
isobutyryl group (Gib). Thymidine is unreactive and does not need a protecting group.
The structures of these nucleosides are shown in Figure 2.1.2. The nucleosides were
prepared as 0.1 M solutions in anhydrous acetonitrile.
The first step of the synthesis, detritylation, removed the DMT group at the
5’-hydroxyl end of the nucleoside attached to the solid support by 3% trichloroacetic
acid (TCA) in dichloromethane to allow the addition of other nucleosides. In the
second step, activation, 0.45 M tetrazole in acetonitrile was added to a nucleoside
phosphoramidite. The tetrazole protonates the nitrogen of the nucleoside, promoting
nucleophilic attack by the 5’-hydroxyl group of the bound nucleoside. The third step,
18
addition, allowed for reaction of the 3’-end of the activated nucleoside with the 5’hydroxyl of the attached nucleoside. In the fourth step, capping, any oligonucleotide
chain which did not undergo an addition reaction was capped with an acetyl group at
the 5’-hydroxyl group using two capping reagents, tetrahydrofuran (THF) / 2,6lutidine / acetic anhydride and 10% 1-methylimidazole in THF. The final step,
oxidation, converted the internucleotide-linkage trivalent phosphorus to pentavalent
phosphorus with 0.1 M iodine in tetrahydrofuran/pyridine/water. After this step, the
DMT group was removed and the cycle was repeated until the desired DNA
oligonucleotide was formed.
The RNA oligonucleotide was synthesized by the same method as the DNA
oligonucleotide except the cyanoethyl phosphoramidites were protected by a 2’ -Otriisopropylsilyloxymethyl (TOM) group and acetyl groups on the exocyclic amines
of A, C, and G (Aac, Cac, Gac). Uridine is unreactive and does not need a protecting
group. The structures of the nucleosides are shown in Figure 2.1.3. The synthesis
cycle for RNA is the same as for DNA except that RNA synthesis requires longer
coupling time.
The oligonucleotides were removed from the solid support by soaking the
solid support in 3 mL fresh concentrated ammonium hydroxide at room temperature
for one hour. The ammonium hydroxide solution was collected and this procedure
was repeated three times until all oligonucleotide products were removed. The
protecting groups on the amines were removed by heating the collected ammonium
hydroxide solution at 55 C for 8-12 hours. To prevent the loss of the dimethoxytrityl
group at the 5’-end, 200 µL triethylamine was added after deprotection. The volume
19
of the sample was reduced with a water-driven aspirator and then evaporated with a
lyophilizer. The final nucleotide sample was dissolved in 1 mL of 50 mM
triethylammonium acetate buffer (pH 9).
20
Figure 2.1.1. Base sequences of synthesized RNA and DNA strand.
21
Figure 2.1.2. Structures of protected nucleosides used in synthesis of DNA strands.
22
Figure 2.1.3. Structures of protected nucleosides used in synthesis of RNA strands.
23
2.2. Nucleic Acid Purification
The sample was purified by Yuegao Huang using reverse-phase high-pressure
liquid chromatography (HPLC) on a PRP-1 column (Hamilton) with the trityl-on
program in 50 mM triethylammonium acetate buffer at pH 7 with a linear gradient of
5 to 32% acetonitrile in 46 minutes at 60 C. A maximum of 200 O.D.260 units of
nucleic acid can be injected for each HPLC run. The purified sample was frozen in
dry ice and then dried in a lyophilizer. To remove the trityl group, 30 µL 80% acetic
acid solution was added to the nucleic acid per O.D. unit for 20 minutes. Distilled
water was added to the solution to dilute the acetic acid concentration to 1/3. To
remove the acetic acid, the sample was frozen in dry ice and lyophilized.
24
2.3. Sample Preparation
The counterions in the sample were replaced with Na+ ions by repeated
centrifugation through Centricon YM-3 tubes (Amicon Inc., Bedford, MA) using 1 M
NaCl solution. Excess Na+ ions were removed by adding water and centrifuging 8 to
10 times. The concentrations of the nucleic acids were determined from the maximum
absorbance at 260 nm using calculated extinction coefficients (Cantor et al., 1970).
The single strands of nucleic acid were annealed in a 80 C water bath for 30 minutes
and then cooled down to room temperature. The sample contained 0.5-2 mM
triethanolamine, which was used to determine the pH of the sample directly in the
NMR tube (Section 2.4.5.). Small amounts of stock ammonia solutions (0.5-1 M) of
pH 7.8 were added to the sample to obtain increasing concentrations of ammonia for
imino proton exchange experiments.
25
2.4. NMR Spectroscopy
The NMR experiments were performed on a Varian INOVA 500 spectrometer
operating at 11.75 T.
2.4.1. 1D Proton Experiments
The imino proton resonances of the RNA-DNA hybrid were observed with the
Jump-and-Return pulse sequence (Plateau and Gueron, 1982). The pulse sequence
(Figure 2.4.1.a.) consists of two nonselective 90 pulses with opposite phase (for
example, the first on y and the second on –y) with a delay (d1) before the first pulse
and a delay (d2) after the first pulse. The delay time d2 is chosen to maximize the
intensities of the resonances of interest. The dependence of intensity of the resonance
of interest on d2 is:
I  I 0 sin[ 2 ( f  f 0 )  d 2]
(5)
where I is the measured intensity of the resonance, I 0 is the intensity of the
resonance at full excitation, f is the frequency of the resonance, and f 0 is the
frequency of water. The resonance has maximum intensity when ( f  f 0 )  d 2  0.25 .
The Inversion-Recovery pulse sequence (Figure 2.4.1.b.) was used to measure
the longitudinal relaxation rate of water protons. The magnetization of water is
selectively inverted by a 180 Gaussian pulse, followed by a delay time  (arrayed
from 2 ms to 16 s). To prevent rapid relaxation due to radiation dumping, a weak
gradient pulse (0.03 gauss/cm) is applied during the delay. After the delay, a 90
26
Gaussian pulse is applied and the water signal is detected using a single low-power
pulse. The intensity of water, I w , as a function of the delay time  is given by:
I w ( )  I w (0)  [1  2 exp(  R1w )]
(6)
where I w (0) is the intensity of water resonance after the first pulse, and R1w is the
longitudinal relaxation rate of water. The intensity of water resonance is fitted against
the various delay times (τ) with Equation 6 to calculate the longitudinal relaxation
rate of water protons.
27
a) Jump-and-Return
b) Inversion-Recovery
c) Transfer of Magnetization from Water
d) Watergate NOESY
Figure 2.4.1. Pulse sequences used in the NMR experiments. Rectangles represent
non-selective 90 pulses. Wave-like shapes represent selective 90 or 180 Gaussian
pulses on water. The gradients are labeled gt1 and gt2. In transfer of magnetization
experiments (panel c), the exchange delay τ was varied from 1 to 800 ms. In the
Watergate NOESY experiment (panel d), the mixing time was 250 ms.
