Synthesis of Guanidinylated-Substituted Polymers that bind Trans-activation Responsive Region of Human Immunodeficiency Virus Type-1 RNA Master’s Thesis Presented to The Faculty of the Graduate School of Arts and Sciences Brandeis University Department of Biochemistry Jason Pontrello, Advisor, Department of Chemistry Melissa Kosinski-Collins, Advisor, Department of Biology In Partial Fulfillment of the Requirements for Master’s Degree by Shakara Lavisha Scott May, 2013
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Synthesis of Guanidinylated-Substituted Polymers that bind Trans-activation Responsive Region of Human Immunodeficiency Virus Type-1 RNA
Master’s Thesis
Presented to
The Faculty of the Graduate School of Arts and Sciences
Brandeis University
Department of Biochemistry
Jason Pontrello, Advisor, Department of Chemistry
Melissa Kosinski-Collins, Advisor, Department of Biology
Each copy of RNA is approximately 10, 000 nucleotides in length and encodes for nine genes
(gag, pol, vif, vpr, rev, tat, vpu, env and nef) (F igure 1.2). Of the nine genes, six encode for
viral accessory proteins, which assist in the proliferation of the HIV-1 virus. The HIV viral
proteins vif and vpu influence the assembly and budding of new virions. Env encodes for the
viral envelope glycoprotein SU (gp160) that is essential for the binding of host cell receptors
and co-receptors. Nef, the negative regulator factor protein participates in cell activation, T-
cell apoptosis and the down-regulation of host molecules that are critical for the development
of cellular and humoral immune responses (Ranjan Das et al. 2005). Rev mediates the
transportation of unspliced messenger RNA (mRNA) from the nucleus into the cytoplasm.
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The molecular functions of viral protein R (vpr) include nuclear import of viral pre-
integration complex (PIC), modulation of T-cell apoptosis, transcriptional co-activation of
viral and host genes, and regulation of nuclear factor kappa B (NF-#$B%-93('(34%CD0E-/%
2011). Tat is a Trans-activating protein that regulates viral replication and gene expression.
Taken together, though all the viral proteins contribute to the processes that fuel the HIV-1
infection and evasion of the immune system, the role of Tat, Rev, and Vpr are considered to
be the largest contributors to the morbidity and mortality of HIV/AIDS (Karn 1999; Romani
2010; Kogan 2011). The numerous functions of Vpr, Rev and Tat in the viral life cycle
suggest that they would be attractive targets for therapeutic intervention and development of
HIV antiviral agents. In this manuscript, we focus on the Tat protein.
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F igure 1.2: O rganization of H I V-1 G enome (top) and the V iral L T R Promoter (bottom).
The Tat gene is encoded by two exons (labeled in red). The first exon codes for the first 72aa
are sufficient for transactivation. The second exon encodes for amino acids 73-104. A
detailed structure of the organization of the HIV LTR promoter is shown at the bottom of the
picture. The HIV LTR promoter contains many binding site and resembles promoters
activated by RNA Polymerase II. Immediately downstream of the start of transcription is the
transactivation response region (TAR). TAR encodes a stem-loop RNA structure that acts a
switch during HIV replication. Tat recruits transcription factors on the LTR to up regulate the
transcription of the HIV-1 genome (Karn 1999; Romani 2010). (This image was adapted
from Karn 1999).
The Role of Tat in H IV-1 Replication and L ife Cycle : HIV virions predominantly
target immune cells expressing glycoprotein CD4 (cluster differentiation 4) and thus infect a
variety of immune cells such as dendritic cells, CD4+ T lymphocytes and macrophages
(Stevenson and Crowe et al. 2003). In addition, recent evidence suggests that natural killer T
cells (NKTs) are also an important target of HIV-1 virions during the early course of
infection (Fleuridor et al. 2003). The significance of the HIV virusFCD4 interaction is
underscored by studies that have demonstrated that the HIV virus is able to target vital
anthropomorphic cells. Two main phases dominate the pathogenesis of HIV-1 virus (F igure
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1.3) (Karn 1999). In the first phase, the virus enters the cell via a fusion mechanism between
the glycoprotein 120 SU (gp120) envelope of the virion and the CD4 cell membrane receptor.
This fusion between the virus and the host cell membrane also requires chemokine
coreceptors CCR5 (predominant during acute and asymptomatic phases of the HIV-1
infection) and CXCR4 (Crowe 2003; Stevenson et al. 2003; Mishra 2008). Once in the
cytosol, the virus uncoats and uses its inherent reverse transcriptase (RT) to synthesize
double-stranded viral DNA. This is followed by nuclear import of the viral DNA. The
accessory protein Rev transports the viral DNA into the nucleus where intergrase (IN)
catalyzes the integration to the host genome (Mishra 2008). The second phase involves viral
gene expression, replication, assembly, and virion maturation (Karn 1999).
