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From Department of Laboratory Medicine
Karolinska Institutet, Stockholm, Sweden
BIS-LOCKED NUCLEIC ACIDS: A NEW
TOOL FOR DOUBLE HELIX INVASION
Sylvain Geny
Stockholm 2015
All previously published papers were reproduced with permission from the publisher.
Published by Karolinska Institutet.
Printed by E-Print AB 2015
© Sylvain Geny, 2015
ISBN 978-91-7676-014-7
Bis-Locked Nucleic Acids: a new tool for double helix
invasion
THESIS FOR DOCTORAL DEGREE (Ph.D.)
By
Sylvain Geny
Principal Supervisor:
Prof C. I. Edvard Smith
Karolinska Institutet
Department of Laboratory Medicine
Clinical Research Center
Co-supervisor(s):
Dr Karin Lundin
Karolinska Institutet
Department of Laboratory Medicine
Clinical Research Center
Dr Samir EL Andaloussi
Karolinska Institutet
Department of Laboratory Medicine
Clinical Research Center
Opponent:
Prof Keith Fox
University of Southampton
Centre for Biological Sciences
Examination Board:
Prof Joakim Lundeberg
Kungliga Tekniska Högskolan
School of Biotechnology
Division of Gene Technology
Prof Matti Sällberg
Karolinska Institutet
Department of Laboratory Medicine
Division of Clinical Microbiology
Assoc Prof Björn Högberg
Karolinska Institutet
Department of Medical Biochemistry and
Biophysics
To my family and my friends.
ABSTRACT
Deoxyribonucleic acid (DNA) is a macromolecule that contains the information for all living
organisms to achieve important cellular processes such as growth and replication. DNA
consists of two strands coiled around each other to form a double helix. Several interactions
including Watson-Crick pairing and stacking between the bases stabilize the double helix. To
ensure the integrity of genetic information, the bases are on the inside the double helix
whereas the phosphate and sugar groups are on the outside. Thus, most DNA information is
not accessible until DNA is unwound prior to replication or transcription. In cells, enzymes
called helicases achieve DNA strands separation.
The anti-gene strategy aims to introduce a short single-stranded oligonucleotide (ON) to bind
genomic DNA at a specific site to block the mRNA transcription. As a result, mRNA and
protein expression for a specific gene are expected to be reduced, which could be beneficial
in certain clinical contexts. However, this therapeutic strategy is hampered by the low
stability of the DNA binding, the difficulty to reach the genomic target and to block the RNA
polymerase, suggesting the needs for new DNA binding ONs.
In this thesis, we developed clamp-ONs, bis-locked nucleic (bisLNAs), binding via
Hoogsteen and Watson-Crick interactions. In paper I, we assessed their binding
characteristics and demonstrated their ability to invade supercoiled DNA at intranuclear pH
and salt conditions. In paper II, we investigated the step-by-step mechanism for bisLNA
invasion. Based on this mechanism, we designed a new generation of bisLNAs using nonnatural modifications such as a stacking linker and 2´-glycylamino LNAs. In paper III, we
investigated how the bisLNAs invade in a more complex environment using rolling circle
amplification for detection.
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LIST OF SCIENTIFIC PAPERS
This thesis is based on the following articles
I. Moreno, P.M.D.*, Geny, S.*, Pabon Y.V., Bergquist H., Zaghloul E.M., Rocha C.S.,
Oprea II, Bestas B., Andaloussi S.E., Jorgensen P.T, Lundin K.E, Pedersen E.B,
Wengel J., Smith C.I.E. Development of bis-locked nucleic acid (bisLNA)
oligonucleotides for efficient invasion of supercoiled duplex DNA, Nucleic Acids
Res., 2013, 41, 5, 3257–3273
II. Geny S., Moreno P.M.D.*, Krzywkowski T.*, Andersen N.K., Isse A.J., ElMadani A.M., Lou C., Pabon Y.V., Gissberg O., Anderson B.A., Zaghloul A.M.,
Zain R., Hrdlicka P., Jorgensen P.T., Nilsson M., Lundin K.E., Pedersen E.B.,
Wengel J., Smith C.I.E. Next generation bis-locked nucleic acids with stacking
linker and 2´-glycylamino-LNA show enhanced invasion into duplex DNA
(manuscript submitted)
III. Geny S.*, Krzywkowski T.*, Gissberg O., Lundin K.E., Pedersen E.B., Wengel J.,
Nilsson M., Smith C.I.E. Rolling circle amplification assisted by bis-locked nucleic
acids invasion into supercoiled DNA (manuscript)
*Equal contribution
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CONTENTS
1 INTRODUCTION ........................................................................................................... 1
1.1 Gene therapy ......................................................................................................... 1
1.2 Anti-gene therapy .................................................................................................. 5
1.3 DNA structure ....................................................................................................... 8
1.4 DNA mode of binding......................................................................................... 12
1.4.1 Triple helix formation ............................................................................ 12
1.4.2 Double helix invasion............................................................................. 15
1.5 Oligonucleotide synthesis ................................................................................... 17
1.6 Nucleotide analogues .......................................................................................... 19
1.6.1 TFO analogues ....................................................................................... 19
1.6.2 Peptide nucleic acids .............................................................................. 20
1.6.3 Locked nucleic acids .............................................................................. 22
1.7 Intercalators ......................................................................................................... 24
2 AIMS ............................................................................................................................. 26
3 METHODOLOGY ........................................................................................................ 27
3.1 Fluorescence binding assay................................................................................. 27
3.2 Electrophoresis mobility shift assay ................................................................... 27
3.3 S1 nuclease assay ................................................................................................ 28
3.4 Melting assay....................................................................................................... 29
3.5 Footprinting ......................................................................................................... 30
3.6 Rolling circle amplification ................................................................................ 31
4 RESULTS, DISCUSSION AND PERSPECTIVES .................................................... 33
4.1 Paper I.................................................................................................................. 33
4.2 Paper II ................................................................................................................ 36
4.3 Paper III ............................................................................................................... 39
5 CONCLUDING REMARKS ........................................................................................ 41
6 ACKNOWLEDGMENTS ............................................................................................ 42
7 REFERENCES .............................................................................................................. 45
3
LIST OF ABBREVIATIONS
A
adenine
ag
anti-gene
AGO2
argonaute 2
AR
androgen receptor
C
cytosine
ChIP
chromatin immunoprecipitation
CMV
cytomegalovirus
CRISPR
clustered regularly interspaced short palindromic repeat
CPG
controlled pore glass
Da
Dalton
DNA
deoxyribonucleic acid
DMD
Duchenne muscular dystrophy
DSB
double-stranded break
dsDNA
double-stranded DNA
EMSA
electrophoretic mobility shift assay
glyLNA
2´-glycylamino-LNA
G
guanine
HG
Hoogsteen
HPLC
high pressure liquid chromatography
HIV
human immunodeficiency virus
HDR
homology directed repair
LNA
locked nucleic acid
MALDI
matrix assisted laser desorption ionisation
mRNA
messenger RNA
miRNA
micro RNA
MS
mass spectrometry
4
NMR
nuclear magnetic resonance
nt
nucleotide
ON
oligonucleotide
pc
pseudo complementary
PEI
polyethyleneimine
PLP
padlock probe
PNA
peptide nucleic acid
PR
progesterone
RCA
rolling circle amplification
RCP
rolling circle product
RNA
ribonucleic acid
RNAi
RNA interference
RISC
RNA-induced silencing complex
SCID
severe combined immunodeficiency
siRNA
silencing RNA
ssDNA
single-stranded DNA
SSO
splice-switching oligonucleotide
SNA
spherical nucleic acid
T
thymine
TALEN
transcription activator-like effector nuclease
TFO
triplex forming oligonucleotide
Tm
melting temperature
TINA
twisted intercalating nucleic acid
TISH
triplex in situ hybridization
WC
Watson-Crick
ZFN
zinc finger nuclease
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1 INTRODUCTION
1.1
GENE THERAPY
1.1.1 Introduction
Gene therapy can be defined as the intentional modulation of gene expression in specific cells
for therapeutic purposes. The concept of gene therapy arises from between 1960s and 1970s.
In this decade, it became clear that genetic diseases as well as degenerative and infectious
diseases could be corrected at a genetic level [1, 2]. In 1990, the first authorized clinical trials
were carried out using retroviruses to transfect ex vivo T-cells [3] or cancer cells [4] to treat
severe combined immunodeficiency (SCID), caused by adenine deaminase deficiency (ADASCID) or melanoma without success. In 1999, an 18 years old undergoing a gene therapy
trial for liver disease died due to the treatment, therefore raising question about the feasibility
of gene therapy [5].
In 2000, Fisher et al reported the first clearly successful gene therapy trial [6]. In this study, a
defective retrovirus could complement genetic defects in several boys with X-linked SCID.
However, several treated children developed leukemia like diseases, arising from virus
incorporation raising again concern about safety in gene therapy in particular with viral
vectors [7].
In 2010, more than 1500 clinical trials have been approved worldwide targeting different
diseases and organs [8]. 70% of clinical trials involve the use of recombinant viral vector
such as retroviruses, lentiviruses, adenoviruses and adeno-associated viruses [9]. Promising
results in clinical trials were obtained for β-thalassemia [10], X-linked adrenoleukodystrophy
[11], Leber´s congenital amaurosis [12], Wiskott-Aldrich syndrome [13], hemophilia [14]
and Parkinson´s disease [15]. In addition, patients with choroideremia, an eye inherited
disease had their sight restored after treatment with an adeno-associated virus [16]. In 2012,
Glybera® became the first gene therapy product approved in Western world to treat
lipoprotein lipase deficiency via adeno-associated vector [17]. Although viruses are highly
efficient delivery vectors, issues about safety lead to the development of non-viral vectors.
1.1.2 Non-Viral Gene Therapy
Non-viral vectors include chemical-based delivery methods with nanoparticles [18], cationic
polymers [19] and lipids [20] and physical approaches such as gene gun [21], electroporation
[22], ultrasound [23] and hydrodynamics delivery [24]. Chemical approaches aim to form
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complex between chemical compounds and the ONs to protect from the degradation and
facilitate entry into the cells [25, 26]. Physical approaches use a force to increase the
membrane permeability and thus the gene transfer. These non-viral vectors are of interest
because they are easy to use, do not trigger immune response, and can be produced in large
scale and thus provide attractive alternatives to viral vectors for ON delivery. Currently, nonviral delivery systems are in clinical trials, but none of them have as yet been approved [27].
Recently, the genome editing technologies are gaining a lot of attention due to their ability to
directly correct or remove a mutation in the genome or disrupt viral DNA [28]. They induce
double stranded breaks (DSBs) at specific genomic site. Gene editing occurs subsequently in
the cells due to the activation of the DNA repair machinery via non-homologous end joining
(NHEJ) or homology directed repair (HDR). To date, four types of nucleases have been
reported: meganucleases [29], CRISPR (clustered regularly interspaced short palindromic
repeats) -Cas9 (CRISPR associated protein 9) [30], zinc finger nucleases (ZFN) [31] and
transcription activator-like effector nucleases (TALEN) [32]. Clinical trials with ZFN against
HIV showed promising results [33]. Gene editing technology could offer therapeutic benefits
for other viral or hereditary diseases. However, translating editing technologies into the
clinics involves challenges in term of efficiency and safety.
1.1.3 Oligonucleotide-based approaches
Oligonucleotides (ONs) are short nucleic acids sequences (usually between 8 and 50 bases).