28
2.4.2. Transfer of Magnetization
The exchange rates of imino protons were measured in transfer of
magnetization experiments. The pulse sequence for these experiments is shown in
Figure 2.4.1.c. The magnetization of water protons is perturbed by a selective
inverting pulse. After a delay , the perturbation is transmitted to all exchangeable
protons in the biomolecule of interest. A range of delay times (1 to 800 ms) were
arrayed to measure exchange rates. At the end of the delay , a second selective pulse
is applied to bring the water magnetization back onto z-axis. The observation is with
the Jump-and-Return pulse sequence.
The dependence of the intensity of an exchangeable proton resonance on the
exchange delay  is given by (Russu, 2004):


I ( )  I 0  I (0)  I 0  A  e( R1kex )  A  e R1w
(7)
where I 0 is the intensity at equilibrium, I (0) is the intensity immediately after the
first selective pulse on water, k ex is the exchange rate, R1 is the longitudinal
relaxation rate of the observed proton, and R1w is the longitudinal relaxation rate of
water protons. A is defined as:
 I (0) 
k ex
I0
A   w 0  1 
 I w
 R1  k ex  R1w
(8)
where I w (0) is the intensity of the water proton resonance after the first inversion
0
pulse and I w is the intensity of the water proton resonance at equilibrium.
29
The intensities of imino proton resonances are normalized as:
Normalized intensity 
I ( )  I 0 R1w  I (0)
A
A
e
  0  1  0   e ( R1  kex  R1w )  0
0
I
I 
I
 I
(9)
Or:
y  m1  e  m2  m3
where
(10)
y  normalized intensity,
A
 I ( 0)
m1   0  1  0 
I 
 I
m2  R1  k ex  R1w
m3 
A
I0
y is calculated from the measured intensities ( I ( ) and I 0 ) and the measured R1w
value as described in Section 2.4.1., and is then fitted to Equation 10 as a function of
the exchange delay  using a non-linear least-squares program. An illustration of the
data and their fitting to Equation 10 is shown in Figure 2.4.2. The values generated
for m 2 and m3 are used to calculate the exchange rate:
k ex 
A  ( R1  k ex  R1w )
m m
 2 3
I w ( 0)
 I (0) 
1
I 0   w 0  1
I 0w
 I w

(11)
where I w (0) and I 0 w are measured as described in Section 2.4.1. with the same
acquisition parameters.
30
(rA-dT)5
Normalized Intensity
-0.4
m1
m2
m3
-0.6
0.77515
5.36512
-1.24348
±0.01761
±0.30881
±0.01781
-0.8
-1.0
-1.2
-1.4
0.0
0.2
0.4
0.6
0.8
Exchange Delay (, s)
Figure 2.4.2.
An illustration of NMR magnetization transfer experiments:
dependence of the normalized intensity of (rA-dT)5 imino proton resonance on the
exchange delay at 10 C and 36 mM ammonia concentration.
The curve was obtained by fitting the data to Equation 10.
31
2.4.3. Fast Heteronuclear Single Quantum Coherence (fHSQC) Experiments
Two
15
N-labeled hybrid samples were prepared in order to help assign the
imino proton resonances. The first hybrid sample contains 15N-labeled thymines and
uracil at positions 6, 7, and 10. The second hybrid sample contains
15
N-labeled
thymines at positions 6 and 7. The 1D fHSQC pulse sequence (Mori et al., 1995) in
Figure 2.4.3.a. is used to display on the spectrum only the proton resonances attached
to 15N. This pulse sequence was modified (Chen, 2007), shown in Figure 2.4.3.b., to
display on the spectrum all the proton resonances that are not attached to 15N. Imino
protons attached to
14
N and
15
N can be differentiated through these experiments for
both hybrid samples.
32
Figure 2.4.3. 1D fHSQC pulse sequences used for
15
N-labeled samples. Narrow
white rectangles represent non-selective 90 pulses, wider blue rectangles represent
non-selective 180 pulses, yellow rectangles represent 90 pulses on water with low
power and gray rectangles represent gradients in z-axis. D1 = 6 s,  = 1/2J (J is the
15
N-1H coupling constant, ~ 90 Hz). Gradient: G1 = 4.9 Gauss/cm for 1 ms, G2 = 42
Gauss/cm for 400 µs. (a) fHSQC pulse sequence for detecting protons attached to
15
N. Phase cycle: 1 = x, -x, x, -x; 2 = x, x, -x, -x; 3 = -x, -x, x, x; 4 = x, -x. (Mori
et al., 1995) (b) Modified fHSQC pulse sequence for detecting protons attached to all
other protons except for those attached to 15N. Phase cycle: 1 = x, -x, x, -x; 2 = x, x,
-x, -x; 3 = -x, -x, x, x; 4 = y, -y; 5 = -x, x. (Chen, 2007) Modifications from (a) to
(b) are shown in red.
33
2.4.4. Nuclear Overhauser Effect Spectroscopy (NOESY) Experiments
The imino proton resonances were assigned through 2D 1H-1H NOESY
experiments. The water flip-back Watergate NOESY (Lippens et al., 1995) pulse
sequence (Figure 2.4.1.d.) was used. The relaxation delay D1 was 3 s and the mixing
time was 250 ms. In the first dimension, 4096 data points were acquired over a
spectral width of 11000 Hz. In the second dimension, 512 increments were used in
the phase-sensitive mode, with 32 scans per increment over a spectral width of 11000
Hz.
34
2.5. pH Calculation
The pH of the NMR sample was measured using the NMR resonances of
triethanolamine (TEOA). The chemical structure of TEOA is:
OH
CH2
CH2
HO
CH2
CH2
N
CH2
CH2
OH
TEOA appears on the spectrum (Figure 2.5.1.) as two triplet resonances due to mutual
J-coupling between the two types of methylene protons. Dr. Congju Chen in our
laboratory has shown that the chemical shifts of the triplet resonances are pHsensitive due to the protonation of the N and O atoms. The relationship between the
chemical shifts and pH is given as:
  
0
   0
1  10 pH  pKa (TEOA)
(12)
where  is the chemical shift difference between the two triplet resonances,  0 is the
chemical shift in the non-protonated state, and   is the chemical shift in the
protonated state. Chemical shift vs. pH calibration curves for various temperatures
have previously been generated (Chen, 2007). The pK a ,  0 , and   of TEOA for
various temperatures were also determined through non-linear least square fits to the
data (Chen, 2007).
35
Equation 12 can be rearranged to calculate the pH of the sample:
    
pH  pK a (TEOA)  log  0

   
(13)
The chemical shift difference of the two triplet resonances () was measured
by placing the left cursor in the middle of the downfield triplet and the right cursor in
the middle of the upfield triplet. The difference was obtained in Hertz, which was
then converted to parts per million by dividing by 500, the proton frequency of the
NMR spectrometer. The chemical shift difference was used to calculate the pH of the
sample as shown in Equation 13.