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F igure 1.3: The Essential Steps in L ife Cycle of H I V-1. The process of infection includes
fusion of the HIV envelop with the CD4 receptors on host cell membrane; a mechanism
mediated by viral envelop glycoprotein 160. Subsequently the viral RNA is reverse
transcribed to the corresponding double stranded cDNA using viral RT and integrated into
the host cell genome (red arrows) by the enzyme intergrase. Upon activation of the host cells,
Tat is produced and is shown to simultaneously enhance the processivity RNA polymerase
increasing the production of full-length viral mRNAs (blue arrows). Rev transports the
mRNAs to ribosome where the proteins are transcribed followed by assembly into new
virions at the cell membrane (green arrows). (This image was adapted from Weizman
2003).
Once integration happens, owing to the host cell regulatory machinery, the virus can
either remain dormant (viral gene expression is silent) or become activeFa consequence of
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stimulating infected host cells with mitogens (Karn 1999). The activation of transcription for
the proviral genome is regulated by transcription factors: NF-#$ and Sp1 and the Tat protein
(F igure 1.3). The HIV pre-mRNA that is transcribed from the proviral DNA contains several
splicing signals (Mishra 2008). In the nascent stages of the HIV replication cycle mostly 2 kb
mRNA transcripts to be produced (F igure 1.3). These mRNA transcripts are translated into
regulatory proteins: Tat, Nef and Rev (Mishra 2008; Romani 2010). The Tat protein is
imported in the nucleus where it binds to nascent RNA transcript (TAR RNA) and with the
help of Tat-associated kinases (TAK), dramatically stimulates transcription elongation and
increases the production of mRNA transcripts (Karn 1999; Stevenson 2003; Weizman 2003;
Mishra 2008). In order for the lifecycle to shift to the late phases, the production of unspliced
pre-mRNA transcripts are needed for assembly into the progeny virions. Moreover, in order
for HIV to produce its complete range of structural, accessory enzymatic proteins, unspliced
~9 kb and singly spliced ~4 kb transcripts are required (Mishra 2008). Once these unspliced
and singly spliced transcripts are generated they are translocated to the cytoplasm and
ribosomes by viral protein Rev with the help of host cell nuclear export machinery (F igure
1.3).
At the ribosomes, the unspliced RNA transcripts are translated into Gag and Gal-Pol
proteins, while the unspliced RNA is translated into Env, Vpu, Vif, and Vpr. Finally, new
progeny virions are packaged and released through the cell membrane surface of the host cell
by budding (F igure 1.3). Viral proteins Nef and Env mediate the budding mechanism;
degrading and down regulating cell surface CD4, thus avoiding immune response. This
stealthy release of new progeny into the interstitial of the body allows the virus to be
metastasized to other cells without detection and perpetuates the progression to AIDS and
decimated immune systems.
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T rans-activator of T ranscr iption: Tat is one of the six HIV-1 regulatory protein
products essential for transactivation of viral and cellular genes. It is expressed in both the
early and late stages of the viral replication cycle. Tat that is released in the nascent stages of
replication is found in both the nuclei and nucleolus of HIV infected cells; when it is
produced in the later stages, Tat is predominantly found in the extracellular environment. Tat
has a variable length of 86-104 amino acids and is encoded by two exons Fdepending on the
viral strain. The first-exon form encodes the first 72 amino acids, which are sufficient for Tat
transactivation. The second exon codes for amino acids 73-104. Moreover, the two-exon
form has an additional carboxyl terminal that, based upon the viral isolate varies in length
between 86 and 104 amino acids; the additional amino acids are appended at the carboxyl
terminal (Weissman et al. 1998; Jeang 1996; Aboul-ela et al. 1999). The generation of these
two forms of Tat is regulated during translation via splicing mechanisms: the 86 amino acid
version is produced from completely spliced mRNA and the 104 amino acid version from
partially spliced HIV mRNA transcripts (Weissman et al. 1999; Amendt et al. and Bilodeau
et al. 1999). Consequently, the one-exon form of Tat is expressed predominantly during the
nascent stages while the two-exon version of Tat materializes in the later stages (Amendt et
al. 1994; Romani 2010).
There are five structural regions of the Tat protein: the N-terminal domain, which
contains amino acids 1-20, the cysteine rich region that contains seven high conserved
cysteine residues (residues 22-37), the core region (amino acids 37-48), the basic region
(residues 48-72) and the carboxyl terminal domain (C-terminal; residues 72-86) (F igure 1.4).
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F igure 1.4: T rans-activator of T ranscr iption (Tat). L eft: Organization of Tat peptide. Right: Primary structure of HIV-1 Tat peptide with the Arginine-Rich Motif (ARM) residues
48-57 in red (Yang 2005). L eft: Primary structure of HIV-1 Tat peptide.
(The image on right was adapted and modified from Yang 2005).
Previous work has indicated that deletion and substitution experiments of residues in
the Cys-rich region have resulted in loss of trans-activation; suggesting that it is required for
Tat function (formation of intra-molecular disulphide bonds) but they are not directly
involved in TAR recognition (Aboul-ela 1999; Yang 2005). The basic region, which consists
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of Arginine Rich Motif (ARM), is conserved over several strains of the HIV-1 and regulates
the Transactivation activity of Tat. Furthermore, the basic region is an essential requisite for
the interactions between the protein and its nucleic acid conjugate, TAR RNA (Yang 2005).