Gene therapy through the introduction of ONs is a very exciting approach because both
mRNA and DNA have the potential to be modulated. Depending on the target, ONs can be
classified as antisense, which target mRNA or as anti-gene that target chromosomal DNA. In
the antisense mechanism, RNA-targeting ONs exhibit various inhibition mechanisms.
Antisense-ONs induce the target mRNA degradation via RNase-H, whereas small interfering
RNAs use RISC-mediated complex to destroy mRNA [34]. Gapmers are ONs with modified
nucleic acid analogues inserted at each end to protect from exonuclease degradation. They
can induce RNase-H degradation due to the presence of unmodified DNA nucleotides in the
middle of the ON. Silencing RNA (siRNA) are short dsRNAs with two overhanging
nucleotides and phosphorylated 5´ and hydroxylated 3´ends. After incorporation into RISC
and cleavage of the sense strand, the siRNA-activated RISC complex binds and degrades the
complementary mRNA target. The majority of clinical trials involving siRNAs and antisense
ONs were carried out for treatment of cancers [34]. Steric block ONs bind strongly to mRNA
and prevent translation into proteins without the need for nuclease [35]. Other ON-based
strategies do not require RNase-H. For instance, splice-switching ONs (SSOs) generate
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alternative splicing by targeting pre-mRNA [36]. SSOs showed promising splice correction in
animal model with Duchenne muscular dystrophy (DMD) [37], spinal muscular atrophy [38]
and X-linked agammaglobulinemia.[39]. The first clinical trial with SSOs was initiated in
2007 to treat DMD [40]. AntagomiRs are microRNA (miR)-targeting ONs. miRs are small
noncoding RNAs. They often regulate gene expression in processes such as proliferation,
differentiation and cell death. Disregulation of miR occurs in many cancers, resulting in
overexpression of oncogenes [41]. Thus targeting and blocking the overexpressed miR with
antagomiRs is efficient strategy to regulate gene expression in breast cancer (anti-miR221)
[42] and brain tumor (anti-miR155) [43].
Most ON-based therapeutics strategies are hampered due to the low delivery efficiency [44].
To achieve clinical efficiency, the ONs need to be stable in serum and avoid renal clearance
to reach the target tissues. To enter the cell, ONs need to be internalized by endocytosis and
then released from the endosomes [45]. However, the promise of ON based therapies is very
close to reach a clinical therapy because recent efforts to understand cell uptake mechanisms
have resulted in the design of new delivery methods [9]. In addition, progress in nucleic acid
chemistry has improved stability and potency of ONs available. Recently, an antisense ON
Kynamro® from ISIS has been approved by the Food and Drug Administration (FDA) for
treatment of familial hypercholesterolemia [46].
1.1.4 Oligonucleotide delivery
Naked DNA plasmids and ONs have low uptake via simple diffusion through the cell
membranes due to their hydrophilic structures [47]. Therefore, to improve ON delivery,
several delivery methods have been developed. Cell penetration peptides (CPP) can be
chemically conjugated to ONs to improve the cell uptake with low toxicity. CPPs are usually
short peptides rich in arginine and lysine such as for instance the Tat peptide [48] and
penetratin [49].
As an alternative strategy, nanoparticles are solid particles with a size in the range 1-100 nm
with interesting properties for gene therapy [50]. Due to their small size, they can penetrate
many physiological barriers and serve as a delivery system in vivo. As reported, the
nanoparticle’s ability to cross the membranes depends on the size but also on the physical
properties [51]. In particular, inorganic nanoparticles, prepared form metal, inorganic salts or
ceramic can have their surface coated to attach ONs and ensure an efficient delivery in vivo.
Such constructs called spherical nucleic acids (SNAs) [52] can enter the cells despite their
high negative overall charge. Due to the orientation and the high number of ONs at the
3
surface, SNAs have specific properties compared to regular ONs that cannot enter the cells
without transfection reagents. SNAs also protect ONs from degradation to achieve gene
regulation, show low toxicity and no immune response [53, 54].
Cationic polymers are of particular interest in gene therapy. When combined with negatively
charged ONs, they form complexes, called polyplexes that increase the delivery of ONs into
cells. Polyethyleneimine (PEI) and its derivatives are among the most studied polymers for
delivery [55, 56]. It was demonstrated that PEI promotes gene transfection via enhancing the
endosome escape [57]. Upon systemic administration, the PEI tends to aggregate and form
larger complexes that accumulate in various organs [58].
Similar to polymers, cationic lipids form complexes (lipoplexes) with ONs. Due to their
amphiphilic structure, they assemble themselves into spherical bilayer cavities called
liposomes [59]. Liposomes facilitate the delivery of drugs in particular ONs and protect them
from degradation [60]. The liposomes properties such as size, stability and biodistribution
depend on their preparation and composition in lipids. Because their delivery is safe, the main
limitation for liposome therapy remains their poor stability in serum [61] Finally, dendrimers
are large branched spherical molecules that can be conjugated on their surface with a variety
of molecules and achieve drug delivery [62]
Non-viral gene therapy has shown promising result in animal models and in many clinical
trials [9]. PEI was combined with siRNA for local gene therapy of bladder, ovarian and
pancreatic cancers. As PEI is known to cause cytotoxicity, other polymer-based strategies
(PEG-PEI-cholesterol or PEI mannose-dextrose) have been tested for cystic fibrosis and HIV
and CMV DNA vaccine [9]. Nucleic acids, when encapsulated in very small lipid
nanoparticles (SNALPs) < 200 nm can be efficiently delivered to the hepatocytes as gene
therapy for liver and treatment of hypercholesterolemia [63].
4
1.2
ANTI-GENE THERAPY
1.2.1 Definition
DNA contains the genetic information for the development, functioning and replication of
almost all known living organisms. As the main dogma in molecular biology, DNA is
transcribed into mRNA that is further translated into proteins. In the anti-gene strategy, an
ON hybridizes with dsDNA and therefore inhibits the production of mRNA and proteins for
this particular gene.
These anti-gene ONs can be divided in different categories depending on the mode of binding
to DNA. (i) as a triplex forming ON binding via Hoogsteen (HG) interaction to the DNA
duplex (ii) as an invading ON binding via Watson-Crick (WC) interaction to one strand of the
DNA duplex (iii) as a clamp-ON combining these two interactions. DNA-targeting ONs face
additional challenges in vivo such as finding its ways into the chromatin and achieving a
sufficiently strong affinity to block the RNA polymerase. For this purpose, different types of
ONs have been developed and are described in chapter 1.6.
Figure 1. The different steps in the anti-gene strategy. 1/ A synthetic ON penetrates into
the cellular cytoplasm through the cell membrane. via endocytosis 2/ The ON diffuses into in
the nucleus and binds to a specific site in the genome. 3/ The binding of the ON prevents
transcription and mRNA formation for a specific gene. 4/ Due to the absence of mRNA, no
protein for this gene is detected.
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1.2.2 Differences between anti-gene and antisense therapy
There are fundamental differences between targeting mRNA and genomic DNA [64, 65].
First, DNA is difficult to target because it is double-stranded and highly packaged with
proteins into chromatin whereas mRNA is single-stranded and easily available for direct
hybridization. Also, the target sequence for an anti-gene ON is mostly present once or twice
in the genome whereas each cell contains the target sequence in hundred or thousands copies
for an antisense-ON. Finally, the most efficient antisense ONs induce RNA cleavage via
RNase-H mechanism or via RISC complex whereas an anti-gene ON is only expected to
block the transcription.
1.2.3 Anti-gene triplex forming oligonucleotide
The triplex forming oligonucleotides (TFOs) are ONs that can recognize dsDNA by binding
in the major groove. It is fairly trivial to design TFOs between 16 and 20 nt binding with
enough specificity to target unique sites in the genome [66]. Nevertheless, triplex binding is
limited to polypurine/polypyrimidine sequences. Interestingly, these polypurine sequences
are over represented in the genome particularly in the promoter regions [67, 68]. Therefore,
TFOs have a great potential for control of gene expression including oncogenes myc [69],
BCL2 [70], BCR/ABL [71] and KRAS genes [72]. Interestingly the oncogene down regulation
was demonstrated to correlate with a decrease in tumor growth, improving cancer therapy
[73, 74]. TFOs have also found many applications such as to down regulate or up regulate
transcription in disease-causing genes, to induce mutagenesis or homologous recombination
in cells [75, 76]. TFOs have been reported to inhibit the transcription either by competing
with transcription factor binding during the initiation phase [77, 78] or by physically blocking
the transcription during the elongation phase [79]. The transcriptional up regulation of a gene
was achieved with a TFO linked to activation domain for transcription factors [78, 80].
Chromatin is decondensed in transcriptionally active genes, facilitating the TFO binding to
the genomic DNA [81]. However, the TFOs with highest thermal affinity are not the most
efficient in cell as demonstrated in [82]. One explanation is that increasing the number of
LNA modification correlates with an increase of unspecific targeting in cells, thus reducing
the anti-gene efficiency.
Young et al showed that DNA-ONs block RNA polymerase in vitro in a transitory manner
[83]. However, by crosslinking the TFO to the duplex, he showed that stable binding
completely blocks the RNA polymerase with long-term effect. Supporting this, psoralen
conjugated TFOs displayed successful regulatory effects in HeLa cells [84].
6
Very interestingly, Glazer et al successfully performed TFO-directed mutagenesis in a mouse
model [82, 85] and achieved long-term correction in anti-gene therapy. However, mutation
rate efficiency was very low and thus no anti-gene based therapeutic has reached a clinical
stage [86]. Several limitations need to be overcome to obtain TFOs efficient in vivo. First,
TFOs are negatively charged, difficult to deliver to the target tissue and their uptake into cells
is low. Then, specific delivery into the nucleus is also challenging and has been an active area
of research during the last decades [87, 88]. Finally, TFOs are usually degraded by nucleases
present in the cells and exhibit low stability at intranuclear pH and salt [89]. To enhance
stability and binding affinity, different nucleotide analogues have been synthesized and are
described more in detail in chapter 1.6.1.
1.2.4 Anti-gene-ONs and clamp-ONs
LNA-ONs can bind via WC interactions and invade the double helix although they have
lower affinity than clamp constructs, notably toward the displaced DNA strand. LNA-ONs
can also bind to pre-open genomic regions such as hairpin loops, envisaging a possible antigene effect for linear LNA. Supporting this assumption, Corey et al showed that anti-gene
LNA-ONs (agLNAs) down regulate, in a concentration-dependent manner, the progesterone
(PR) and androgen receptors (AR) [90]. To penetrate the cells, agLNAs were combined with
cationic lipids to enter MCF7 cells. In this study, the LNA binding efficiency was affected by
many factors such as target secondary structure and accessibility, but usually long ONs
>19bp are needed to bind without being kicked out by the displaced strand. Further
investigation with chromatin immunoprecipitation (ChIP) confirmed that the agLNA
mechanism involves binding to genomic DNA [91]. Corey et al also showed similar antigene effect with peptide nucleic acids (PNA) targeting the progesterone receptor [92]. They
noted an alternation in the morphology of the T47D cells, supporting the silencing of the
intended gene. Finally, Corey et al developed anti-gene RNA-ONs. Initially, it was thought
that the RNA interacts directly with DNA. However, 20% of the genes have overlapping
RNA transcript, which complicate the assumption of DNA targeting. Thus after investigation,
RNA was demonstrated to bind to the overlapping RNA transcripts mediated via the protein
argonaute2 (AGO2) [91].