The concentration of ammonia base [ B ] present in the sample can then be
calculated:
[ NH 3 ]total  10  pKa
[ B] 
10  pH  10  pKa
36
(14)
Figure 2.5.1.
Selected region of the NMR spectrum of the RNA-DNA hybrid
investigated showing the two triplet resonances of TEOA (indicated with arrows) that
were used to measure the pH.
37
2.6. Model for Imino Proton Exchange in Nucleic Acids
The imino protons in the canonical A-T/U and G-C base pairs are shown in
Figure 1.1.2. The exchange of these protons with solvent protons occurs through the
transition between “open” and “closed” states of the base pairs. In the closed state,
imino protons are hydrogen bonded and do not have access to proton acceptors in the
solvent. In the open state, imino protons are free to exchange. The observable
exchange rate is (Russu, 2004; Gueron, 1995):
k ex 
k op  k ex,open
k cl  k ex,open
(15)
where k op is the rate of opening of the base pair, k cl is the rate of closing, and k ex,open
is the rate of exchange in the open state.
The rate of exchange in the open state depends on the concentration of proton
acceptor B:
k ex ,open  k B [ B ]
(16)
where k B is the rate constant for the transfer of the imino proton to proton acceptor B
in isolated nucleotides and  is a factor that accounts for differences in proton
transfer rate between isolated nucleotides and open base pairs. For example, when the
proton acceptor is ammonia base (like in the present work), k B  4.082 10 8 M 1 s 1
for thymine and k B  8.797  10 8 M 1 s 1 for guanine and uracil at 10 C, and
k B  3.751 108 M 1 s 1 for thymine, k B  7.186  108 M 1 s 1 for guanine and
k B  7.513  108 M 1 s 1 for uracil at 1 C. α is assumed to be 1.
38
The final equation for the exchange rate is obtained by substituting Equation
16 into Equation 15:
k ex 
k op  k B  [ B]
(17)
k cl  k B  [ B]
Two kinetic regimes for imino proton exchange are distinguished depending
on how the rate of exchange from the open state compares with the rate of closing.
EX1 Regime: When the concentration of proton acceptor is high enough to make the
exchange from the open state very fast ( k ex ,open  k cl ), the exchange is limited by the
rate of base opening. Equation 17 becomes:
k ex  k op
(18)
EX2 Regime: When the concentration of proton acceptor is low, the rate of exchange
from the open state is much smaller than the rate of closing ( k ex ,open  k cl ). The
observed exchange rate depends linearly on the concentration of proton acceptor:
k ex  K op  k ex , open  K op  k B  [ B ]
(19)
where the equilibrium constant of the opening reaction is defined as:
K op 
k op
(20)
k cl
The free energy change for the opening reaction is:
G op   RT ln K op
39
(21)
The enthalpy ( H  op ) and entropy ( S  op ) changes for the base-pair opening
reaction are also calculated:
H  op   R 
S  op  R 
ln K op ,1  ln K op , 2
1 1
  
 T1 T2 
T1 ln K op ,1  T2 ln K op , 2
T1  T2
(22)
(23)
where T1 and T2 refer to the absolute temperature equivalents for 10 C and 1 C,
respectively.
40
Chapter 3. Results
41
3.1. Assignments of Imino Proton Resonances
The base sequence and imino proton resonances of the rA-dT hybrid are
shown in Figure 3.1.1. Three different NMR experimental information were used to
assign the imino proton resonances: temperature dependent 1D 1H NMR spectra, 1D
15
N-edited fHSQC spectra of position-specific labeled hybrid samples, and 2D proton
NOESY.
The 1D imino proton spectra taken at various temperatures (1 to 40 C) are
shown in Figure 3.1.2. At 10 C, twelve imino proton resonances are observed,
ranging from 12.25 to 13.86 ppm. Two resonances from terminal bases are likely too
broad to be observed due to fraying at the ends of the hybrid duplex (positions 1 and
14). Of the twelve observed resonances, resonances at 12.73 ppm and 13.28 ppm
broaden first upon increasing temperature. Therefore, these resonances are likely to
originate from the base pairs near the ends of the hybrid duplex, at positions 2 or 13.
For the 15N-edited fHSQC experiment, two hybrid samples were prepared: the
first hybrid contains
15
N-labeled thymines and uracil at positions 6 and 7, and the
second hybrid contains 15N-labeled thymines at positions 6, 7, and 10. The spectra are
shown in Figures 3.1.3. and 3.1.4., respectively. Comparison of the spectra shows that
the imino proton of (dA-rU)10 resonates at 13.08 ppm. The two other imino proton
resonances at 13.77 ppm and 13.67 ppm are the thymines, (dT-rA)6 and (dT-rA)7, but
this experiment alone is unable to distinguish the two resonances from each other.
Selected regions of the NOESY spectrum are shown in Figure 3.1.5. Two
types of connectivities are used to assign the imino proton resonances: imino protons
42
to imino protons and imino protons to adenine-H2 protons. The adenine-H2 protons,
which are found in the range of 6.2 to 7.7 ppm, are close in space to the paired or
neighboring thymine or uracil imino protons. The close proximity between adenineH2 protons and uracil/thymine imino protons give rise to strong NOE connectivities.
The NOESY spectrum shows seven connectivities between adenine-H2 protons and
imino protons: 6.25 ppm and 12.96 ppm, 7.08 ppm and 13.65 ppm, 7.09 ppm and
13.08 ppm, 7.12 ppm and 13.67 ppm, 7.18 ppm and 13.77 ppm, 7.64 ppm and 13.61
ppm, and 7.52 ppm and 13.86 ppm. These corresponding imino proton resonances
must belong to A-T or A-U base pairs. The five other imino proton resonances at
12.25 ppm, 12.73 ppm, 12.83 ppm, 13.05 ppm, and 13.28 ppm must correspond to GC base pairs.