In addition, some have discovered that the carboxyl-terminal domain (CTD) of Tat represses
the transcription of major histocompatibility class I genes (MCH I), which are the first line of
cell immune defense (Weissman 1998). Overall, Tat is a multifunctional protein that has
significant effects on both the virus and the host cell genes.
Extracellular Tat: In addition to intracellular Tat that activates HIV LTR, Tat is also
found in the extracellular matrix. Extracellular Tat along with helper gp120, are viral
products secreted by HIV-1 infected T-cells in the extracellular environment (Bugatti 2007;
Romani 2010). Cohesively, they act as immune-suppressors, activating quiescent T-cells and
targeting HIV-nonpermissive cells/non-HIV-infected cells for progression of the HIV-1
infection (Litovchick 2001; Bugatti 2007). A compilation of research studies elucidates the
entrance of extracellular Tat into cells via an endocytic pathway by binding to an invariable
amount of cell surface receptors, including vascular endothelial growth factor, heparan
sulfate proteoglycan chemokine receptors CCR2, CCR3 and CXCR4 (Xiao et al. 2000;
Bugatti 2007), and heparan sulfate proteoglycans (HSPGs) (Tyagi et al. 2001; Bugatti 2007).
The bindings of Tat by these receptors increase its local concentration in the extracellular
matrix (ECM) and mediate its internalization and trans-activating activity (Noonan 1996;
Vendeville 2004; Bugatti 2007; Miyauchi 2009). Studies of Tat-derived peptides have
demonstrated that residues 48-60 from the basic domain (protein transduction domain or
PTD) accounts for the functional internalization into cells (Buggati 2007; Romani 2010).
Furthermore, Tat contributes to the development of AIDS and other AIDS-associated
pathologies by concomitantly inducing oxidative stress in the blood-brain barrier cells (ECs)
(Price 2005) and causing apoptosis in cardiomyocytes (Fiala 2004), neurons (Singh 2004)
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and other immune cells. For example, Tat enters host macrophages and inhibits nitric oxide
synthase gene activity. This inhibitory effect of Tat on the production of nitric oxide renders
the host vulnerable to infections, since nitric oxide provides the first line of defense against
opportunistic pathogens (Romani 2010).
T rans-activation Response E lement (T A R) RN A : Replication of HIV-1 LTR
requires Tat to bind to trans-activation response element (TAR) RNA, a conserved 59-base
mRNA transcripts F igure 1.2 (Yang 2005). Several studies performed using mutant HIV-1
variants indicate that the Tat protein and the TAR RNA sequence are necessary for viral
replication and pathogenesis (Jeang et al. 1999; Karn 1999; Harrich et al. 1995). The
structural components of TAR RNA, spanning from nucleotides +1 to +57 (F igure 1.5)
includes: the stem-loop, upper arm, 3-base bulge, and the lower stem (Karn 1999; Yang
2005). The 3- base-bulge along with two base pairs above and below the bulge constitutes the
core elements for Tat binding (Yang 2005). Research has shown that the U-rich 3 base-bulge
residues (U 23, C24, U25 or UUU) near the apex of the TAR RNA stem are necessary for
specific binding and recognition of the Tat protein in vivo trans-activation. The mutations in
TAR RNA that affect the structure and base pairing in the U-rich bulge completely abolish
Tat association.
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F igure 1.5: T rans-activation Response E lement (T A R) RN A . (A) Secondary Structure and
Sequence of HIV-1 TAR RNA with critical trinucleotide residues essential for Tat binding
circled in red and the hexanucleotide loop elements squared in blue. This is the 29 base
residue used and presented in this study. Tat specifically binds and recognizes TAR RNA
through the 3-base-bulge (UCU). In the presence of cyclin T1, conformational rearrangements
in Tat permit interactions above in the apical loop and below in the lower stem (Karn 1999;
Aboul-ela 1996). (B) Molecular schematic of TAR RNA showing the bulge and stem-loop
regions (Ellis et al. 2011).
Binding of Tat to trinucleotide bulge of T A R RN A : It is generally understood that
electrostatic interactions modulate the RNA-protein complexation of TAR-RNA to Tat, a
finding elucidated by!Nuclear Overhauser Effect (NOE). NMR NOE experiments also
showed that upon association with basic residues in the Tat ARM, the configuration of TAR
RNA changes tremendously, allowing Tat to further interact with residues in the stem-region
and loop region (F igure 1.6) (Karn 1999; Anand 2008). Understanding of the dynamics of
Tat-TAR-RNA binding enables the design of drugs that would target the Tat peptide, or
alternatively, the TAR RNA. Furthermore, rational inhibitor designs that mimic the structural
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requirements and specificity for the recognition and binding of Tat to the 3 base-bulge are of
great interest for strategy aimed at controlling HIV-1 replication.
F igure 1.6: Interactions of T A R with the A R M of Tat. (A) The overall three-dimensional
view of Tat binding to the three-base bulge as well as parts of the stem-loop region of TAR.