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1.3
DNA STRUCTURE
1.3.1 Early research in nucleic acids
Unraveling the DNA structure is the result of a work over a long period from many scientists.
In 1868, the Swiss physician Friedrich Miescher isolated an unknown substance common to
many cell types, containing large amounts of phosphorous and nitrogen [93, 94]. He deduced
that it was not a protein or a lipid. Since this substance was found in the nucleus, he called it
nuclein. In 1881, Albrecht Kossel, was able to identify the 5 nucleobases: adenine thymine,
cytosine, guanine and uracil [95]. In 1919, Phoebus Levene identified the nucleotide
structure including the sugar, the base and the phosphate. He also suggested that DNA
contains a chain of nucleotides linked through phosphate bonds [96]. In 1944, Oswald T.
Avery, Colin MacLeod and Maclyn McCarty suggested that DNA was the support of
genetic information [97]. In 1950, Erwin Chargaff discovered that the base composition in
DNA varies between the species but in all species, the amount of cytosine is the same as
guanine and the amount of adenine is same as thymine [98]. Shortly thereafter, Maurice
Wilkins and Rosalind Franklin had amassed X rays results showing that the double helix had
an helix with 2 periodicity along the major axis [99, 100]. Based on this key piece of
evidence, James Watson and Francis Crick proposed, in 1953, a double helix structure for
DNA, now accepted as the first correct model [101].
1.3.2 Structure of the double helix
DNA is a double helix composed of two long polynucleotide chains held together by base
pair interactions between A-T and C-G [101]. The strands, complementary and oriented with
opposite polarity, form an antiparallel duplex. As the strands are not directly opposite to each
other, DNA contains 2 grooves not equal in size to each other: a major groove and a minor
groove. In the major and minor grooves, the edges of the DNA bases are exposed to the
outside of the double helix, which enables proteins or nucleic acids to recognize specific
sequences. However, the major groove, wider, contains edges of the bases that are more
accessible and is therefore richer in sequence information than the minor groove, favoring
binding in the major groove [102].
DNA duplexes can form several structures: right-handed A-DNA and B-DNA, left-handed ZDNA. However, under physiological conditions, B-DNA is the most prevalent form of DNA.
8
•
B-DNA is a right-handed helix with 10.5 bases pair per turn and a 23.7 Å diameter.
B-form is narrower and more elongated than A-DNA. The major groove is wide and
the minor groove is narrow but both grooves are quite deep. B-DNA is also more
symmetrical and less distorted than A-DNA. The change of structure in DNA is
largely due to the conformation change of the sugar from C2´-endo in B-form to C3´endo in A-form because it affects the degree of rotation per base and the distance
between the bases [103].
•
A-DNA is also a right-handed helix with 11 bases per turn and a 25.5 Å diameter.
This is due to the C3´-endo sugar conformation, resulting in an exaggerating major
groove and diminished minor groove. The base pairs are also tilted rather than
perpendicular to the helix axis. Due to this structure, less water molecules can bind
and therefore dehydration favors A-DNA conformation.
•
Z-DNA is a deformed left-handed helix. The double helix remains but the structural
parameters are different. Both C2´ and C3´ endo conformations are observed. ZDNA’s role in cells is unclear.
Figure 2. Standard conformation of DNA, A-DNA, B-DNA, isolated duplex and triple
helix. The triplex forming strand is displayed as a blue shade. Reproduced from (85) with
permission
9
1.3.3 Factors determining the DNA structure and stability
There are several interactions affecting the DNA structure. DNA is not a static structure but
more an equilibrium of various states between different dynamic structures. The structure of
the double helix results from a balance of non-covalent stabilizing and destabilizing forces in
aqueous solution.
Stacking force: The base stacking refers to the interactions between aromatics molecules in
juxtaposition that results in a contact with the π interactions. Recent studies have shown that
base stacking is the most important stabilizing factor for dsDNA and favors a double helix
organization with 3.4 Å between the bases [104]. Base stacking occurs mainly between bases
within the same strand but also between the two strands due to the helical twist. The stacking
force results from the interaction of several forces dipole-induced dipole-interactions. In
general, purines stack stronger than the pyrimidines due to a larger surface and higher
polarizability [105].
Base pairing: The hydrogen bonds can be defined as an electrostatic interaction between an
acidic hydrogen and an electron donor group. The hydrogen bonds between WC base pairs
stabilize the double helix. Longer sequences of polynucleotides are therefore more stable than
shorter sequences due to a higher number of hydrogen bonds. Cytosine-guanosine base pair
via 3 hydrogen bonds whereas adenosine-thymine base pair via two hydrogen bonds. As a
result, G-C rich sequences are more stable than A-T rich sequences [106].
Charge repulsion: The DNA backbone is negatively charged. Destabilization of the double
helix occurs via the electrostatic repulsion of phosphates along one strand as well as the
repulsion between the phosphates of the two strands of the double helix [105]. The cation
concentration is directly proportional to the duplex stability [107]. A high ionic concentration
stabilizes the double helix, increasing the melting temperature because the negative charges
from the phosphate backbone are shielded by the cations, reducing the electrostatic repulsion.
However, at low ionic strength, the unshielded negative charges from the phosphate
backbone render the helix less stable.
DNA hydration: Water molecules contribute to the duplex stability by adding additional
hydrogen bonds with exposed functional groups in the duplex [105]. It is an important factor
that also to determines the DNA conformation (A-DNA or B-DNA).
Steric effects vary in their contribution and can stabilize or destabilize the double helix.
10
1.3.4 DNA organization in eukaryotes and prokaryotes
In eukaryotes, DNA is divided between different chromosomes, each containing a single
DNA molecule. The cells accommodate the very long genome by folding chromatin into
several levels of organization and by wrapping it into nucleosomes. The nucleosome is the
unit of the chromatin containing 8 proteins histones and wrapped approximately about 146
base pairs. In the chromatin, the nucleosomes are packed to form a 30 nm fiber. The 30 nm
fiber forms loops that folds to form a 250 nm fiber, which further organizes itself into a 700
nm chromatid. The left handed wrapping of the nucleosomes creates a negative supercoiling
in DNA in eukaryotes [108], similar to the supercoiling present in circular dsDNA. This
supercoiling effect increases DNA breathing - a transient base pair opening in DNA that
favors unwinding for proteins and other cellular processes necessary for life [109]. As a result
of the constraint, a supercoiled DNA is shorter than a relaxed DNA and this topological
constraint is very important in cellular processes. In cells, the supercoiled DNA is stressed,
therefore breaking of one strand leads to immediate relaxation that can be detected by the
DNA repair machinery. Because DNA supercoiling affects transcription, it was reported to be
a rapid and efficient cellular mechanism to control gene expression [110]. During replication
or transcription, the two strands are separated by the DNA or RNA polymerase [111]. This
local opening during replication or transcription creates positive supercoiling ahead of the
RNA polymerase and negative supercoiling behind it [112].
In bacteria, the genome consists of a single circular double stranded DNA (4.6 106 bp in E.
coli) [113]. Bacteria can also contain one or several shorter circular DNA molecules called
plasmids that contain non-essential genes. To characterize DNA topology, the twist number is
the number of time the double helix make by rotation a full turn. Adding or removing one
twist will not have any effect in linear DNAs because they have free extremities to
accommodate by rotation the change in the number of twist. Therefore, linear DNAs are
relaxed and thus very stable. However, when the two extremities are bound to each other,
plasmids have a different situation when adding or removing a twist [114]. The number of
twists is absolute and cannot be changed unless one of the strands is interrupted. This
constrain creates a tension, that favors either DNA unwinding (negative supercoiling) or
overwinding (positive supercoiling). This explains why negatively supercoiled plasmids and
DNA in prokaryotes and eukaryotes are easier to invade than linear DNAs.
11
1.4
DNA MODE OF BINDING
1.4.1 Triple helix formation
The triplex helix was first reported in 1957 [115], only 4 years after the DNA double helix.
The triplexes have been studied for their structures and their physic-chemical properties
during three decades [75]. Then, synthetic ONs became available due to progresses in ON
synthesis. ONs were also demonstrated to bind in a sequence specific manner to form a triple
helix [116, 117]. In 1963, Karl Hoogsteen reported a crystal structure where A and T base
pair with a different geometry than WC pairing [118] Triple helixes form base triplets in a
HG or reverse-HG conformation (figure 3). These triplex forming ONs (TFOs) have raised
interest as they allow the rational design of specific ligands that could interfere with gene
expression in therapeutical applications [82, 86, 119]. TFOs also found applications in
biotechnology [120], due their ability to recognize double-stranded sequences. Although
triple helixes were first characterized in vitro, recent evidences have now confirmed their
functional roles in cells [86]. Two types of triplexes are possible: the intermolecular triplexes
when the third strand originates from different DNA duplexes and the intramolecular
triplexes when the third strand come from the same DNA helix. These structures also called
H-DNA form at the mirror repeat regions in the genome and are involved in the development
of certain diseases [86, 121].
In vitro, several methods use TFOs as mean of detection. For instance, triplex in situ
hybridization (TISH) is a detection method that uses fluorescently labeled probes to stain α
satellite regions [122]. Each of these regions contains 500-1000 repeats that could be targeted
with a 16bp fluorescently labeled TFO and detected via fluorescence microscopy. Another
approach is the TFO-padlock that allows detection via rolling circle amplification [123],
further described in chapter 3.6.
•
Triple helix structures
Triplex formation is based on the recognition of polypurine/polypyrimidine sequences
present in DNA. The TFO recognizes the purine strand by forming hydrogen bonds with the
donor and acceptor groups of the purine bases. TFO formation was demonstrated to occur via
nucleation zipper, starting form the 3´-extremity, forming transient intermediates with 2-3
nucleotides until the TFO finds its complementary sequence and binds in a zipper manner
[124, 125].
12
It was initially thought that the underlying duplex adopts an A-DNA conformation upon
triplex formation in contrast to normal DNA duplex that exhibits B-DNA conformation.
More recent studies have now shown that triplex conformation differs from A- and B-forms
[126, 127]. The major groove becomes slightly wider to accommodate a supplementary
strand and the minor groove becomes narrower. Numbers of triplex structures have been
investigated by NMR, X-ray crystallography or with bioinformatics models [128-132].
Figure 3. Base triplets for parallel and antiparallel TFOs. Hydrogen bonds are indicated
in doted lines. Triplets are referred as H*R-X. The symbols * and – represent respectively
HG- and WC-interactions. C+ indicates protonation for the nitrogen N3 in cytosine.
13
Triplexes can be divided into 3 motifs, depending on their base composition and the
orientation of the phosphodiester backbone [75, 133] (figure 4).
Figure 4. Orientation of the triplex motifs according to the base composition. TFO is
drawn in red. The base compositions within the duplex strands are indicated in the legend
box.
•
(T,C)-motif: The third strand binds in a parallel orientation to the duplex, forming
HG triplets T*A-T and C+*G-C triplet. To produce stable triplex, the imino group of
the cytosine must be protonated, rendering the triplex formation pH dependent and
therefore unstable at neutral pH [134-136]. These (T,C)-TFOs are usually referred in
the literature as pyrimidine-motif
•
(G,A)-motif: The third strand binds in an antiparallel orientation, forming reverse HG
triplets, G*G-A and A*A-T.