Imino proton to imino proton NOESY connectivities are observed between
successive base pairs. The connectivities are outlined in Table 3.1.1. Two NOE
crosspeaks are observed that correlate thymine imino protons to guanine imino
protons. One connectivity is that between the G-C resonance at 12.25 ppm and the AT resonance at 13.61 ppm, and the other is that between the G-C resonance at 12.83
ppm and the A-T resonance at 13.86 ppm. According to the base sequence of the
hybrid, the A-T resonances must be (dT-rA)5 and (dT-rA)11 and the G-C resonances
must be (dC-rG)4 and (dC-rG)12. Since the resonance at 13.08 ppm is assigned to (dArU)10 by the
15
N-edited fHSQC experiments, the connectivity observed between the
resonance at 13.08 ppm and the resonance at 13.61 ppm assigns 13.61 ppm to (dTrA)11. The G-C resonance at 12.25 ppm is assigned to (dG-rC)12 because, as
mentioned above, it is connected to (dT-rA)11 at 13.61 ppm. The resonance at 13.86
43
ppm is assigned to (dT-rA)5 and the resonance at 12.83 ppm is assigned to (dC-rG)4
by default. The connectivity observed between (dC-rG)4 (12.83 ppm) and the
resonance at 13.05 ppm assigns the resonance to (dC-rG)3. (dC-rG)3 is also connected
to the resonance at 13.28 ppm, which is assigned to (dC-rG)2. The connectivity
between the adenine-H2 proton of (dT-rA)5 at 7.52 ppm and the resonance at 13.77
ppm assigns the resonance to (dT-rA)6. The remaining resonance at 13.67 ppm in the
15
N-edited fHSQC spectrum can be assigned to (dT-rA)7 by default. A strong NOE is
observed between the resonance at 12.96 ppm and the resonance at 13.65 ppm. These
resonances must be (dT-rA)8 and (dT-rA)9, though more information is needed to
distinguish the two from each other. By default, the resonance at 12.73 ppm is
assigned to (dG-rC)13. The imino proton assignments are summarized in Table 3.1.2.
44
Figure 3.1.1. (a) Base sequence and (b) imino proton resonances of rA-dT hybrid
investigated at 10 C.
45
Figure 3.1.2. Temperature dependent 1D 1H NMR spectra of rA-dT hybrid. The
broadened resonances are indicated by dashed lines.
46
Figure 3.1.3.
15
N/14N-editing of NMR resonances at 10 C of imino protons for a
sample of rA-dT hybrid in which thymines in positions 6 and 7 are labeled with 15N.
(a)
15
N-edited imino proton resonances. (b)
14
N-edited imino proton resonances. (c)
All imino proton resonances on the unlabeled sample.
47
Figure 3.1.4.
15
N/14N-editing of NMR resonances at 10 C of imino protons for a
sample of rA-dT hybrid in which uracil in position 10 and thymines in positions 6 and
7 are labeled with 15N. (a) 15N-edited imino proton resonances. (b) 14N-edited imino
proton resonances. (c) All imino proton resonances on the unlabeled sample.
48
A9/8-H2
A6-H2
A7-H2,A 10-H2
A8/9
-H2
A11-H2
A5-H2
(dC-rG)12
(dT-rA)
(dC-rG)103
(dT-rA)9/8
(dC-rG)4
(dG-rC)13
(dG-rC)2
(dT-rA)5
(dA-rU)6
(dT-rA)7
(dT-rA)8/9
(dT-rA)11
F1
(ppm)
12.4
12.6
12.8
13.0
13.2
13.4
13.6
13.8
14.0
14.0
13.8
13.6
13.4
13.2
13.0
12.8
12.6
12.4
12.2
F2 (ppm)
8.6
8.4
8.2
8.0
7.8
7.6
7.4
7.2
7.0
6.8
6.6
6.4
6.2
Figure 3.1.5. Expanded regions of the 1H-1H NOESY spectrum at 10 C of the rA-
dT hybrid showing two types of connectivities: (1) adenine-H2 proton to imino
proton (dashed lines) and (2) imino proton to imino proton (full lines).
49
6.0
Table 3.1.1. Imino Proton Connectivities of rA-dT Hybrid
Chemical
Shift
(ppm)
13.86
13.86
---
13.77
13.77
13.67
13.65
13.61
13.05
12.96
X
12.83
12.73
12.25
X
---
13.65
---
13.61
X
---
13.28
X
---
X
X
12.96
X
---
13.05
12.83
13.08
---
13.67
13.08
13.28
---
X
X
---
X
X
12.73
-----
12.25
X
---
Table 3.1.2. Chemical Shifts of Imino Proton Resonances of rA-dT Hybrid at 10 C
Base-Pair
(dG-rC)2
(dC-rG)3
(dC-rG)4
(dT-rA)5
(dT-rA)6
(dT-rA)7
(dT-rA)8/9
(dT-rA)9/8
(dA-rU)10
(dT-rA)11
(dC-rG)12
(dG-rC)13
Chemical Shift for
Imino Proton
Resonance (ppm)
13.28
13.05
12.83
13.86
13.77
13.67
13.65
12.96
13.08
13.61
12.25
12.73
50
3.2. Imino Proton Exchange
The energetics and dynamics of the base pairs in the rA-dT hybrid are
determined by the exchange rates of the imino protons. The imino proton exchange
rates have been measured at 1 C at various concentrations of ammonia (0 to 76 mM)
and at 10 C at various concentrations of ammonia (0 to 78 mM). Figure 3.2.1. shows
the imino proton resonances at 10 C at selected ammonia concentrations. No
significant change in chemical shift is observed for imino proton resonances
indicating that the hybrid structure does not change upon increasing ammonia
concentration.
As discussed in the preceding section, at 10 C there are twelve imino proton
resonances clearly observed in the NMR spectrum since the two base pairs at the 5’
and 3’ ends of the hybrid are affected by fraying. These two resonances from terminal
bases, (dG-rC)1 and (dC-rG)14, are resolved at 13.28 ppm and 12.98 ppm at 1 C as
shown in Figure 3.1.2. As the amounts of ammonia concentrations are increased,
these two resonances are broadened enough such that they do not affect the intensities
of the other resonances, (dG-rC)2 and (dT-rA)8/9. However, at zero and low
concentrations of ammonia, the two resonances have some intensity and may affect
the intensities and exchange rates of (dG-rC)2 and (dT-rA)8/9. The fitting of exchange
rate against ammonia concentration for (dT-rA)8/9 at 1 C is not reported because of
large errors, possibly due to this overlapping.
There are two regions on the NMR spectrum where overlapping of imino
proton resonances is observed: (dT-rA)7 at 13.67 ppm with (dT-rA)8/9 at 13.65 ppm
51
and (dA-rU)10 at 13.08 ppm with (dC-rG)3 at 13.05 ppm. For these resonances, the
intensities and exchange rates are measured as averages of the two contributing
resonances.
Due to fast exchange, the imino proton resonance of (dG-rC)2 broadens
quickly with increasing ammonia. The intensity for this resonance is very close to the
baseline for many of the measurements, which produces large errors in calculations.
Therefore, the fitting of exchange rate as a function of ammonia concentration is not
reported.
The imino proton exchange rates are plotted against various ammonia base
concentrations in Figures 3.2.2. (10 C) and 3.2.3. (1 C). For 10 C, (dT-rA)5, (dTrA)6, (dT-rA)7,8/9, (dT-rA)11, and (dG-rC)13 are fitted according to the EX1 regime
(Equation 18), and (dC-rG)3/(dA-rU)10, (dC-rG)4, (dT-rA)8/9, and (dC-rG)12 are fitted
according to the EX2 regime (Equation 19). For 1 C, (dC-rG)3/(dA-rU)10, (dT-rA)5,
(dT-rA)11, and (dG-rC)13 are fitted according to the EX1 regime (Equation 18), and
(dC-rG)4, (dT-rA)6, (dT-rA)7,8/9 and (dC-rG)12 are fitted according to the EX2 regime
(Equation 19). The kop and kcl values are calculated for base pairs that are fitted to
EX1 regimes, and the Kop and Gop are calculated for base pairs fitted with both EX1
and EX2 regimes (Tables 3.2.1. and 3.2.2.). The enthalpy ( H  op ) and entropy
( S  op ) changes for the opening reaction are also estimated based on Equations 22
and 23 for each base pair for which the equilibrium constant could be calculated. The
values are listed in Table 3.2.3.