Tat ARM residues Lys 50, Gln 54 and Arg 55 are highlighted as well as the stem-loop
residues of TAR (Anand 2008). (B) Highlights the Watson-Crick conformation of the
interactions of Lys51 and Arg55 with U10-G17. The guanidinium group of Arg55 is
coordinated to O2 and O4 of U13, O6 of G16, O6 of G17 and O4 of U10. In addition, Lys51
also coordinates to O6 of G17. These interactions are mediated by H-bonds. (C) Schematic
representation of the interactions between TAR nucleotides and residues of Tat (magenta).
(D) Provides a detailed view of Arg55 interactions with bases U13 and G16 in the TAR loop
region (Anand 2008). (This image was adapted from Anand 2008).
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Mutational studies have identified that in addition to acting as the binding site for
Tat, the TAR acts as the recognition signal for Tat cellular cofactor cyclin T1 (CycT1) a
component of the Tat-associated kinase (TAK)/Positive Elongation Factor (P-TEFb) CTD
kinase complex (Garber 1998b; Karn 1999; Raghunathan 2006). The CylcT1 once recruited
by Tat binds the apical stem loop sequence of TAR. It is important to note that, the binding of
the stem-loop sequence by the cofactor cyclin T1 (F igure 1.7) is required only for trans-
activation, but not for Tat binding (Karn 1999). Therefore, interfering with the interaction
between the Tat/CycT1 complexes can also be an attractive target for developing HIV-1
antiviral agents.
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F igure 1.7: The recognition of H IV-1 T A R RN A by Tat and Cyclin T1. The interaction of
HIV-1 Tat with CycT1 is critical for high-affinity, loop-specific binding to TAR RNA
(Garber et al. 1998a-b). The full length HIV-1 Tat protein binds very weakly to TAR RNA in vitro. The apical stem-loop and 3-base bulge sequence of TAR are critical for the highly
cooperative binding of Tat and CycT1 to TAR RNA. Additionally, high-affinity binding of
Tat to TAR RNA can also be achieved upon truncation of the trans-activation domain,
leaving the arginine-rich motif (ARM) of Tat to bind to the bulge of the RNA structure
1998a). Moreover, it has been proposed that binding of Tat to CycT1 induce a
conformational change in Tat, which promotes binding to TAR RNA as well as
concomitantly induces a conformational rearrangement in the apical loop of the TAR RNA
36)0*E6%-%@196-/(+@%0A%.(/:*91:%A(35%CM-);1)%13%-2N%&OOP;BN%(This figure was extracted from
Garber et al. 1998b).
Activation of H I V-L T R by Tat: Some concede that the host cellular transcription
machinery sustains basal levels of HIV-1 transcription (i.e. both short non-polyadenylated
and long polyadenylated mRNA transcripts). However, in the presence of Tat, increased
levels of long favorable HIV-1 mRNA transcripts predominate (Jeang 1996; Mischiati 2001).
The original conclusion to this observed phenomenon was that short transcripts resulted from
aborted transcripts and that TAR acts as a terminator sequence, forcing premature release of
the elongation polymerase in the absence of Tat. At the same time, there has been no
evidence to support this conclusion. Furthermore, in-depth studies have shown that this
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phenomenon occurs because TAR acts as a pause site that result in a brief kinetic block to
transcription (Muesing et al. 1987; Selby et al. 1989; Karn 1999). In the presence of Tat the
kinetic block is deactivated and transcription of viral LTR occurs.
In HIV-1 infected cells, the first step in activation of the HIV-1 LTR is the
recruitment of RNA polymerase II (RNA Pol II) (F igure 1.8). Once the RNA Pol II, along
with its mediators that regulate the carboxyl-terminal domain (CTD) of the enzyme is bound,
several downstream events must occur. The phosphorylation of the CTD by the Cylcin-
Dependent Kinase-7 (CDK-7) component of the Transcription factor II H (TFIIH) complex
allows the RNA POL II to clear the promoter and begin the transcription of TAR. Soon after
initiation and transcription of TAR, RNA Pol II is stalled by the repressive Negative
Elongation Factor (NELF), another component of basal transcription factor TFIIH. The
nascent RNA chain folds into the TAR RNA structure constituted of the 3-base bulge and
apical stem-loop. In order to reinitiate transcription, the HIV regulatory protein Tat is
recruited to the three-base bulge sequence of TAR and subsequently recruits the positive
transcription elongation factor complex b (P-TEFb)/Tat-associated kinase (TAK). The P-
TEFb complex consists of CDK9 and Cyclin T1. Tat interacts directly with the cyclin T1
subunit of P-TEFb through zinc (Zn2+) cation to induce the cooperative binding of Cylcin-
Dependent Kinase-9 (CDK-9) (F igure 1.8). This recruitment enables the phosphorylation of
the negative elongation factors as well as the CTD of RNA Pol II, which allows the RNA Pol
II to transcribe the remainder of the HIV-1 genome (Karn 1999). Furthermore, Tat binding
enhances the processivity of the RNA Polymerase II (RNA Pol II) elongation complex,
which induces transcription of HIV-1 long terminal region (LTR) (Karn 1999).