•
(G,T)- motif: the third stand binds either in an antiparallel or parallel orientation
forming HG/ reverse HG triplets G*G-C and T*A-T. Both (G,A)- and (G,T)-TFOs
are referred as purine motif [137]. With these motifs, competing structures such as Gquadruplexes can prevent triplex formation at intranuclear conditions [138].
However, purine TFO binding is pH-independent due to the absence of cytosine.
14
1.4.2 Double helix invasion
The invasion into dsDNA occurs when an ON binds to one of the strands via WC-pairing and
displaces the second strand, resulting in a DNA opening. However, due to the relatively high
duplex stability, the displacement of hydrogen bonding is rather infrequent [104]. Among the
DNA targeting strategies, the double helix invasion has raised interest due the simplicity of
design with WC-pairing rules. Several DNA invasion strategies have been tested and can be
classified depending of the structure of the invasion complex.
Figure 5. DNA invasion. (A) PNA- and LNA-ONs invade the duplex (B) PNAs and LNAs
bind homopurine sequences via triplex invasion. (C) Pseudo complementary PNA (pcPNA)
and invader-LNAs can bind overlapping sequences in both strands of the duplex without selfinteraction due to the presence of modified bases in pcPNA and 2-N-(pyren-1-yl)methyl-2amino-alpha-L-LNA in Invader-LNAs (D) Clamp-ONs (bisLNAs and bisPNAs) bind
homopurine via a combination of HG- and WC interactions (E) Tail clamp-ONs with an
extended WC-region bind via HG and WC-interactions (F) Zorro-LNAs also bind via double
stranded invasion two targeting adjacent sites in both DNA strands. Due to their Z-shape,
Zorro-LNAs avoid self-interaction.
Peptide nucleic acid (PNA) was the first invading ON reported to displace a DNA strand in a
duplex. PNA invasion is described more in details in chapter 1.6.2. In particular, linear PNAONs displace one strand via duplex invasion (figure 5A) when targeting a mixed sequence
and via triplex invasion (figure 5B) when targeting a homopurine sequence.
15
Pseudo complementary (pc) PNAs are a pair of PNA-ONs that contains base analogues that
form weak bases with each other (for example 2,6-diaminopurine and 2-thiouracil). [139] The
PNAs are formally sequence complementary but due to the presence of modified bases, the
PNA strands form an unstable duplex. Thus they retain high affinity to complementary DNA
and are able to double strand invade DNA (figure 5C). To further improve this strategy,
efforts have been made to develop new pseudo complementary base pairs [140]. pcPNAs
protect DNA from the action of restriction enzymes and DNA methyltransferases [141].
Clamp-ONs are single-stranded ONs with a WC- and a HG-binding region tethered by a
linker. Due to the linker, the clamp invasion is favored by turning a trimolecular reaction into
a dimolecular one, as compared to the unlinked arms [142]. Clamp PNAs, also called
bisPNAs, show efficient invasion (figure 5D). In order to decrease the TFO length, the WCarm was extended into mixed sequences to form a bisPNA tail-clamp (figure 5E).
Zorro-LNAs, are short ONs, in the first generation, annealed via a complementary double
stranded region and are able to bind on both strands in a duplex (figure 5F) [143].
Interestingly, Zorro-LNAs block the transcription when targeting multiple sites in the 5´ UTR
region of the promoter within a plasmid reporter gene. In addition, Zaghloul et al reported
improved invasion mechanism for Zorro-LNAs invasion with single stranded Zorro-LNA
when the arms are connected via a synthetic linker [144]. In 2011, Ling et al used ZorroLNAs to block the transcription of neurofibromatosis 1 gene (NF1) in fibroblast cell line
[145].
Invader-LNAs are double-stranded probes that contain hotspots destabilizing the duplex,
reported by Hrdlicka et al [146]. The destabilization is achieved through the presence of
pyrenyl-methyl-2´-amino-α-L-LNA monomers forced to intercalate in the same duplex
region. Thus, Invader-LNAs exhibit a high affinity to ssDNA due to the intercalating
properties of pyrene. As a result, the Invader-LNAs invade and displace the strands of an
isosequential DNA duplex under physiological conditions without sequence restriction.
However, the efficiency of Invader-LNAs remains to be investigated in longer duplex targets.
Finally, the Invader-LNAs were efficient to achieve fluorescent in situ hybridization (FISH)
in cells [147].
16
1.5
OLIGONUCLEOTIDE SYNTHESIS
A phosphoramidite nucleoside is a derivative of a natural or synthetic nucleotide, with a
N´N´-diisiopropyl phosphoramidite group attached to the 3´-hydroxyl. It is more reactive
than the naturally occurring nucleotides and commonly used during ON synthesis. To prevent
undesired reactions, the phosphoramidite nucleosides have their functional groups protected
by an acid-labile dimethoxyltrityl or base-labile 2´-cyanoethyl groups. The synthesis proceeds
via cycles containing the following steps: deprotection, coupling, capping and stabilization.
Each cycle results in the attachment of an additional nucleotide from the first nucleotide
attached to the solid support made of controlled pore glass (CPG). First, during deprotection,
the dimethoxytrityl group protecting the 5´-hydroxyl is removed with trichloroacetic acid in
an inert solvent (toluene or dichloromethane), leaving a reactive hydroxyl group to attach the
following nucleotide. In the coupling step, reaction between the hydroxyl group and a
tetrazole-activated phosphoramidite creates a 5´-3´ linkage to attach the additional nucleotide.
After reaction, excess of reagent and tetrazole are washed out. Although this step is usually
99% efficient, a small amount of hydroxyl group remains freely reactive. To avoid further
reaction in the following coupling steps, a capping step is performed with acetic anhydride to
block the free hydroxyls. The last step in the cycle is the stabilization that results in the
oxidation of the phosphite into a phosphate using iodine and water.
Figure 6. Phosphoramidite ON synthesis cycle. Each cycle consists of 4 steps:
(deprotection, coupling, capping and oxidation) and results in the addition of one nucleotide
to the polynucleotide chain.
17
The synthesis proceeds in a 3´ to 5´ direction. At the end of the synthesis, the ON is attached
via its 3´ extremity to the solid support CPG with protecting groups that remain on the
nucleobases. The ON is usually deprotected and cleaved from the solid support by
ammonium hydroxide treatment at high temperature to release a functional single-stranded
ON. Although deprotection removes the protecting groups, they will remain present as salt. A
desalting step is then required to remove these contaminants.
Because each coupling step has an efficiency of about 99%, the synthesis of long ONs
requires purification to remove shorter truncated ONs. This purification is also particularly
important for ONs containing fluorophores or other non-natural modifications that result in
lower coupling efficiency and therefore contain more contaminants. For purification, highpressure liquid chromatography (HPLC) is a method of choice in which the ON is dissolved
in a liquid phase that will go through a solid phase and separated from other impurities. In
ON synthesis, two types of HPLC are commonly used depending on the nature of the ON, the
mobile phase and the composition of the liquid phase. In ion exchange HPLC, the separation
occurs based on the net charge of the ONs. In reverse phase HPLC, the separation occurs
based on a difference of solvent affinity.
The final step in the ON synthesis is the quality control. For that, MALDI-ToF is a mass
spectrometry method to determinate with precision the mass of ON synthesized. Briefly, in
MALDI-ToF, the samples are ionized into a potential energy field. By measuring the time the
ions reach the detector, it is possible to determinate the mass of the ions and by consequence,
the mass of the final ON.
18
1.6
NUCLEOTIDE ANALOGUES
1.6.1 TFO analogues
The first anti-gene experiments were done with unmodified DNA-ONs [148, 149]. It was
soon realized that new chemistry would be needed to reach clinical efficiency. Efforts have
been done to design novel nucleotide analogues that can be incorporated during ON
synthesis. The new analogues were developed through modifications in the nucleobase, in the
sugar ring or in the phosphodiester backbone. The aim of these modifications is to improve
certain properties, such as the synthesis efficiency, the affinity and specificity to their targets
and the nuclease resistance. Due to the very large number of nucleotide analogues, this thesis
will focus on the nucleotide analogues reported to increase triplex formation.
Figure 7. Chemical modifications favoring TFO binding Structure of A/ 2´deoxypseudoisocytidine, 5´-propargylamino-cytosine, deoxycytosine, 5´-methylcytosine, B/
phosphorothioate, phosphoramidate, peptide nucleic acids, γ-peptide nucleic acids C/ 2´-Omethyl-RNA- 2´-O- aminoethyl-RNA, locked nucleic acid (from left to right).
19
•
Nucleobase modifications have been developed to stabilize triple helix structure. For
example 5´-methylcytosine (mC) increases the triplex formation at neutral pH [150],
due to the steric effect of the methyl group. To circumvent the pH dependence of
(C,T)- TFO, pseudoisocytidine (ψC) is an isomer of cytidine (C) where the base is
linked to the sugar via a carbone-carbone bond. Due to its structure, the
pseudoisocytidine has a hydrogen atom to form a second bond and does not need
protonation to form parallel triplex, favoring triplex formation. Recently, pyrimidines
with 5´-attached positive charge such 5´-propargylamino-cytosine (pC) were reported
to have a TFO stabilizing effect [151].
•
Backbone modifications have been tested to alter the negative charge of the
phosphodiester backbone in order to change the properties of the ON. TFOs with
cationic backbone are expected to bind better due to less electrostatic repulsion [152,
153]. Substituting the phosphodiester linkage by a phosphorothioate increases the
biodistribution and the stability toward nucleases. Peptide nucleic acids are nucleic
acid analogues with pseudopeptide backbone described in chapter 1.6.2.
•
Sugar modifications were also shown to improve the triplex formation. First, RNA
with the 2´-OH hydroxyl additional forms more stable triplex than DNA. 2´OMeRNAs were also shown to further stabilize the TFO because the methoxy substituent
stabilizes the C3´-endo sugar conformation [154, 155]. Locked nucleic acids are
analogues with sugar modifications reported to improve the triple helix formation and
are discussed more in detail in chap 1.6. 3.
Recognition of pyrimidine bases is hard to achieve but different groups have developed new
base analogues, extending TFO binding to pyrimidine bases, [89, 156-158] The TFOs
including modified nucleobases showed affinity and selectivity to pyrimidine interruption
[157, 159] but stability at intracellular conditions remains a challenge, which is a limitation
for further studies on these modified TFO in vivo [160]. Due to instability of the triplexes
under physiological pH, TFO stabilization is crucial to improve its therapeutic potential.
1.6.2 Peptide nucleic acids
Peptide nucleic acids (PNAs) were first synthesized as a DNA analogue that retains the
nucleobases but replaces the phosphodiester backbone by a pseudo-peptide backbone [161,
162]. The PNA neutral backbone improves their affinity to DNA due to the lack of
electrostatic repulsion. PNA was successfully synthesized using the peptide solid phase
synthesis. First, it was demonstrated that two poly-thymine PNAs could form a triplex with
20
one strand of the DNA duplex, displacing the poly-thymine strand of the duplex [163]. This
was the first example of double helix invasion with synthetic ONs. After that, it became clear
that mixed-sequence PNAs were able to bind any complementary ssDNA forming duplexes
more stable than the native DNA/DNA duplex. PNA can bind DNA in a parallel and
antiparallel orientation [164]. But the antiparallel orientation is preferred. Although PNA can
be considered as a DNA analogue, the structure and the physical properties are more similar
to proteins. Solubility can be a problem with PNA > 15 bp especially for G-rich PNAs or in
presence of a label (fluorophore-biotine) [165]. Due to their pseudo-peptide backbone, PNAs
are resistant to nucleases and proteases.