52
Figure 3.2.1.
Imino proton resonances of rA-dT hybrid at various ammonia
concentrations and at 10 C.
53
Figure 3.2.2.
Dependence of imino proton exchange rates on ammonia base
concentration in the rA-dT hybrid at 10 C.
54
Figure 3.2.3.
Dependence of imino proton exchange rates on ammonia base
concentration in the rA-dT hybrid at 1 C.
55
Table 3.2.1. Base-Pair Opening Parameters in the rA-dT RNA-DNA Hybrid at 10 C
a
b
Base Pair
kop (s-1)
kcl ( x 10-6, s-1)
Kop ( x 106)
(rC-dG)2
a
a
a
Gop
(kcal/mol)
a
(rG-dC)3
b
b
b
b
(rG-dC)4
d
d
0.0005 ± 0.0011
12.02 ± 1.36
(rA-dT)5
33 ± 14
44 ± 23
0.8 ± 0.5
7.90 ± 0.38
(rA-dT)6
12 ± 3
58 ± 17
0.20 ± 0.08
8.62 ± 0.20
(rA-dT)7
b
b
b
b
(rA-dT)8/9
d
d
2.0 ± 0.1
7.36 ± 0.20
(rU-dA)10
b
b
b
b
(rA-dT)11
(rG-dC)12
(rC-dG)13
18 ± 4
d
d
10 ± 3
d
d
2 ± 0.6
0.020 ± 0.003
1.00 ± 0.03
7.38 ± 0.17
9.94 ± 0.10
7.75 ± 0.02
The data were not fitted due to large errors in intensity measurements.
Opening parameters could not be determined because the imino proton resonance
overlaps other resonances.
c
No definitive assignment for imino proton resonances of (rA-dT)8 and (rA-dT)9 (see
Section 3.1.).
d
The EX1 regime could not be reached at the highest ammonia concentrations
investigated in this work.
56
Table 3.2.2. Base-Pair Opening Parameters in the rA-dT RNA-DNA Hybrid at 1 C
a
b
Base Pair
kop (s-1)
kcl ( x 10-6, s-1)
Kop ( x 106)
Gop (kcal/mol)
(rC-dG)2
a
a
a
a
(rG-dC)3
b
b
b
b
(rG-dC)4
d
d
0.030 ± 0.003
9.48 ± 0.08
(rA-dT)5
8±2
20 ± 5
0.4 ± 0.3
7.98 ± 0.41
(rA-dT)6
d
d
0.100 ± 0.005
8.54 ± 0.02
(rA-dT)7
b
b
b
b
(rA-dT)8/9
a, b, c
a, b, c
a, b, c
a, b, c
(rU-dA)10
b
b
b
b
(rA-dT)11
(rG-dC)12
(rC-dG)13
11 ± 3
d
6.0 ± 0.6
14 ± 6
d
11 ± 3
0.8 ± 0.5
0.010 ± 0.002
0.5 ± 0.2
7.61 ± 0.35
9.79 ± 0.08
7.83 ± 0.24
The data were not fitted due to large errors in intensity measurements.
Opening parameters could not be determined because the imino proton resonance
overlaps other resonances.
c
No definitive assignment for imino proton resonances of (rA-dT)8 and (rA-dT)9 (see
Section 3.1.).
d
The EX1 regime could not be reached at the highest ammonia concentrations
investigated in this work.
57
Table 3.2.3. Estimation of Enthalpy and Entropy Changes for BasePair Opening in the rA-dT RNA-DNA Hybrid at 1 C and 10 C
a
Base Pair
Hop (kcal/mol)
Sop (cal/mol)
(rC-dG)2
a
a
(rG-dC)3
a
a
(rG-dC)4
b
b
(rA-dT)5
11.8
41.3
(rA-dT)6
11.8
38.6
(rA-dT)7
a
a
(rA-dT)8/9
a
a
(rU-dA)10
a
a
(rA-dT)11
15.6
56.6
(rG-dC)12
11.8
34.0
(rC-dG)13
11.8
41.8
The enthalpy and entropy changes were not estimated for these base pairs because
the associated equilibrium constants of opening reactions were not calculated at both
temperatures.
b
The enthalpy and entropy changes were not estimated for this base pair because the
errors in the equilibrium constants of the opening reactions are very large (Tables
3.2.1. and 3.2.2.).
58
Chapter 4. Discussion
59
Circular dichroism and optical melting experiments have previously been
done (Martin and Tinoco, 1980) on oligomers of rA-dT hybrid, rU-dA hybrid, dA-dT
duplex, and rA-rU duplex. The results of the circular dichroism experiments find that
the dA-dT duplex is in the B-form and the rA-rU duplex is in the A-form. Both
hybrid oligomers have structures similar to the A-form but are unstable relative to the
duplexes because the DNA strands in the hybrids are unstable in the A-form. The
optical melting experiments reveal the order of stability for these oligomers from
most unstable to most stable: rU-dA hybrid (melting temperature Tm < 0 C), rA-rU
duplex (Tm = 19.1 C), rA-dT hybrid (Tm = 22.9 C), dA-dT duplex (Tm = 27.5 C).
Analogous molecules, rU-dA hybrid (Section 4.3.), dA-dT duplex (Section 4.3.), and
rA-dT hybrid (current investigation, Section 4.1.), have been studied in our
laboratory. The imino proton exchange method allows for the investigation of the
relative stabilities of these molecules at each base pair.
4.1. Analysis of Base-Pair Energetics in the rA-dT Hybrid
Imino proton exchange is used to measure the stabilities of the base pairs on
the rA-dT hybrid. The stabilities are evaluated from the opening equilibrium constant
( K op , Equation 20) and the free energy change for the opening reaction ( Gop ,
Equation 21). The rate of opening ( k op ) and rate of closing ( k cl ) are also calculated
for the resonances which reach the EX1 regime. More stable base pairs are indicated
by smaller values of K op and k op and larger values of Gop and k cl .
60
The K op values for the base pairs in the rA-dT hybrid at 10 C range from
0.0005 x 10-6 for (rG-dC)4 to 2.0 x 10-6 for both (rA-dT)8/9 and (rA-dT)11. In general,
the G-C base pairs are observed to be more stable than the A-T base pairs. The K op
values for A-T base pairs range from 0.2 x 10-6 to 2.0 x 10-6. The K op values are
0.0005 x 10-6 for (rG-dC)4, 0.02 x 10-6 for (rG-dC)12, and 1.0 x 10-6 for (rC-dG)13.