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F igure 1.8: Model for the activation of RN A polymerase I I by Tat and cellular co-factors. (a) Initiation. The RNA Pol II is recruited to the HIV LTR promoter through
interactions with TFIID and other basal transcription factors such as TFIIH, which contains
CDK7. The CTD of the RNA polymerase is phosphorylated by CDK9 kinase, allowing the
RNA Poll II to clear the promoter and begin transcription. (b) Promoter C learance. The
aborted transcript of RNA folds into stem-loop structure, TAR. In the absence of Tat RNA
pol II (grey) synthesized short non-polyadenylated RNAs (black squiggly line). (c) Tat binds T A R RN A and T A K . Association of Tat to the 3-base-bulge promotes the recruiting of P-
TEFb/TAK, forming a ternary complex by direct binding to Cyclin T1. The interface
between Tat and cyclin T1 is believed to involve cysteine residues from each protein that
participate in zinc binding (Wei et al. 1998 and Karn1999). After p-TEFb is bound, CDK-9
phosphorylates the two negative elongation factors as well as the carboxyl-terminal domain
of RNA Pol II. (d) Tat-activated elongation. The TAR is displaced from the polymerase and
transcription of the remainder of the HIV genome occurs (i.e. HIV LTR region). Tat-TAR
association increases the processivity of the RNA polymerase II. (This image was adapted
from Karn 1999).
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Summary: Altogether, the data available in the literature suggest that inhibition of
Tat/TAR RNA interactions and CyclinT1/TAR interaction could be of great interest for
controlling HIV-1 replication. Accordingly, this knowledge has catalyzed the search for
molecular compounds that specifically block Tat/TAR interactions. In this study we focus on
elucidating the binding of synthetic to the TAR-RNA to further develop a TAR-RNA drug
that may warrant pharmaceutical development.
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Section I I . Cur rent Drug T reatments:
There is currently no cure for HIV. Yet, the HIV pandemic remains one of the most
deadly threats to world health and presents a significant development challenge (Karn 1999;
UNAIDS 2012). There are approximately thirty-three million people living with HIV/AIDS
worldwide. However, in the thirty-two years since the discovery of HIV, only twenty-five
antiviral drugs are available, for mass use and production. These drugs have been able to
reduce HIV prevalence rates but they are by no means effective preventions or cures for the
disease.
Typically the regulation of HIV includes antiretroviral therapy (ART) and Highly
Active Antiretroviral Therapy (HAART). There are over twenty-three U.S. Food and Drug
Administration (FDA) approved antiretroviral drugs that are used to treat the disease. The
function of ARTs is to repress the growth and reproduction of HIV as well as allow people
infected to live longer, healthier lives. Using several of these drugs in combination also
allows for the rebuilding of the immune system. These drugs are classified by the phase of
the retrovirus life cycle that the drug inhibits; the seven categories are as follows: Entry
G eneral Succinimidyl Ester Substituted Polymeric Scaffold: The succinimidyl substituted
polymers was synthesized under inert nitrogen (N2) atmosphere by ring-opening-metathesis
polymerization using bispyridine-carbene catalyst (6) (Scheme 4). Control over average
polymer length was accomplished by variation of the monomer-to-initiator (M/I) catalyst
stoichiometric ratio. Ratios of 10:1 monomer: catalyst, 25:1, 50:1, and 100:1 were used to
obtain polymers of average lengths n~10, n~25, n~50 and n~100 (7a-d). A solution of NHS
ester monomer (3) dissolved in anhydrous (degassed) dichloromethane was cooled in a dry
ice/isopropanol bath to -`U%rQN%H61%8024@1)(\-3(0/%)1-93(0/%7-+%(/(3(-31:%*80/%-::(3(0/%0A%
solution of ruthenium catalyst (6) in degassed dichloromethane and termination after
complete consumption of the substituted norbornene (3) with ethyl vinyl ether, an electron
rich olefin. The excess ethyl vinyl ether undergoes metathesis with the living polymer chain
end carrying Grubbs catalyst to generate a metathesis-inactive Fischer carbene. After 12
hours, the polymer was precipitated into falcon tube from vortexing diethyl ether (Et2O).
After centrifuging and decanting the tubes, the precipitate was dried under high vacuum,
leaving a greenish solid for the 10-mer and grey solid for the 25-mer, 50-mer and 100-mer.
Polymerization yields ranged between 27 and 37%. The low percent yields were presumably
due to too much dicholoromethaneFa solvent which our polymers was soluble inFin the
vortexing tubes. Note that using more diethyl ether and/or more falcon tubes can optimize the
yields. The resulting polymers were characterized by 1H NMR spectroscopy to afford the
average length (Mn) values. The Mn values were determined by comparing the 1H-NMR
integration signals of the polymer alkene protons to that of the terminal phenyl protons
(Puffer 2007). The calculated Mn values more or less corresponded to the expected values
from the monomer initiator ratios, M:I =10:1, 25:1, 50:1, 100: 1.