PNAs can bind as TFOs via HG-interaction to polypurine/polypyrimidine stretches. In
addition, two poly-pyrimidine PNAs can be tethered together via a linker to form a clamp
PNA, also called bisPNA. These clamp constructs form highly stable PNA:DNA:PNA
triplexes at the polypurine sites. bisPNA tail-clamps have an extended tail region, forming a
duplex in addition to the triplex, reducing the need for a long polypurine stretch [166, 167].
Different modifications have been used to improve the PNA invasion [168]. First,
pseudoisocytidine is a modified nucleobase, mimic of a protonated cytosine that renders
Hoogsteen binding pH independent for PNA. Thus, pseudoisocytidine became commonly
used in clamp constructs –bisPNA-, to improve HG-binding in G-rich sequence [169].
Interestingly, bisPNAs can invade both relaxed and supercoiled DNA and are an efficient tool
for targeting homopurine/homopyrimidine sites in buffers containing low salt concentration.
Linear PNAs invade mixed sequences only in supercoiled DNA but not in relaxed DNA
duplexes [170]. To circumvent this limitation, γPNAs are pre-organized in a right-handed
helix [170, 171]. Due to their pre-organization, they are able to invade mixed sequences in
relaxed dsDNA. Remarkably, when containing G-clamp nucleobases, γPNAs invade mixed
sequences under physiological salt and pH conditions [172]. In addition, cell penetration and
strand invasion with PNAs can be improved by addition of positively charged lysine residues
or cell penetrating peptides (CPPs) [173, 174].
PNAs were demonstrated to arrest translation for eukaryotic polymerase when targeting the
template strand [175]. Mutations were detected in PNA binding in a 10 fold increase [176],
suggesting PNA may bind to its cellular target but no specific evidence have shown anti-gene
effect with PNA in living animals. Cell uptake and ionic strength are the main limitations for
the anti-gene strategy with PNAs [177].
21
1.6.3 Locked nucleic acids
Locked nucleic acids (LNA) are nucleotide analogues containing a methylene bridge between
the 2´O and 4´C atom in the ribose (figure. 8). Due to this constraint, the ribose is locked in a
C3´-endo conformation that reduces the flexibility of the ribose and increases the local
organization of the phosphate, as shown by NMR studies and crystallography [178]. LNAs
were independently developed by Wengel´s and Imanishi´s groups [179, 180] and contain the
4 common nucleobases (adenine, guanine, 5-methylcytosine, thymine) that form base pairs
according to Watson-Crick rules. LNA-ONs can be synthesized using conventional
phosphoramidite chemistry allowing automated synthesis of fully LNA ONs or DNA/LNA
mixmers. LNA phosphoramidites and LNA-ONs are commercially available from Exiqon
[181]. LNA chemistry is also compatible with the introduction of non-natural modification,
such as intercalators described in chapter 1.7.
Due to the preorganization of the sugar, LNA possesses high affinity to their complementary
DNA or RNA target. Thermal denaturation studies showed an increase of melting temperature
Tm of +2°C to +8°C per LNA incorporation compared to the unmodified duplexes [182].
LNA-ONs also exhibit very high specificity to their target and improved mismatch
discrimination compared to other nucleic acids [183]. In addition, LNA-ONs are resistant to
nuclease and therefore display high stability in vivo. Together, the benefits from LNA have
allowed the design of shorter ONs efficient in vivo [182-185]. Upon LNA incorporation, the
B-DNA duplex changes conformation to A-DNA [186, 187].
LNA-containing TFOs have been studied extensively because triplexes are less stable than
duplexes due to the repulsion of three negatively charged strands [188]. Due to the sugar
conformation, LNA-TFOs exhibit higher stability than DNA-TFOs. Kinetic studies showed
that LNA-TFOs have similar association rate but significantly lower dissociation rate than
regular TFOs [189]. Although fully LNA-ONs were unable to form triplex, alternating LNA
every 2-3 nucleotides seemed to give the most stable TFOs [190]. In addition, the most stable
TFO at neutral pH contains a majority of thymine than cytosine [190].
22
Figure 8. The structures of nucleotide analogues LNA/α-L-LNA and 2´-amino-LNAs.
LNAs exhibit a β-D-ribose in C3´endo furanose conformation. α-L-LNAs display an α-Lribose in C3´exo furanose conformation. 2´-amino-LNA can be functionalized on the 2´nitrogen with positively charged substituents or a pyrenyl moiety.
Among the various LNA analogues, 2´-amino-LNA is particularly interesting because it
retains the properties due to the N-type furanose conformation such as high thermal stability
[191]. More interestingly, 2´-amino-LNAs can be functionalized with various chemical
substituents [192]. In addition, the synthesis for 2´-amino-LNAs has been adapted for large
scale and makes this modification and its derivatives more available [191] Wengel et al
attached positive moieties to 2´-amino-LNAs in particular amino acid [193] or piperazino
substituents [194] to decrease electrostatic repulsion and thereby increasing affinity.
Although 2´-amino-LNAs destabilize the triplex, 2´-glycylamino-LNAs (glyLNAs) enhance
the triple helix stability by 1.7-3.5°C per modification as compared to native TFO [195].
Other 2´-amino-LNAs modified with a pyrenyl moiety have been used as probes [196-198].
Hdrlicka et al have also used pyrene moieties to form intercalating moieties that will be
discussed in chap 1.6. Many of these modified LNAs were shown to improve triplex
formation, as shown in [199].
α-L-LNAs are diasteroisomers of LNAs with α-L-ribose in C3´-exo conformation [200,
201]. Due to the sugar configuration, LNAs show a RNA-like structure whereas α-L-LNAs
display a DNA like-structure. Similar to LNA, α-L-LNA exhibits higher melting temperature
when hybridized to complementary DNA. 2´-amino-α-L-LNAs and their derivatives have
been developed as diagnostic probes [202, 203] and intercalators for DNA invasion [204,
205]
23
1.7
INTERCALATORS
Intercalation is described as the insertion between two adjacent base pairs in DNA double
helix without breaking the WC hydrogen bonds. Intercalators have specific characteristics
such as size and shape but also polarity and ability to interact non-covalently with DNA [206208]. Intercalators are usually small planar molecules with a cationic charge that enhance
DNA binding. Partial intercalation can also occurs with large polyaromatic molecules with
bulky substituents that prevent a full intercalation. Intercalators usually bind in a non-specific
way to any sequence, based on
π-π
interaction, together with hydrophobic and electrostatic
interactions.
Intercalators can be divided into 3 groups depending on their structure [209].
•
Typical intercalators contain fused aromatic rings.
•
Non typical intercalators contain non fused aromatic rings.
•
Bis-intercalators are molecules containing 2 intercalating moieties connected by an
alkyl linker. In natural compounds, a polypeptid can serve as a linker to substitute to
the alkyl linker and stabilizes further the DNA binding [210]. Interestingly, bisintercalators show higher association and lower dissociation constants than regular
intercalators, decreasing the doses in cancer treatments [210-212].
Based on Lerman, first model in 1961, intercalation changes the double helix structure, by
decreasing the helical twist, stiffening, unwinding and extending the double helix [213].
Based on the structural changes, using UV spectra, circular dichroism or fluorescence assays,
it is possible to determine the mode of intercalation. Many substances or chemically
synthesized compounds were found to intercalate into the double helix.
One of the oldest intercalator is acridine [213]. Acridine was shown to have an anticancer
activity. The most studied intercalator is ethidium bromide. Over the last decades, many
intercalators have been developed in different families for instance: anthracenes, acridines,
anthraquinones, phenanthridines, phenanthrolines and ellipticines. In 2007, four
intercalators were approved by FDA for cancer treatment [103].
However, one limitation of DNA intercalators is their low specificity. An intercalator
connected to an ON can increase affinity but might also increase the affinity for non-desired
target. Covalent attachment of psoralen or acridine or other intercalators has been reported to
enhance the TFO binding and therefore their potential to modulate gene expression.
24
In 2005, Petersen et al developed a new class of intercalators called twisted intercalating
nucleic acids (TINA) covalently attached and inserted as a bulge in the ONs [214]. Unlike
the terminal intercalators, this bulge intercalator could be inserted at several positions in the
sequence, which could considerably increase duplex and triplex stabilities compared to the
single insertion. TINA contains an aromatic group that stacks with the nucleobases and
therefore stabilizes the binding. A triple bond connecting the phosphate and the pyrenyl
moiety allows the aromatic residue to be accurately placed.
TINAs can be placed either at the termini or internally as a bulge in an ON and their effect
depending on their position in duplex or a triplex has been studied thoughtfully. Based on
thermal stability analyses [215], both para- and ortho-TINA molecules should be placed
terminally in the nucleotide sequence to obtain a maximum increase in Tm for antiparallel
duplex. When placed internally, the 2 isomers of TINA affect differently the duplex
stability due to the position of the ethynylpyrene functional group. As a result, ortho-TINA
moiety increased Tm or had no effect whereas para-TINA residue decreased Tm in all
positions in the duplex [215]. Also, ONs including several TINAs exhibited higher stability
when the intercalators were located at both 5´ and 3´ extremities or placed internally
separated by a half or a whole helix turn [215].
Figure 9. Chemical structures of various DNA intercalators.
25
2 AIMS
The aim of this PhD thesis is to develop the bis-locked nucleic acids, (bisLNAs), a new class
of DNA-binding ONs for the anti-gene strategy and DNA detection.
I- Develop bisLNAs with efficient invasion to a single target site at intranuclear pH and
salt conditions
II- Investigate the invasion mechanism and improve the efficiency via the incorporation
of chemical modifications in the bisLNAs
III- Develop methods for the detection of DNA invasion in an intracellular environment
26
3 METHODOLOGY
This section covers the methods used in this thesis.
3.1
FLUORESCENCE BINDING ASSAY
This assay aims to detect and quantify fluorescently labeled ONs hybridized to plasmid DNA.
Under electrophoresis, plasmid-bound ONs migrate slower than free ONs. In our work, most
of the bisLNAs are fluorescently labeled with Cy3 or Cy5. Therefore, using a scanning
system and a software, we measure the signal arising from the ONs bound to the plasmids. To
quantify the signal, we used a titration curve generated by a known amount of fluorescent
bisLNAs. We noticed that the hybridization impacts the fluorescence properties of the
cyanine dyes in the titration curve. Therefore, we mimic the plasmid environment by
hybridizing the bisLNA to a short DNA-ON. Nevertheless, by doing so, we obtained good
approximation in agreement with other methods such as S1 nuclease assay and
electrophoretic mobility shift assay. However, as a limitation, it is challenging to quantify
binding for ONs that contain fluorescent residues such as TINAs or stacking linkers. In
addition, this method also requires that the ON hybridization can withstand electrophoresis.
3.2
ELECTROPHORESIS MOBILITY SHIFT ASSAY
The electrophoretic mobility shift assay (EMSA) is a method used to detect DNA complexes
with a variety of ligands [216]. In our project, we used EMSA to assess the binding of
bisLNAs to the target plasmid. First, plasmid and bisLNA are hybridized under intranuclear
pH and salt conditions. Subsequently, the plasmid is digested using restriction enzymes to
generate short fragments that contain the binding site (150 bp). The linearization reduces the
fragments length and allows the separation between bound and unbound fragments via gel
electrophoresis. In addition, it removes the negative supercoiling and provides a “kick out”
effect, thus we can assess the stability of the complex formed. Then, we performed DNA
staining using ethidium bromide or SYBR gold prior quantification with the Versadoc
software. EMSA is robust and accommodates a wide range of nucleic acid size and binding
under various pH and salt conditions. EMSA is also compatible with a wide range of
structures (single stranded, duplex, triplex and quadruplexes…).