Although this last equilibrium constant for (rC-dG)13 is higher than the rest, the base
pair is close to the end of the hybrid which may account for its higher opening
equilibrium constant. The same is true for (rC-dG)2, the K op and Gop of which
cannot be calculated due to large errors in the intensity measurements. The only A-U
base pair, (dA-rU)10, overlaps with (dC-rG)3 and cannot be compared to the rest of the
base pairs.
A-T base pairs have been shown to be more unstable than G-C base pairs in
other nucleic acid structures. The base-pair stabilities of a 10-mer DNA duplex (5’CCAACGTTGG-3’/3’-GGTTGCAACC-5’) have previously been studied in our
laboratory (Every, 2009). The K op values for the dA-dT base pairs in this structure at
15 C are 117.1 x 10-6 and 38.6 x 10-6, both of which are much higher than the K op
for the dG-dC base pair, 0.30 x 10-6. The differences in stability of the base pairs may
be attributed to the number of hydrogen bonds associated with the canonical WatsonCrick structures. An adenine-thymine base pair has two hydrogen bonds between the
bases, whereas a guanine-cytosine base pair has three hydrogen bonds (Figure 1.1.2.).
In the present work, we find that the stability of rA-dT base pairs depends on
their location in the base sequence. The rA-dT base pair in the 11th position, (rA-
61
dT)11, is in a mixed base-pair sequence, since it is next to a rU-dA base pair and to a
rG-dC base pair. This rA-dT base pair has a K op value of 2.0 x 10-6 at 10 C. The rAdT base pair in the 6th position, (rA-dT)6, is next to two other rA-dT base pairs and
has a K op value of 0.2 x 10-6, smaller than that of (rA-dT)11 by ten-fold. Comparison
of these data suggests that the rU-dA base pair may have a destabilizing effect on its
neighboring base pair or that a tract of rA-dT base pairs has a stabilizing effect. On
the other side of the rU-dA base pair is the rA-dT base pair in the 9th position.
Although the assignment for the (rA-dT)8/9 resonance is not definitive, if this
resonance corresponds to the rA-dT base pair in the 9th position, the rU-dA base pair
also affects this base pair, since its K op is close to that of (rA-dT)11. In addition, the
(rA-dT)5 base pair, which is next to a rG-dC base pair and a rA-dT base pair, has a
K op value of 0.8 x 10-6, which is greater than that of the central (rA-dT)6, but still
smaller than that of (rA-dT)11. This suggests that the (rA-dT)11 in the mixed base
sequence is likely destabilized by the presence of the rU-dA rather than the rG-dC.
62
4.2. Comparison of the rA-dT Hybrid with Two Homologous GC-rich RNADNA Hybrids
The stability of base pairs in two GC-rich RNA-DNA hybrids corresponding
to the hybrids formed at the transcription termination site tR2 of phage  have
previously been studied in our laboratory (Huang et al., 2009) at 10 C. The base
sequences of the two hybrids are shown in Figure 4.2.1. and the base-pair opening
parameters are shown in Tables 4.2.1. and 4.2.2. The rA-dT hybrid currently
investigated can be compared to these two other hybrids to determine the stability of
base pairs in two different sequence contexts: (i) in a GC-rich sequence and (ii) in a
rA-dT tract.
Like for the rA-dT hybrid, the K op values for the A-U/A-T base pairs are
higher than those of the G-C base pairs in the GC-rich hybrids. The K op values for
the central G-C base pairs (positions 7 to 10) of the GC-rich hybrids range from 0.030
x 10-6 to 1.0 x 10-6. The K op values for the central G-C base pairs (positions 4 and
12) in the rA-dT hybrid are 0.0005 x 10-6 and 0.02 x 10-6. The G-C base pairs of the
rA-dT hybrid are slightly more stable than those in the GC-rich hybrids. A large
difference is observed between the A-U/A-T base pairs in the GC-hybrids ( K op range
from 10.5 x 10-6 to 270 x 10-6) and the A-T base pairs in the rA-dT hybrid ( K op range
from 0.2 x 10-6 to 2.0 x 10-6). These data suggest that the A-T base pairs are more
stabilized in the structural context of an A-T tract than in a GC-rich sequence.
63
For the two GC-rich hybrids, Huang et al. found that neighboring A-U base
pairs affect the stabilities of the guanines located on the DNA strand. This is similar
to the effect the A-U base pair has on its neighboring A-T base pairs in the rA-dT
hybrid mentioned above. Even in different structural contexts, the destabilizing role
of the A-U base pair is not changed. Also, the proximity of A-T base pairs, (rA-dT)5
and (rA-dT)11, in the rA-dT hybrid is not shown to destabilize rG-dC base pairs, (rGdC)4 and (rG-dC)12. The two G-C base pairs next to A-T base pairs, the guanines of
which are on the RNA strand, have the highest stabilities. It is not clear whether this
is due to the presence of A-T instead of A-U or to the location of the guanines. There
are no A-T base pairs in proximity to G-C base pairs where the guanine is on the
DNA strand, and so a comparison cannot be made.
64
Figure 4.2.1. Base sequences of GC-rich RNA-DNA hybrids.
65
Table 4.2.1. Base-Pair Opening Parameters in the GC-rich RNA-DNA
Hybrid I at 10 C (Huang et al., 2009)
Base pair
(dG-rC)2
(dC-rG)3
(dG-rC)4
(dC-rG)5
(dA-rU)6
(dG-rC)7
(dG-rC)8
(dC-rG)9
(dC-rG)10
(dT-rA)11
(dT-rA)12
(dC-rG)13
kop (s-1)
c
48 ± 2
4.3 ± 0.3
5.1 ± 1.4
a
2.7 ± 0.1
c
1.7 ± 0.1
1.5 ± 0.1
a
a
38.3 ± 0.8
kcl ( x 10-7 s-1)
c
4.8 ± 0.3
1.8 ± 0.2
8.1 ± 2.7
a
0.9 ± 0.1
c
1 ± 0.1
0.9 ± 0.1
a
a
0.37 ± 0.03
Kop ( x 106)
c
1.0 ± 0.1
0.24 ± 0.03
0.06 ± 0.02
26.4 ± 0.3
0.32 ± 0.03
c
0.16 ± 0.02
0.18 ± 0.02
10.5 ± 0.1
21.1 ± 0.2
10.4 ± 0.8
Gop (kcal/mol)
c
7.77 ± 0.05
8.59 ± 0.07
9.33 ± 0.22
5.93 ± 0.01
8.42 ± 0.05
c
8.79 ± 0.08
8.74 ± 0.06
6.45 ± 0.01
6.06 ± 0.01
6.46 ± 0.04
a
The exchange rates in the EX1 regime are too fast to be measured by NMR.
b
The EX1 regime could not be reached at the highest ammonia concentrations.
c
Imino proton resonance is not resolved in the NMR spectrum.