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Scheme 4: Synthesis of Succinimidyl Ester Substituted Polymer Scaffolds. Using
Ruthenium Carbene-Initiator Catalyst (6), succinimidyl ester polymers (7a-d) with the
average lengths n~10, n~25, n~50 and n~100 were derived from monomer units of N-succinimidyl ester substituted exo-norbornene (3). Reagents and conditions: i) 10-mer: Amine reactive ester (3) (200.0 mg, 0.8502 mmol 10 eq), Ruthenium Carbene-Initiator
Summary: Altogether, we hypothesized that we would be able to compare the
arginine derivatives of arginine and agmatine to assess the role of the carboxylic acid in the
inhibition of Tat/TAR-RNA interactions. In addition, the variability in polymer length will
also allow us to identify whether the valency increases or decreases the affinity of the
potential RNA-binding drugs. To test this hypothesis, we will assay the RNA-binding
activities of our guanidinylated polymers using an EMSA-based approach. We expect that
since the polymers display guanidiniums an essential requisite for the electrostatic interaction
between the RNA and its endogenous cognate, we should observed some level alteration in
the electrophoretic mobility shift of TAR RNA especially at high concentrations of the
polymer.
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Section I I : E lectrophoretic Mobility Shift Assays The identification of multivalent oligomers that bind TAR RNA with great affinity
and specificity could potentially serve as a potent therapeutic antiviral agent in the fight
against HIV/AIDS. To this end, our laboratory collaborated with Dr. Melissa Kosiniski-
Collins to develop a quantitative assay that allows us to analyze the effects of our ROMP-
derived synthetic polymers on TAR RNA. To investigate whether our synthetic polymeric
guanidinium compounds possess desired reactivity, we performed binding studies using the
Electrophoretic Mobility Shift Assay (EMSA) technique. To implicate TAR RNA folding
and activity, we first dissolved the lyophilized RNA in Tris/EDTA buffer, pH 7.5. In our
protocol, we incubated the wild type HIV-1 TAR RNA at biological cesQ, followed by
subsequent gel electrophoresis (4oC, 130V, 90 mins) to determine the optimal concentration
required for visualization with commercially acquired SYBR® Green Nucleic acid EMSA
stain. By titrating varying concentrations of RNA we observed the gel mobility shifts as
illustrated in F igure 3.3. Using the Typhoon 6410 Variable Mode Scanner, we concluded
that the SYBR® GREEN stain was sensitive to 43 ng of TAR RNA or more. We also
concluded that for future binding/inhibition experiments, TAR RNA concentrations between
200 ]300nM would be ideal.
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F igure 3.3: Native Gel E lectrophoresis of control T A R RN A samples with increasing concentrations. Increasing concentrations of TAR RNA was incubated at 37oC in a total
volume mixture of 15 µL including 32.7% DMSO and 0.67% D2O (final concentration v/v)
to control against polymer solvents. Subsequently, the reaction mixture was electrophoresed
into a 16% native PAGE gel. The gel was visualized using a Typhoon Variable Mode
Scanner at ?n$I%E)11/"+%excitation wavelength of 520 nm. The dark concentrated bands
correspond to the labeled TAR RNA (red arrow). We suspect the band underneath free RNA
is an RNA degradation product (black bracket). TAR RNA concentrations of 200 and 300nM
gave the cleanest band of the group.
Binding of Guanidinylated R O MP-derived Polymers to the H I V-1 T A R RN A in
the absence of Magnesium: In our experimental protocol for monitoring binding between
the arginine 10-mer and TAR RNA, we incubated TAR RNA (300nM) with an increasing
concentration of polymer ranging from &%hV,%30%fUU%hV%CF igure 3.4). After incubation, the
RNA-polymer complexes were resolved by electrophoresis along with control solutions of
strictly polymer and strictly TAR RNA. The various bands (F igure 3.4) suggest that the
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arginine conjugated 10-mer binds the TAR RNA, decreasing its mobility through the gel.
Furthermore, the juxtaposition of the RNA-polymer complex band with the free RNA band
provided convincing evidence that arginine 10-mer is an RNA-binding molecule active in the
1-fUUhV%)-/E1N%The number of bands and their respective intensities are not sensitive to
small changes in polymer concentration, but large changes show an increase in the number of
visualized RNA-polymer complexes and a decrease in free RNA (Table 3.1). In the
concentration range of 1F10 µM, all the retarded band shifts were similar. In contrast, in the
range 200F400 µM we observed three or more high density oligomeric precipitates (F igure
3.4). The fluorescence in the retarded bands was quantified using a fluorescent Image Quant
software.
Table 3.1: Quantified A rginine 10-mer binding exper iment. This table corresponds to the
gel shifts in F igure 3.4. The gel shifts seen in the figure were categorized into free RNA and
complexed/bound RNA. The fluorescence is strictly a measurement of the SYBR stain on the
RNA, which was quantified using Imager Quant software. The values of Fluorescence are
reported in generic Absorbance Units (AU) as obtained from the Typhoon scanner. % bound
was calculated as complexed RNA AU divided by total absorbance times 100%.