One limitation of EMSA is that during electrophoresis, dissociation of the complex can
prevent the detection. In this case, short electrophoresis time or lower temperature can help to
27
stabilize the complexes. Another major limitation is that migration through the gel depends
on the size but also on the structure. Polyacrylamide gel offers a better resolution than the
agarose gel. However, agarose is easier to use, and less toxic. Finally, EMSA does not
indicate the location of the ligand. For this purpose, another method is required: the chemical
footprinting, which is described in chapter 3.5.
3.3
S1 NUCLEASE ASSAY
S1 nuclease is a 32kDa metalloprotein, purified from Aspergillus oryzae. This enzyme has
the ability to degrade single-stranded DNA/RNA but do not degrade double-stranded DNA or
RNA-DNA duplexes. S1-nuclease hydrolyzes ssDNA 5 times faster than RNA and 75,000
times faster than dsDNA [217], rendering this enzyme particularly useful for certain
applications. The S1 nuclease assay has been widely used to map open regions in the genome
such as inverted repeated sequences [218], hairpins [219] and gene promoter regions [220].
S1 nuclease is also used to detect the displaced DNA strand during invasion of PNAs [221],
bisPNAs [222] and zorro-LNAs [144].
Figure 10. Specificity of S1 nuclease. The bis-locked nucleic acid is drawn in red. (A) Upon
triplex binding, no digestion is observed. (B) Upon triplex-invasion, S1 nuclease cleaves the
displaced strand.
S1 nuclease enzymatic activity is achieved at pH 4.0- 4.3 in presence of divalent Zn2+ ions.
To stop the reaction, S1 nuclease activity is strongly inhibited by chelating agents such as
EDTA or citrate ions. The S1 nuclease assay is of interest due to its simplicity, its robustness
and requires very short enzymatic incubation time. Also, it indicates that binding occurs via
strand displacement and is therefore complementary to the fluorescence binding assay and
EMSA. Importantly, this method can be applied to the bisLNAs including non-natural
modifications.
28
3.4
MELTING ASSAY
A melting assay is a thermal denaturation experiment to assess the stability of a complex. In
this thesis, we used the melting assay to determine the stability of complex formed between
bisLNAs and a DNA target ON [223, 224]. The melting assay is performed using a UVvisible spectrophotometer recording absorbance as a function of the temperature.
Due to the aromaticity of the bases, nucleic acids absorb in the ultraviolet spectrum. By
measuring the absorbance at 260 nm, it is possible to estimate the concentration and the
quantity of nucleic acid in an aqueous solution. In a single-stranded conformation, the DNA
bases are unstacked and absorb more light at 260 nm than in a duplex conformation. Thus,
melting of duplex coincides with a higher absorption (hyperchromicity). Reciprocally,
hybridization of the 2 strands corresponds to a lower absorption decrease (hypochromicity).
The absorption change between duplex at low temperature and dissociated ONs at high
temperature occurs usually in a narrow range of temperature. The melting temperature (Tm)
corresponds to the temperature at which 50% of the complex is associated and 50% is
dissociated.
For nucleic acids, the denaturation is reversible and cooling the samples allows the nucleic
acids to re-hybridize with each other. The association and dissociation via WC-interaction are
relatively fast compared to the temperature gradient (0.2°C/min). Thus, cooling and heating
curves are usually superimposable. However, when the heating and the cooling curves are not
superimposed, the phenomenon is called hysteresis, which means that neither of the heating
nor cooling curves correspond to the true equilibrium curve [223, 225].
It is also possible to observe triplex transition without any inference with duplex transition.
At 295 nm, only triplex formation is associated with a hyperchromism, due to the increase of
number of protonated cytosines [226] whereas no change can be detected for duplex-single
stranded ON transition. However, triplex formation is reported to be slower, 10-100 times
slower than WC-interaction [227]. Due to its slow kinetics, triplex dissociation can thus lead
to a hysteresis with Tm overestimation during the heating and underestimation during the
cooling.
In melting assays, the choice of the buffer is crucial and affects the Tm determination. First,
the buffer should not absorb at the recorded wavelength. Also, the pKa of the buffer should
be close to the desired pH and should not be too temperature dependent because pH changes
may happen during the temperature increase, affecting the stability of DNA structures.
Therefore, we used the sodium phosphate buffer. Temperature gradient should also be
29
adequate, depending on the kinetics of binding in the assays. In addition to the thermal
melting temperature, the study in detail of the melting curves can yield many thermodynamic
data such as enthalpy and entropy, which will not be discussed in this thesis.
3.5
FOOTPRINTING
Footprinting is a method developed for the study of DNA-binding, first reported in 1978
[228]. This method was used with a wide range of ligands (proteins [229], TFOs [230, 231],
polyamides [232], DNA intercalators [233]). Footprinting can be used to determinate the
sequence selectivity, the kinetics and the affinity of a ligand [234, 235].
In footprinting, a ligand protects or allows the double helix to be digested by a cleaving agent.
These products will be missing or detected from the all reactions products, usually observed
as a footprint in the gel. The ideal cleaving agent should cleave independently of the
sequence. (DNase1, S1 nuclease, chloroacetaldehyde). If the cleaving agent digests
randomly, it produces a random distribution of fragment. These fragments vary in length and
thus can be separated in a denaturating polyacrylamide gel. However, in presence of a ligand,
the digestion will produce a specific set of fragments from footprinting in which the region
bound by the ligand creates a ladder of cleavage products. To predict the exact location of
the binding site, footprinting is run together with Sanger´s chain termination method [236].
Sanger sequencing relies on dideoxynucleotides that lack the 3´-hydroxyl substituent. When
incorporated in a polynucleotide chain, the dideoxynucleotides block the elongation and
terminate the chain.
Labeling of the digestion fragments is performed via primer extension. For this purpose, we
used a primer 5´-fluorescently- or radioactively labeled. Radioactivity is the most common
labeling for detection in footprinting [221, 222]. However, there have been significant recent
advances in fluorescence detection that allow separation and detection via capillary
electrophoresis [237]. Because the capillaries have a small diameter (50 µm), they can
dissipate heat very quickly and be run with much higher voltage, thus reducing the run time.
Finally, this method can be automated and is thus suitable for high-throughput [238].
30
Figure 11. Footprinting via polyacrylamide gel or capillary electrophoresis. First, the
plasmid is digested via S1 nuclease or chloroacetaldehyde (CAA) treatment. Then, the
fragments from S1 nuclease or CAA digestion are labeled using a 5´-radioactively- or
fluorescently labeled primer extension. Detection and separation of the fragments are
achieved via polyacrylamide gel or via capillary electrophoresis.
3.6
ROLLING CIRCLE AMPLIFICATION
The rolling circle amplification (RCA) is a method that can amplify the binding to a single
molecule making this method particularly suitable for very sensitive detection applications.
[239]. RCA has been used in many applications to detect DNA [240], mRNA in situ [241243], DNA methylation [244], proteins [245, 246], single nucleotide polymorphisms [247]
and small molecules [248, 249].
The RCA protocol has several steps. The first step is the binding of a padlock probe (PLP).
PLPs are linear ONs designed to have complementary ends to a specific single-stranded
target. When properly hybridized, the padlock extremities can be joined via enzymatic
ligation using T4 ligase to form a circular molecule [250]. Interestingly, the T4 ligase is able
31
to discriminate single nucleotide mismatch [251], rendering the method very sequencespecific. As a second step, after ligation, the circular padlock is linearly amplified by primer
extension to produce a long repetitive ssDNA strand. Finally, hybridization with short
fluorescent ONs produces large fluorescent complexes called rolling circle products (RCPs)
easily detectable. Interestingly, the RCA can be conducted at isothermal reaction, usually at
37°C, in solution, on a solid support, in cells, tissues or bacteria.
Figure 12. RCA protocol. The padlock probe recognizes via its 5´ and 3´ extremities the
target sequence. After ligation of the padlock extremities, the assembled circular DNA
serves as a template for the RCA reaction, which generates a long, single-stranded fragment
that contains thousands of copies of the target sequence. For the detection, fluorophorelabeled probes are hybridized to the RCA products (RCPs).
The RCA step is required to distinguish between fluorescent complexes and free detection
probes in solution. In order to quantify the RCPs, the solution containing RCPs is pumped
through a micro channel and illuminated by laser light. The image data are then collected and
are processed by the Q-Linear software to count the RCPs. However, RCA in solution lacks
the spatial information about the target.
Due to its design, PLP hybridization and RCA can be performed in situ in more complex
environment such as cells or bacteria and provide information about distribution, and location
of the targets. Nilsson et al have employed RCA in cells to detect genomic DNA and mRNA
[252]. Thus, using this method, they detect centromeric regions due to the repetitions of
numerous PLP binding sites in chromosomes [253]. Another method to target DNA single
site was reported, using enzymatic digestion to create ssDNA region thus available for
padlock ligation and RCA amplification [254]. For mRNA detection, the RCA-based method
use an additional reverse transcription step to generate ssDNA, subsequently amplified via
RCA. More recently, Smolina et al used a pair of bisPNAs to pre-open the double helix in
genomic DNA for RCA detection of single genomic sites in the sexual chromosomes [255]
but also single sites in bacterial genomes [256-258].
32
4 RESULTS, DISCUSSION AND PERSPECTIVES
4.1
PAPER I
Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient
invasion of supercoiled duplex DNA
This work is based on previous studies achieved in our group about the anti-gene properties
of LNA-ONs. In our lab, we previously showed that Zorro-LNAs successful invade two
adjacent binding sites [143]. With this design, the Zorro-LNAs were able to stop the
transcription of a reporter gene transfected into cells. However, when targeting one single
binding site, the invasion was severely reduced and no inhibition of the transcription was
observed. In addition, we noticed that Zorro-LNAs and linear LNA-ONs invade supercoiled
DNA but do not withstand linearization of the DNA due to a kicking effect. In order to target
a genomic site, we expect that the transcription will unwind the double helix and therefore we
need to develop LNA constructs able to remain bound to the double helix to block the
transcription process.
To this purpose, we investigated a clamp type of ONs – bisLNAs – to overcome the
limitations described earlier and achieve strong binding to the DNA target. The bisLNAs are
ONs containing DNA and LNA nucleotides, designed so that one region binds via HGinteraction and the other binds via WC-interaction to a specific polypurine sequence.
Because this clamp-concept has not been reported with LNAs, we tested whether it is
beneficial to have LNA modifications in both Hoogsteen arm (HG-arm) and Watson-Crick
arm (WC-arm). This was achieved using short 25mer bisLNAs, containing 10 bases in each
arm and connected via a 5nt linker (bisLNA-20 a/b/c/d/e). These 25mer bisLNAs were
designed to target a plasmid containing six consecutive binding sites. The hybridizations were
performed at pH 5.8 to favor triplex formation and at low salt condition to enhance DNA
opening. Using fluorescence binding assay, our results indicate that bisLNAs have higher
invasion than the constructs lacking LNA in the WC-arm (bisLNA-20a) or in the HG-arm
(bisLNA-20b). The control clamp-construct without LNA nucleotides did not show any
invasion (data not shown). We confirmed these results using S1 nuclease assay, which shows
that the bisLNA binding results from DNA invasion and not only from triplex binding.