Table 4.2.2. Base-Pair Opening Parameters in the GC-rich RNA-DNA
Hybrid II at 10 C (Huang et al., 2009)
Base pair
(dG-rC)2
(dC-rG)3
(dG-rC)4
(dC-rG)5
(dA-rU)6
(dG-rC)7
(dG-rC)8
(dC-rG)9
(dC-rG)10
(dT-rA)11
(dT-rA)12
(dC-rG)13
kop (s-1)
33.2 ± 1.4
6.6 ± 0.7
18.8 ± 3.9
2.6 ± 0.2
a
b
b
b
9.9 ± 1.8
a
a
a
kcl ( x 10-7 s-1)
0.65 ± 0.08
2.4 ± 0.5
10 ± 3
2.0 ± 0.5
a
b
b
b
3.5 ± 1.2
a
a
a
Kop ( x 106)
5.1 ± 0.6
0.27 ± 0.06
0.19 ± 0.06
0.13 ± 0.03
21.8 ± 0.4
0.030 ± 0.002
0.035 ± 0.002
0.085 ± 0.003
0.3 ± 0.1
51 ± 2
270 ± 3
221 ± 13
Gop (kcal/mol)
6.85 ± 0.07
8.50 ± 0.13
8.71 ± 0.19
8.92 ± 0.14
6.04 ± 0.01
9.75 ± 0.03
9.65 ± 0.03
9.16 ± 0.02
8.49 ± 0.22
5.56 ± 0.02
4.62 ± 0.01
4.74 ± 0.03
a
The exchange rates in the EX1 regime are too fast to be measured by NMR.
b
The EX1 regime could not be reached at the highest ammonia concentrations.
c
Imino proton resonance is not resolved in the NMR spectrum.
66
4.3. Comparison of rA-dT Hybrid with Its Homologous rU-dA Hybrid and dAdT DNA Duplex
The base sequences of the rU-dA hybrid and dT-dA duplex is shown in Figure
4.3.1. and the base-pair opening data are shown in Tables 4.3.1. and 4.3.2. The rU-dA
hybrid contains the same base sequence as the rA-dT hybrid except that the DNA and
RNA strands are interchanged and thymines are replaced with uracils. The dA-dT
duplex contains the same base sequence as the rA-dT hybrid except that both strands
are DNA and the uracil is replaced with a thymine. The two molecules have been
studied in our laboratory (Huang et al., 2010) at 10 C. The NMR results on these two
molecules can be compared to those in the rA-dT hybrid investigated in the present
work to reveal how the energetics of the same base sequence varies in different
structural contexts. Overall, the relative stabilities of the three structures from the
NMR experiments are consistent with previous melting experiments (Martin and
Tinoco, 1980).
The rU-dA hybrid contains a contiguous sequence of rU-dA base pairs from
positions 5 to 9 and a single rU-dA base pair at position 11 in a mixed sequence. The
K op values for the contiguous A-U base pairs, ranging from 79 x 10-6 to 497 x 10-6 at
10 C, are much higher than the value for the lone A-U base pair, 9.8 x 10-6. This
shows that the contiguous A-U base pairs are much less stable than the A-U base pair
in a mixed sequence (Table 4.3.1.). Since this A-U tract is present at the tR2
terminator site of phage , the data suggest that the A-U tract plays a role in the
separation of the DNA and RNA strand during intrinsic transcription termination. The
67
instability of the RNA-DNA hybrid at this site allows for the release of the RNA
transcript from the DNA template. The rA-dT hybrid similarly contains a contiguous
sequence of rA-dT base pairs and a lone rA-dT base pair in a mixed sequence.
However, the lone rA-dT base pair is observed to have more or equal stability than
the contiguous rA-dT base pairs. This shows that it is the presence of uracils instead
of thymines that greatly affects the stability of the hybrid.
The dA-dT duplex contains a contiguous sequence of dA-dT base pairs from
positions 5 to 9, a dT-dA base pair in position 10, and another dA-dT base pair in
position 11 in a mixed base sequence. The K op values for the contiguous dA-dT base
pairs range from 0.1 x 10-6 to 1.0 x 10-6. These values are slightly smaller than the
values for the rA-dT base pairs in the rA-dT hybrid, indicating that the dA-dT base
pairs in the DNA duplex are more stable than the rA-dT base pairs in the hybrid. The
nature of the strands plays a role in the stability of each base pair. Furthermore, the
K op value of 1.9 x 10-6 for the lone dA-dT base pair in the mixed sequence is greater
than the values for the contiguous dA-dT base pairs, which shows that a tract of dAdT base pairs is much more stable than an isolated dA-dT base pair.
The K op for the (dA-dT)11 base pair in the mixed sequence is also close to
that of the analogous mixed sequence (rA-dT)11 base pair in the rA-dT hybrid (2.0 x
10-6). Both of these mixed sequence A-T base pairs are more stable than the mixed
sequence (rU-dA)11 base pair in the rU-dA hybrid. This further shows that the
destabilization of a nucleic acid complex depends on the presence of uracil, even in a
mixed base sequence context.
68
Figure 4.3.1. Base sequences of rU-dA RNA-DNA hybrid, dA-dT DNA duplex, and
rA-dT RNA-DNA hybrid.
69
Table 4.3.1. Equilibrium Constants and Free-Energy Changes for Base-Pair
Opening in the rU-dA RNA-DNA Hybrid at 10 C (Huang et al., 2010)
Base pair
(rU-dA)5
(rU-dA)6
(rU-dA)7
(rU-dA)8
(rU-dA)9
(rA-dT)10
(rU-dA)11
a
Kop ( x 106)
79 ± 2
462 ± 14
497 ± 19
228 ± 7
228 ± 5
a
9.8 ± 0.4
Gop (kcal/mol)
5.30 ± 0.01
4.31 ± 0.02
4.27 ± 0.02
4.70 ± 0.02
4.70 ± 0.01
a
6.47 ± 0.02
Imino proton resonance is not resolved in the NMR spectrum.