10-mer Arginine:
d024@1)%ChVB
Free RNA
Fluorescence
Complex
Fluorescence % Bound
1 3449106 1704197 33.1
5 3510325 1685399 32.4
10 2761667 1260555 31.3
200 2423354 2090077 46.3
400 1708616 3653452 68.1
0 2793172 0 0
After confirming that the developed polymers bind TAR RNA, we inquired about the
binding mechanism of TAR RNA to the polymers. Initially, we hypothesized that since the
10-mer polymer is approximately the same length as the arginine rich domain in Tat, then the
multiple gel shift bands suggest that RNA-polymer interactions were non-specific. In
addition, such bindings could compel the RNA to fold into various configurations that could
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be accounted for in the different gel bands. This hypothesis would further suggest that the
increase in the polymer length and molecular weight may decrease the number of gel shift
bands. To make convincing conclusions about the binding mechanism as well as the effect of
polymer length in the RNA-binding assays, we moved forward with the arginine-conjugated
compounds.
F igure 3.4: T itration of T A R-RN A with R O MP-der ived A rginine Peptidomimetic (10-mer). Increasing amounts of arginine 10-mer polymer were mixed with 25 ng of TAR RNA,
incubated for 30 minutes at 37oC and then separated into a 16 % non-denaturing
polyacrylamide gel for 130 V, for 90 minutes, at 4oC. The gel was stained with SYBR®
Green EMS stain components of the Electrophoretic Mobility-Shift Assay Kit. After staining,
the image was scanned using a Typhoon 9410 Variable Mode Scanner at an excitation
?n$IoE)11/"+%1m9(3-3(0/%wavelength of 520 nm. This native electrophoresis gel shows the
TAR RNA-polymer complexes. The leading band is free TAR (red arrow) while the trailing
bands are hetero-complexes of the arginine 10-mer and TAR (black arrow). In addition, the
absence of the polymer showed no inhibition of the TAR RNA, as was expected.
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In contrast to the arginine 10-mer, the bindings of arginine conjugated 25-mer and
50-mer to the wild type RNA in the 1F&U%hV%)-/ge resulted in only one shifted RNA band
(F igure 3.5-6). The results suggested that the 25-mer and 50-mer specifically recognize the
TAR RNA and retards the migration of the RNA. However, at considerably higher
concentrations (approximately 200!M), we begin to observe multiple retarded band shifts
suggesting less specific binding exist between the TAR RNA and the polymers. Given the
fact that the 25-mer and 50-mer are longer in length than the 10-mer, it is possible that
alternative polymer-RNA stoichiometries exist. The length would attribute multiple RNA
binding sites for a single polymer unit.
Table 3.2: Quantified A rginine 25-mer binding exper iment. This table corresponds to the
gel shifts in F igure 3.5. The gel shifts seen in the figure were categorized into free RNA and
complexed/bound RNA. The fluorescence is strictly a measurement of the SYBR stain on the
RNA, which was quantified using Imager Quant software. The values of Fluorescence are
reported in generic Absorbance Units (AU) as obtained from the Typhoon scanner. % bound
was calculated as complexed RNA AU divided by total absorbance times 100%.
25-mer Arginine:
d024@1)%ChVB
Free RNA
Fluorescence
Complex
Fluorescence % Bound
1 3448799 1202956 25.9
5 3705031 1002198 21.3
10 4131736 993494 19.4
200 2210197 1521484 40.8
0 4112013 0 0
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F igure 3.5: T itration of T A R RN A with R O MP-der ived A rginine Peptidomimetic (25-mer). Increasing amounts of arginine 25-mer polymer were mixed with 25 ng of TAR RNA,
incubated for 30 minutes at 37 oC and then separated into 16 % non-denaturing
polyacrylamide gel at 130 V, for 90 minutes, at 4oC. The gel was stained with the SYBR®
Green EMSA stain component of the Electrophoretic Mobility-Shift Assay Kit. After
staining, the gel was analyzed using a Typhoon 9410 variable mode imager at SYBR®
E)11/"+%excitation wavelength of 520 nm. This native electrophoresis gel shows the TAR
RNA-polymer complexes. The leading band is free RNA (red arrow) while the trailing bands
are hetero-complexes of the arginine 25-mer and RNA (black arrows).
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Table 3.3: Quantified A rginine 50-mer binding exper iment. This table corresponds to the
gel shifts in F igure 3.6. The gel shifts seen in the figure were categorized into free RNA and
complexed/bound RNA. The fluorescence is strictly a measurement of the SYBR stain on the
RNA, which was quantified using Imager Quant software. The values of Fluorescence are
reported in generic Absorbance Units (AU) as obtained from the Typhoon scanner. % bound
was calculated as complexed RNA AU divided by total absorbance times 100%.
50-mer Arginine:
Polymer (hVB
Free RNA
Fluorescence
Complex
Fluorescence % Bound
1 1393080 50890 3.52
5 2865762 82811 2.80
10 2099457 56872 2.64
200 1513969 406472 21.17
0 2815080 0 0
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F igure 3.6: T itration of T A R-RN A with R O MP-der ived A rginine Peptidomimetic (50-mer). Increasing amounts of arginine 50-mer polymer were mixed with 25 ng of TAR RNA,
incubated for 30 minutes at 37 oC and then separated into 16 % non-denaturing
polyacrylamide gel at 130 V, for 90 minutes, at 4oC. The gel was stained with the SYBR®
Green EMSA stain component of the Electrophoretic Mobility-Shift Assay Kit. After
staining, the gel was analyzed using a Typhoon 9410 variable mode imager at SYBR®
E)11/"+%excitation wavelength of 520 nm. This native electrophoresis gel shows the TAR
RNA-polymer complexes. The leading band is free RNA (red arrow) while the trailing bands
are hetero-complexes of the arginine 25-mer and RNA (black arrows).