33
Our initial aim was to design stable constructs to block the transcription. Therefore, we tested
the stability of these constructs after linearization by electrophoretic mobility shift assay
(EMSA). Remarkably, these 25mer bisLNAs were able to withstand linearization and
electrophoresis as we observed different shift in EMSA in agreement with the number of
bisLNAs bound to the plasmid (between one and six bisLNAs). As expected, WC-LNA-20
fell off during linearization/electrophoresis. Very interestingly, the two unlinked arms TFOLNA-20/WC-LNA-20 formed a stable DNA-binding complex, able to withstand the
linearization,
When tested at intranuclear conditions, bisLNA invasion was severely decreased. We assume
that the higher pH disfavors the triplex binding due to the short length of the HG-arm in the
25mer bisLNAs. Moreover, higher salt concentration increases the duplex stability.
Furthermore, we also tested target plasmids containing one or a two binding sites. Reducing
the number of site correlates with a decrease in bisLNA invasion even at low pH and low salt
concentration (data not shown). Based on this result, we hypothesized that the bisLNAs,
when binding consecutive sites, have a cooperative effect that pre-opens the double helix for
each other, therefore achieving higher invasion.
To address the challenge of targeting a single binding site at intranuclear pH and salt
conditions, we synthesized bisLNA-m30 that contains 15 bases in each region connected via
a similar 5nt linker. Very interestingly, bisLNA-m30 showed trace of binding and invasion,
detectable by fluorescence binding and S1 nuclease assay on a single binding site plasmid. To
further improve efficiency, we designed the tail-clamp bisLNA-m44 with a 15 nt HG-arm
and a extended 29 nt WC-arm, similar to the previously reported bisPNA tail-clamp.
Interestingly, with this new design, the bisLNA-m44 invaded efficiently after 16h and
achieved almost complete binding after 3 days.
Because the length of the HG- and WC arms may affect the bisLNAs efficiency, we tested
the bisLNA-m44-3t with shorter HG-arm, which resulted in higher invasion. Our
interpretation is that the HG-arm, though shorter, exhibits higher stability due to the absence
of 2 cytosines at its 5´ extremity, and thereby results in enhanced invasion. We designed also
the bisLNA-m44-7w with a tail half of the length, resulting in lower invasion than bisLNAm44 tail clamp with 14 nt tail, but higher than the clamp bisLNA-m30. Also supporting the
importance of the tail, a bisLNA–m44e with an LNA-rich tail showed enhanced invasion,
compared to the regular bisLNA-m44.
34
Although triplex helix LNA:DNA:LNA structure has not been studied, we assume that the
positioning of the LNA may affect the invasion. Therefore, we tested bisLNAs with parallel
LNA positioning, meaning that LNAs are in the same bases triplets (bis-m44a/c/d) and
antiparallel bisLNA with LNAs placed in alternating bases triplets (bis-m44b/e). Our results
point out that that efficient invasion requires the highest number of LNA nucleotides, but is
rather independent on the parallel/antiparallel positioning.
The stability of the tail-clamp bisLNAs was assessed by EMSA. Similar to the 25mer
bisLNAs, the bis-LNAm44 and its derivatives could withstand linearization. The unlinked
arms (TFO-LNA-m44 + WC-LNA-m44) could withstand linearization whereas the WCLNA-m44 alone fell off. Interestingly, the stability after linearization seems to correlate with
the efficiency of bisLNA invasion, supporting that all the constructs have same ability to
withstand linearization.
Using the melting assay, we demonstrated that bisLNA-m20 has higher thermal stability than
the clamp-constructs, lacking LNAs in one of the arms (bisLNA 20a -20b). As expected, the
clamp bis-m30 showed significantly higher stability than its respective WC-LNA-m30 arm,
due to the absence of TFO stabilization. For the tail-clamp bis-m44, the difference was less
pronounced with its respective WC-LNA-m44 arm because the WC-LNA-m44 showed
already very high stability.
Finally, the specificity to mismatches of the construct was assessed using plasmids containing
mutations in the triplex forming region. Interestingly, the bisLNA-m44 tail-clamp showed
high discrimination as one or two bases mutations decrease drastically the invasion. The
specificity to the target site was further tested using footprinting method. This method shows
the binding in the sequence and indicated DNA opening at the expected polypurine site.
Altogether, the results in this paper indicate that bisLNAs are a new efficient type of LNAONs able to invade single polypurine site with high specificity and affinity at intranuclear pH
and salt conditions. Our results demonstrate also that tail clamp bisLNAs are more efficient
than clamp bisLNAs. Although the bisLNAs require relatively high concentration and long
incubation to achieve complete invasion, we expect that further studies about bisLNA
binding mechanism and the introduction of modifications will increase the bisLNAs potency,
thus reducing the bisLNA concentration required to achieve invasion in a cell.
35
4.2
PAPER II
Next generation bis-locked nucleic acids with stacking linker and 2´-glycylamino-LNA
show enhanced invasion into duplex DNA
This paper is a follow-up study of paper I. The synthesis of bisLNAs, based on
phosphoramidite chemistry, is compatible with non-natural modifications. Due to recent
development of bisLNAs, none of the modifications other than LNAs have been tested yet.
To introduce the suitable modification, we investigated the invasion mechanism for the
bisLNAs. In our previous study, although we demonstrated the advantage of having a
clamp-construct as compared to a linear ON, it was unexpected that the WC-arm and bism44 invade to the same extend at high ON concentration (12 µM). Thus, in this work, we
assessed the effect of the TFO-arm by comparing the invasion efficiency between the
unlinked arms and the WC-arm alone. Using S1 nuclease assay, we found that the HG-arm
hampers the WC-invasion. A similar effect was observed also in a bisLNA with TFO
depleted of LNAs. The bisTFO-1 and-2 including less LNA nucleotides in the TFO-arm
exhibited lower invasion than their respective WC-m44 arm presumably due to the faster
TFO dissociation kinetics. Very interestingly, the observation of this effect has allowed us
to propose a binding mechanism for the bisLNA. First, bisLNAs form a transient complex
via Hoogsteen binding-first until invasion starts via the tail region duplex. Then, several
experimental results support that the invasion starts in the tail region. First, the bisLNA
with tail clamp are more efficient than the bisLNA clamp. Then reducing the tail in a
bisLNA correlate with a decrease in term of invasion. Finally, when mutations were placed
closed to the triplex/duplex junctions, invasion for bis-m44 was severely decreased.
We introduced the intercalator known as TINA to stabilize either the HG-arm or the WCarm, When introduced in the TFO arm, the TINA stabilizes HG triplex, as expected but did
not enhance invasion, forming triplex with a dangling tail, as observed with bis-TINA1.
However, when placed in the WC-arm at the junction, TINA enhances the bisLNA-4
invasion but destabilizes the final complex. This shows that the TINA cannot be easily used
in a bisLNA context. Although, we tested different linkers in paper I, our results pointed
out a particular role for the linker to improve the cooperativity between the WC- and the
HG-arms. Therefore, we thought to introduce into the linker aromatic moieties that allow
stacking to the adjacent nucleobases in the triplex to favor the invasion into the triple helix.
Several stacking linkers were tested (linker N2, M2 and M3). According to S1 nuclease
36
assay, the linker structure is crucial to improve the invasion. Our results from S1 nuclease
assay and our model showed a better stacking for the linker M3 due to the higher rigidity of
this linker compared to other linkers M2 and N2. Although the linker M3 improves the
invasion in a tail-clamp construct bis-m44-M3, reducing the tail renders the linker
inefficient to improve the invasion as shown with bis-m37-M3. This strategy based on the
stacking linker is particularly specific to the clamp structure because it seems to improve
only the proper triplex structure and does not have any effect with a WC-arm.
After testing the linkers, we tested different LNA modifications. We found it interesting to
use positively charged analogues because the triplex is destabilized due to the electrostatic
repulsion of the three negatively charged strands. First, we tested bisLNAs containing
modified LNA nucleotides with a positive charge in the 2´ position of the sugar with glycyl
(glyLNA) and piperazino substituents (pipLNA) or directly attached to the base (5´propargylamino-LNAs). Improvement was observed only with glyLNAs most likely due to
a favorable conformation for the interaction between the positive charge and the negatively
charged backbone. Interestingly, both the bisLNAs clamp and tail-clamp exhibit higher
invasion due to the presence of glyLNAs.
We tested bisLNAs directed to different sites in the genome to assess whether the bisLNA
can be used as a general strategy to target polypurine sites. As expected, the base
composition in the target site influences the stability of the HG-arm and by consequence the
invasion efficiency. Using S1 nuclease assay, we showed that T-rich bisLNAs or mixed
(T,C) bisLNAs can invade with high efficiency, whereas the C-rich bisLNAs are less
efficient presumably due to the protonation required for the cytosine in the TFO. These
results further support the Hoogsteen-first binding mechanism and the need for stable TFO
to achieve efficient invasion.
In order to generate next generation bisLNAs, we assessed whether we can combine the
modifications. With this in mind, we designed a tail-clamp bisLNA with both 2glyLNAs
and linker M3, resulting in efficient invasion at low concentration (<500 nM). In addition,
for this combination, it becomes really interesting to design shorter bisLNAs. With 2
stacking linkers at both extremities, the bis-2M3 without tail shows invasion, which was
not expected. We assume that the M3 moieties are able to stack at both extremities,
stabilizing the triple helical structure. Interestingly, the bis-2M3gly, combining glyLNAs
and two residues M3 achieved even higher invasion after 3 days incubation.
37
Because the linker M3 increases the triplex stability, we were concerned that it affects the
specificity of the invasion. Therefore, we first assessed the invasion using a non-radioactive
S1 nuclease footprinting assay. The method confirmed invasion at the expected polypurine
sites. In addition, no significant difference in the S1 nuclease cleavage was observed
between bis-m44-M3 and the control constructs bis-m44 and the WC-m44. Also, we tested
the bisLNAs specificity in plasmids containing mutations either in triplex or in the tail
region. When the mutations were located in the tail region, the bis-m44-M3 discriminated
less than unmodified bis-m44. However when mutations were placed in the triplex region,
the bis-m44-M3 showed superior discrimination to its corresponding WC-arm. This is most
likely due to the specificity of triplex binding, which is important as first step for the
bisLNA to invade. Finally, we assessed the binding of the bisLNA in more complex
environment. To this purpose, we grew E. coli bacteria containing the target plasmid
pUC19-2BS. After fixation on a microscope slide, the bacteria were incubated with the
bisLNAs. Subsequently, invasion was detected via RCA. In absence of bisLNA, no RCP
was detected, indicating that the RCA does not occur without duplex preopening by
bisLNAs. RCA does not arise from other bacteria components because in presence of the
control plasmid pUC19, very little amount of RCPs was detected, which indicates that the
RCPs come specifically from the target plasmid. This results further supports that the
invasion occurs under physiological salt and pH conditions in bacteria.
As a summary, we successfully designed bisLNAs with significantly more efficient
invasion than WC-LNA-ONs at low ON concentrations (<500 nM). The results are in
agreement with a HG-binding first mechanism. In a cellular environment, we therefore
expect that the bisLNAs reach their genomic target due to the HG recognition. With this in
mind, we designed shorter bisLNAs (30 nt) less prone to unspecific binding in a cellular
environment. In addition, we envisage the use of nucleotide analogues in C-rich bisLNA
that further stabilize the TFO (for instance pseudoisocytidine). The bis-m44-gly-M3 that
contains a stacking linker M3 and glyLNAs has the potential to invade DNA at low ON
concentration (<500 nM) in a sequence specific manner and we predict many applications
in gene therapy.