Table 4.3.2. Equilibrium Constants and Free-Energy Changes for Base-Pair
Opening in the dT-dA DNA Duplex at 10 C (Huang et al., 2010)
Base pair
(dT-dA)5
(dT-dA)6
(dT-dA)7
(dT-dA)8
(dT-dA)9
(dA-dT)10
(dT-dA)11
Kop ( x 106)
0.5 ± 0.2
0.3 ± 0.1
0.1 ± 0.1
0.3 ± 0.1
1.0 ± 0.2
1.4 ± 0.2
1.9 ± 0.3
70
Gop (kcal/mol)
8.1 ± 0.2
8.4 ± 0.2
9.0 ± 0.6
8.4 ± 0.2
7.7 ± 0.1
7.6 ± 0.1
7.4 ± 0.1
4.4. Dependence of Base-Pair Openings on Temperature
The enthalpy ( H  op ) and entropy ( S  op ) changes for the base pairs in the
rA-dT RNA-DNA hybrid are estimated from the equilibrium constants of the opening
reaction at 1 C and 10 C using van ‘t Hoff equation (Equations 22 and 23 in Section
2.6.). The values, which are summarized in Table 3.2.3., are calculated for the base
pairs for which the equilibrium constants of the opening reaction have been
calculated. (rG-dC)4 was not included in this estimation because the errors in the
equilibrium constants of the opening reactions are very large. The enthalpy changes
range from 11.8 to 15.6 kcal/mol and the entropy changes range from 34.0 to 56.6
cal/mol.
Enthalpy changes have been reported previously in our laboratory (Coman
and Russu, 2005) for two DNA duplexes found in the origin of replication of bacterial
chromosomes: (I) 5’-GCGATCTATTTATTTGC-3’/3’-CGCTAGATAAATAAACG5’ and (II) 5’-GCGATCTGTTCTATTGC-3’/3’-CGCTAGACAAGATAACG-5’. The
values are summarized in Table 4.4.1. The enthalpy changes range from 10 to 26
kcal/mol. The study found that the enthalpy changes for base pairs are influenced by
the surrounding base pairs. For example, the G-C base pairs in position 3 for both
DNA duplexes, which are both surrounded by other G-C base pairs, are about 25
kcal/mol. Conversely, the G-C base pairs in position 6 in duplex I and position 11 in
duplex II, which are both surrounded by A-T base pairs, are both about 12 kcal/mol.
Analogous values for the rA-dT hybrid are only seen for an A-T base pair (position
11) next to an A-U base pair (position 10). Most of the base pairs in the hybrid have
71
enthalpy changes of about 11.8 kcal/mol, including an A-T base pair surrounded by a
G-C and an A-T, an A-T base pair surrounded by other A-T base pairs, a G-C base
pair surrounded by a G-C base pair and an A-T base pair, and a G-C base pair
surrounded by other G-C base pairs. The exception is (rA-dT)11 (15.6 kcal/mol),
which is next to the A-U base pair. This finding suggests that the effect of a
neighboring A-U base pair is much greater than those of A-T or G-C in the hybrid.
A similar study has also been done on a DNA duplex containing the same
sequence of the TATA box in the adenovirus major late promoter: 5‘GCTATAAAAGGG-3’/3’-CGATATTTTCCC-5’ (Chen and Russu, 2004). The
enthalpy changes are shown in Table 4.4.2. The values range from 17 to 29 kcal/mol.
The A-T base pairs in the TATA sequence were found to have lower enthalpy
changes than the A-T base pairs in the A-T tract, indicating that the opening of base
pairs depends greatly on the location and base sequence context. This is demonstrated
as well for the rA-dT hybrid. The hybrid A-T base pairs in the A-T tract have the
same enthalpy changes (11.8 kcal/mol), whereas the A-T base pair in the mixed
sequence context has a different, higher enthalpy change (15.6 kcal/mol).
72
Table 4.4.1. Enthalpy changes for base-pair opening in two DNA duplexes
(Coman and Russu, 2005)
Position
3
4
5
6
7
8
9
10
11
12
13
14
15
Duplex I
Base Pair Hop (kcal/mol)
GC
25 ± 2
AT
19.3 ± 0.3
CG
12 ± 1
AT
22.9 ± 0.7
TA
TA
AT
24.2 ± 0.9
19.2 ± 0.2
22.9 ± 0.8
TA
TA
24 ± 1
24.0 ± 0.1
Duplex II
Base Pair Hop (kcal/mol)
GC
26 ± 2
TA
TA
CG
16.7 ± 0.4
14.0 ± 0.8
10 ± 2
TA
18 ± 4
Table 4.4.2. Enthalpy changes for base-pair openings in a DNA
duplex containing a TATA box sequence (Chen and Russu, 2004)
Position
3
4
5
6
7
8
9
10
Base Pair
TA
AT
TA
AT
AT
AT
AT
GC
73
Hop (kcal/mol)
23 ± 1
17 ± 2
17.5 ± 0.9
21 ± 2
29 ± 2
28 ± 1
25 ± 2
17.6 ± 0.8
Conclusions and Future Work
Intrinsic transcription termination relies on the base sequence at the
termination site. The tR2 termination site contains a guanine/cytosine-rich inverted
repeat motif, which forms a hairpin loop structure in the transcribed RNA, and an
adenine-rich tract, which yields a uracil-rich sequence at the 3’ end of the RNA
transcript. It is believed that the presence of this adenine-uracil tract destabilizes the
RNA-DNA hybrid formed in the transcription complex.
The G/C-rich DNA-RNA hybrids, rU-dA RNA-DNA hybrid and dA-dT
DNA-DNA duplex involved in transcription termination have previously been studied
in our laboratory. This thesis characterizes the energetics and stability of the
homologous rA-dT RNA-DNA hybrid through analysis of base-pair opening and
proton exchange. We have found that G-C base pairs are more stable than A-T base
pairs, the stabilities of base pairs are dependent on their locations in the sequence, and
the A-U base pair contributes to destabilization of its neighboring base pairs.
Comparison with previous findings shows that A-U base pairs in the G/C-rich
hybrids similarly destabilize neighboring base pairs. The A-T base pairs in the rA-dT
hybrid are also found to be more stable than the A-T base pairs in the G/C-rich
hybrids. The A-T base pairs in the rA-dT hybrid have also been found to be more
unstable than the A-T base pairs in the dA-dT duplex, suggesting that the nature of
the nucleic acid strands affects structural stability. Comparison of the rA-dT hybrid
with the rU-dA hybrid reveals that it is the presence of a tract of uracils, not thymines,
that contributes to destabilization. The relative stabilities of the complexes are
74
consistent with previous melting experiments (Martin and Tinoco, 1980). The use of
proton exchange allowed us to analyze the stabilities at the base-pair level.
To obtain a more definitive analysis of the rA-dT hybrid, the exchange rates
for all base pairs, including those with overlapping NMR resonances, need to be
measured. This will be done by performing transfer of magnetization experiments
with a hybrid sample containing 15N-labeled atoms. Analysis of the analogous rA-rU
RNA-RNA duplex, along with the data for the G/C-rich DNA-RNA hybrids, dA-rU
DNA-RNA hybrid, dT-rA DNA-RNA hybrid, and dA-dT DNA-DNA duplex, will
provide a complete characterization of the transcription termination site.
75
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Cantor, C.R., M.M. Warshaw, and H. Shapiro. 1970. Oligonucleotide interactions. III.
Circular dichroism studies of the conformation of deoxyoligonucleotides.
Biopolymers 9(9):1059-1077.
Chen, C. 2007. Structural energetics and dynamics of nucleic acids and their
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