Due to the success of the preliminary Native-PAGE experiments, our group was
convinced that the equilibrium-binding constant of the arginine polymers to the HIV-1 TAR
RNA could be increased. That is, we believed the binding was being perturbed by the
presence or absence of an important binding cofactor. Initial concerns led to the discussion of
whether the binding environment was optimal. There was a general agreement that the
presence of salt ions and pH were essential for any reputable binding reaction. In fact, the
addition of too much salt may have destabilized the binding interactions between the polymer
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and RNA, decreasing the interactions actually observed on the gel. We concluded that the
initial 750 mM KCl binding buffer was concentrated enough to destabilize the binding
interactions. By lowering the salt concentration, we observed increased binding slightly, but
this enhancement was not significant enough to warrant the end to development of a better
binding reaction buffer. In fact, this lack of significant increase in binding led us to question
whether binding system promotes and maintains the native folding of the TAR RNA.
Incorrect folding may also have influenced the presence other bands in the gel.
To alleviate our concerns about the TAR RNA folding, as well as the hypothesized
multiple binding states of the various polymers, we decided to explore binding buffer systems
that contained both biological KCl and magnesium ion (Mg2+) concentrations. We then
studied the effects of introducing 50 mM KCl and 1 mM MgCl2 in the binding reaction and
same no evidence of misfolded or degraded RNA (Figure 3.7a). The effects of the Mg2+ ion
on the electrophoretic mobility of TAR RNA are shown in F igure 3.7b. Evidently, the
presence of the divalent Mg2+ cation in solution allowed for more sensitive binding between
TAR RNA and the polymers, with interactions being observed within polymer concentration
ranges of 1-10!M.
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Table 3.4: Quantified polymer binding experiment. This table corresponds to the gel shifts
in F igure 3.7b. The gel shifts seen in the figure were categorized into free RNA and
complexed/bound RNA. The fluorescence is strictly a measurement of the SYBR stain on the
RNA, which was quantified using Imager Quant software. The values of Fluorescence are
reported in generic Absorbance Units (AU) as obtained from the Typhoon scanner. % bound
was calculated as complexed RNA AU divided by total absorbance times 100%.
ARG 10-mer:
d024@1)%ChVB Free RNA Complex % Bound
1 1463685 266890 15.4
5 1811174 358795 16.5
10 1675748 274198 14.1
ARG 25-mer
1 1600827 306245 16.1
5 1698572 312540 15.5
10 1698572 312540 15.5
ARG 50-mer
1 1657244 191358 10.4
5 1644738 347365 17.4
10 1550906 253107 14.0
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F igure 3.7: T itration of A rginine-Conjugated polymers with H I V-T A R after pre-incubation in Mg2+
binding buffer . (A) A sample
Native-PAGE gel of TAR RNA
control and Arginine 10-mer
reaction. (B) The titration of RNA
with increasing concentration of
arginine-10-, 25-, 50-mer in the
presence of Mg2+. The titration of
RNA with increasing concentration
of arginine-The large streaking may
be attributed to RNA degradation as
a result a result of increased RNase
activity due the presence of Mg2+ in
solution. The presence of both
degraded and misfolded RNA may
explain the lack of multiple shifts in
RNA mobility since we assumed
that the polymers should bind 100%
of the folded/active RNA.
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To make further conclusions regarding whether our ROMP-derived polymers
possessed unique binding to the TAR RNA, we decided to evaluate the RNA-binding effects
of the free arginine and the arginine-conjugated norbornene monomer (9). The results of
these experiments under the optimized Mg2+ buffer conditions revealed multiple band shifts
for both the arginine-conjugated monomer and the free arginine (F igure 3.8). These results
suggest that the monomers may be affecting the mobility shift of the RNA in two ways: (1)
varying stoichiometric ratios of polymer and RNA, or (2) binding of the monomers forces the
TAR RNA to form a variety of tertiary structures. Both possibilities of large stoichiometries
or multiple configurations could cause the alteration of electrophoretic migration of TAR
RNA. In comparison, the separation of both the free arginine and the conjugated-arginine
norbornene monomer from free TAR RNA is orthogonal to those observed for the arginine
10-mer without the Mg2+. These results allow us to conclude that the arginine 10-mer
polymer binds more specifically to the HIV-1 TAR RNA than the monomers.
,&#
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F igure 3.8: T itration of T A R RN A with arginine conjugated 10-mer (A), norbornene-arginine monomer (B) and free arginine (C). At all concentrations we observed only one
retarded band in lanes labeled A , while in the monomer and free arginine lanes we observed
multiple bands. These results may suggest that the polymeric guanidinium binds the TAR