38
4.3
PAPER III
Identification of bis-locked nucleic acid invasion into duplex DNA by rolling circle
amplification.
This manuscript is another approach to use the bisLNAs invasion. Instead of targeting to
DNA in plasmids to block transcription, we developed a method to detect invasion in a more
complex environment. The detection method is based on rolling circle amplification (RCA),
described in chapter 3.6. However, RCA requires a single stranded sequence to perform
padlock ligation. So far, bisPNAs have been used with success to pre-open DNA duplexes for
RCA. Therefore, we sought to use bisLNAs due to their extended length and their ability to
unwind larger DNA regions under physiological conditions.
We developed the RCA detection in solution of the plasmid pUC19-2BS containing two
bisLNA binding sites. The RCA detection in solution is performed in a more controlled
environment but is also challenging due to the remaining excess of reagents from the
previous enzymatic steps. However, after optimization, we detected production of RCPs in
presence of plasmids pUC19-2BS invaded by the bisLNAs. Interestingly, the RCPs are
specific to invasion and do not arise from ligation on the bisLNAs because no RCPs were
detected with a plasmid control pUC19-0BS co-incubated with bisLNAs. This was as
expected because the padlock and the bisLNAs are not complementary at the ligation site.
Interestingly, small amount of RCPs were detected with the target plasmid alone, which
suggests that due to DNA breathing, padlock probe can bind and produce RCA. Based on this
RCA detection in solution, we also observed that high padlock concentration blocks the
RCA.
Similar approach was used with a single binding site plasmid pDel-1BS. This approach is less
straightforward due to the hybridization between the padlock and the bisLNA. It was
unknown whether a padlock could be ligated to on an LNA-rich ON. Therefore, it was
interesting that both control and target plasmids co-incubated with bisLNAs produced RCPs
despite that no RCPs were detected in target plasmid alone. These results confirm that the
RCPs come from the ligation of the padlocks on the bisLNAs.
To circumvent this limitation, we synthesized bisLNAs incorporating ortho TINA, a specific
duplex intercalator. As we showed previously in paper II, intercalators can affect the stability
and the invasion. We synthesized bisLNA-2 and -3 with the ortho TINA placed respectively
in the clamp or in the tail region. Based on S1 nuclease assay, the bisLNA-2 showed
39
moderate invasion into plasmid pDel-1BS, similar to the unmodified bis-m44. In contrast, the
bisLNA-3 exhibited a lower invasion presumably due to negative contribution from the ortho
TINA in the triplex structure. Then, we assessed bisLNA-2’s ability to pre-open a single site
plasmid for RCA detection in solution. Remarkably, in presence of bisLNA-2, we detected
significantly more RCPs in the target plasmids than in the control plasmids. This result
indicated that the ortho TINA reduces the undesired ligation between the bisLNA and the
padlock extremities when targeting the single binding site plasmid pDel-1BS. Finally, we
tested the one binding site strategy in HEK293 cells, transfected with the plasmid pDel-1BS.
Statistical analyses are ongoing to assess the significance of the varistion of RCP numbers
between the samples.
As reported before, plasmids are circular dsDNA with a topological constraint that favors
DNA supercoiling. However, upon padlock hybridization and ligation, the plasmid and the
padlock form a topological link that might affect the RCA efficiency. As reported, the DNA
polymerase Φ29 from the Bacillus subtilis phage has a high strand displacement activity. In a
linear ssDNA, the polymerase can dissociate the padlock-plasmid hybrid and efficient RCA
is initiated when the circularized padlock is released from the target when reaching one
extremity. However, in a closed ssDNA, it was demonstrated that the polymerase replicates
the probes only a few times. But in our context, we observe that this topological constraint
does not completely prevent RCA. Thus, we hypothesize that RCA depends on additional
parameters such as the length of the DNA open region. Therefore, further investigation is
needed to optimize the RCA conditions for efficient detection of plasmid invasion.
Although further investigation is needed, we expect, with this strategy to detect invasion into
a wide range of supercoiled DNA targets. We envisage the use of this bisLNA-assisted RCA
to target binding sites in mitochondrial or genomic DNA in various cell lines or tissues. In
addition, this method can assess whether a site is accessible for targeting in an anti-gene
strategy.
40
5 CONCLUDING REMARKS
The most crucial finding in this thesis is that the bisLNAs achieve efficient invasion at
intranuclear salt and pH conditions, forming extremely stable complexes. Another interesting
finding is that the TFO-arm in a bisLNA renders the underlying duplex less accessible for
invasion. Based on this finding and in agreement with our experimental results, we suggest
that the bisLNAs bind first with HG-arm, followed by WC-arm invasion initiated in the tail
region. These results indicate the possible use of bisLNAs as anti-gene-ONs.
Our results indicate that DNA invasion depends on many parameters. The base composition,
the linker and the length of the arms strongly impact the invasion in bisLNA. There are no
predicting models to guide the design of invading ONs. Thus, trial and error is so far the only
approach to develop more efficient ONs. However, we found out how to design efficient
bisLNAs. As a first design rule, bisLNAs require a stable TFO with slow dissociation. Then,
to circumvent possible limitation arising from the TFO, bisLNA should incorporate a new
linker stacking to the adjacent nucleobases. Further invasion can be obtained by incorporating
glyLNAs. However, many other modifications available have not been tested, thus bisLNAs
may become even more potent.
41
6 ACKNOWLEDGMENTS
I would like to thank in particular
My supervisor Professor Edvard Smith for giving me the opportunity to do a PhD at
Karolinska Institutet, for all the passionate discussions about the bisLNAs, his help to become
independent and organized scientist and his ability to stay available even during travels and
holidays.
My co-supervisor dr Karin Lundin for many interesting discussions, correcting so many
times my manuscripts, the nice company in the office and the help to solve all the problems
along the PhD
My co-supervisor dr Samir EL Andaloussi for coming in our office, to start great
discussions sharing many advises taking me to the gym.
My collaborators from University of Southern Denmark, professors Jesper Wengel, Erik
Pedersen and Per Jørgensen. Thanks you for the synthesis of so many oligonucleotides. It
was a pleasure to visit you in Odense to have these intensive but productive discussions.
Professor Mats Nilsson thanks so much for accepting me in your laboratory and sharing your
great knowledge about rolling circle amplification. In your laboratory, I had the chance to
meet Tomasz Krzywkowski, a very talented and nice guy.
Professor Lennart Nilsson and Alessandra Villa for the discussions about triplex stability
and Mauricio Esguerra for the very nice illustrations of bisLNAs.
Professor Jean Louis Mergny for having me in your laboratory in Bordeaux. It has been a
great week. Special thanks for Aurore Guédin teaching me all these new methods.
Professor Roger Stromberg, Alice Ghidini and Martina Herrera for letting me using the
spectro-analyzer and giving help whenever needed.
Thanks to Rula Zain and Helen Bergquist, from Stockholm University for their help with
footprinting and radioactivity work.
The member of the PhosChemRec, meeting you every time in a different city has been a great
experiences and a great learning. Thanks in particular to Lieselot.
Thanks to the people in Novum that have made this building a nice workplace.
42
People in my office: Burcu for your support during these years and for reminding me that is
going to be fine and also for making fun of everything. Thanks Dara for always-relaxed
attitude, for your massages, for taking care of our office and helping me through the last steps
finish my PhD. Thanks Eman for your kindness and also your always interesting discussions
about Egypt.
In the MCG group, many thanks to the people in the nitrogen filling club. Thanks Abdul for
all the candies and always nice company, Thanks Janne for gladly sharing the bench with me
although I was taking all the space, Vladimir for your help with radioactivity and for sharing
the same passion than for gels, Olof for following me during the most stressful part of my
PhD and for all your jokes. Joel the gourmet, always funny and serious at the same time,
Oscar the perfect person to have lunch outside and Cristina for your good company in the
PhosChemRec travels. Thanks Emelie for very good teaching of Swedish. Manuela for
always saying hi in the corridor, Ana for mind opening discussions about arts and
demotivation speeches when it was needed, Sofia for discussion about sci-fi and books,
Thanks for people who have joined the laboratory to make it even better place, Helena that is
very funny in many languages, Giula that can solve any of my problems in the lab, Xin for
your help with the dishes, Niels for your valuable company during weekends and late
evenings. Thanks Dhanu for being a cool dude, Mamiko for your help with my japanese
dropbox, and Fiona, very recently arrived in the lab. Thanks to Hannah, Kathrin and
Kristin always there to help with administration or many other issues.
Thanks to former colleagues, Pedro my old master for all the methods you taught me, Abdi
for amazing discussions in the Fika-room, Alambar for the stories about Patans and
mysteries of Pakistan. Iiulian who helped me to find an apartment, Joanna, Per always very
friendly, Lotta for PCR help, Leo and Jason. Thanks to the nice interships students Izzy and
Fatin, Sophie and Xu. Thanks to Noriko for nice walks and good company in Stockholm.
Many thanks to the people from professor Paolo Macchiarini´s group: Sebastian the boat
party guy and Mei-Ling, hard working but also hard partying, Johannes and Philip for all the
football games, Risul (my general!!!), Neus for letting me play your music instrument once.
Special thanks for Ylva for being such a good friend, Nathalie (very good quizz teammate),
Annika and Antonio (miss you guys)! Thanks to people from the Anesthesia lab, Birgitte
for nice housewarming party, Eva and Kristina and Nicolas for your jokes, it was nice to
hear some french accent!!! Thanks to Caroline at HERM for being such a nice person! And
the japonese team Kazuhiro, Makoto and Taichi!!! Sandra and Rami for your friendship in
the corridor! Thanks Professor Moustapha Hassan and his group with Mona, Maria,
43
Fadwa, Heba and Ibrahim always there at unexpected hours and always funny, Thanks you
to professor Karolina Kublickiene for your support during my PhD. Gracias Nelson! We
managed to communicate without swedish during the 3 first years. Thanks to other people in
Novum for the friendly attitude in the corridor. Many thanks to the staff in restaurant Tango
for the food and the friendship.
In Solna campus, thanks to Soazig for the diner session and Paola my dear flat-mate,
Matteus and Gonzalo for their help in organizing the ski trip, Giulo for nice company during
your internship. Thanks to very nice SciLife group amazingly good at playing curling and
Krubs (how do you write it?). Thanks for letting me participate to your lab fika in particular I
want to thanks Malte (nice party), Yvan, XiaXiong, Amal, Pervan, Anja, Annika, Jessica,
and Thomas.
Thanks to all my flat mates that made my home special: Mike, Mimmi, Heli. Ana, Kim,
Laurent, Matthieu, Chrizz and Emilie for nice time. I also want to thanks other people from
Stockholm: Sia, Charlotte, Anna, Erasmus, Frida, Parviz, Kalle, Krika and Alex. Special
thanks to Hedvig, for all the things together, the concerts and all your talks about social
sciences!!!
Thanks to my family, my brother Valérian for sharing his travel stories, to my sister Alice,
for being so special, to my mum Jacqueline that makes sure I do not get lost during my trips,
to my dad Christian that always plans my holidays in the best way…
44
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