Sterochemically modified polyamides for recognition in the minor groove of DNA

The design of synthetic molecules that recognize specific sequences of DNA is an ongoing challenge in molecular medicine. Cell-permeable small molecules targeting predetermined DNA sequences offer a potential approach for offsetting the abnormal effects of misregulated gene-expression. Over the past twenty years, Professor Peter B. Dervan has developed a set of pairing rules for the rational design of minor groove binding polyamides containing pyrrole (Py), imidazole (Im), and hydroxypyrrole (Hp). Polyamides have illustrated the capability to permeate cells and inhibit transcription of specific genes in vivo. This provides impetus to identify structural elements that expand the repetoire of polyamide motifs with recognition properties comparable to naturally occurring DNA binding proteins. Through the introduction of chiral amino acids, we have developed chiral polyamides with stereochemically regulated binding characteristics. In addition, chiral substituents have facilitated the development of new polyamide motifs that broaden binding site sizes targetable by this class of ligands.

Binding site size limitations of imidazole-pyrrole polyamides for recognition in the minor groove of DNA

The discovery that the three ring polyamide Im-Py-Py-Dp containing imidazole (Im) and pyrrole (Py) carboxamides binds the DNA sequence 5'-(A,T)G(A,T)C(A,T)-3' as an antiparallel dimer offers a new model for the design of ligands for specific recognition of sequences in the minor groove containing both G,C and A,T base pairs. In Chapter 2, experiments are described in which the sequential addition of five N- methylpyrrolecarboxamides to the imidazole-pyrrole polyamide Im-Py-Py-Dp affords a series of six homologous polyamides, Im-(Py)_2-7-Dp, that differ in the size of their binding site, apparent first order binding affinity, and sequence specificity. These results demonstrate that DNA sequences up to nine base pairs in length can be specifically recognized by imidazole-pyrrole polyamides containing three to seven rings by 2:1 polyamide-DNA complex formation in the minor groove. Recognition of a nine base pair site defines the new lower limit of the binding site size that can be recognized by polyamides containing exclusively imidazole and pyrrolecarboxamides. The results of this study should provide useful guidelines for the design of new polyamides that bind longer DNA sites with enhanced affinity and specificity.

In Chapter 3 the design and synthesis of the hairpin polyamide Im-Py-Im-Py-γ-Im- Py-Im-Py-Dp is described. Quantitative DNase I footprint titration experiments reveal that Im-Py-Im-Py-γ-Im-Py-Im-Py-Dp binds six base pair 5'-(A,T)GCGC(A,T)-3' sequences with 30-fold higher affinity than the unlinked polyamide Im-Py-Im-Py-Dp. The hairpin polyamide does not discriminate between A•T and T•A at the first and sixth positions of the binding site as three sites 5'-TGCGCT-3', 5'-TGCGCA-3', and 5 'AGCGCT- 3' are bound with similar affinity. However, Im-Py-Im-Py-γ-Im-Py-Im-PyDp is specific for and discriminates between G•C and C•G base pairs in the 5'-GCGC-3' core as evidenced by lower affinities for the mismatched sites 5'-AACGCA-3', 5'- TGCGTT-3', 5'-TGCGGT-3', and 5'-ACCGCT-3'.

In Chapter 4, experiments are described in which a kinetically stable hexa-aza Schiff base La³⁺ complex is covalently attached to a Tat(49-72) peptide which has been shown to bind the HIV-1 TAR RNA sequence. Although these metallo-peptides cleave TAR site-specifically in the hexanucleotide loop to afford products consistent with hydrolysis, a series of control experiments suggests that the observed cleavage is not caused by a sequence-specifically bound Tat(49-72)-La(L)³⁺ peptide.

Structure-, stereochemistry-, and metal-regulated DNA binding/cleaving molecules

In the cell, the binding of proteins to specific sequences of double helical DNA is essential for controlling the processes of protein synthesis (at the level of DNA transcription) and cell proliferation (at the level of DNA replication). In the laboratory, the sequence-specific DNA binding/cleaving properties of restriction endonuclease enzymes (secreted by microorganisms to protect them from foreign DNA molecules) have helped to fuel a revolution in molecular biology. The strength and specificity of a protein:DNA interaction depend upon structural features inherent to the protein and DNA sequences, but it is now appreciated that these features (and therefore protein:DNA complexation) may be altered (regulated) by other protein:DNA complexes, or by environmental factors such as temperature or the presence of specific organic molecules or inorganic ions. It is also now appreciated that molecules much smaller than proteins (including antibiotics of molecular weight less than 2000 and oligonucleotides) can bind to double-helical DNA in sequence-specific fashion. Elucidation of structural motifs and microscopic interactions responsible for the specific molecular recognition of DNA leads to greater understanding of natural processes and provides a basis for the design of novel sequence-specific DNA binding molecules. This thesis describes the synthesis and DNA binding/cleaving characteristics of molecules designed to probe structural, stereochemical, and environmental factors that regulate sequence-specific DNA recognition.

Chapter One introduces the DNA minor groove binding antibiotics Netropsin and Distamycin A, which are di- and tri(N-methylpyrrolecarboxamide) peptides, respectively. The method of DNA affinity cleaving, which has been employed to determine DNA binding properties of designed synthetic molecules is described. The design and synthesis of a series of Netropsin dimers linked in tail-to-tail fashion (by oxalic, malonic, succinic, or fumaric acid), or in head-to-tail fashion (by glycine, β-alanine, and γ-aminobutanoic acid (Gaba)) are presented. These Bis(Netropsin)s were appended with the iron-chelating functionality EDTA in order to make use of the technique of DNA affinity cleaving. Bis(Netropsin)-EDTA compounds are analogs of penta(N-methylpyrrolecarboxamide)-EDTA (P5E), which may be considered a head-to-tail Netropsin dimer linked by Nmethylpyrrolecarboxamide. Low- and high-resolution analysis of pBR322 DNA affinity cleaving by the iron complexes of these molecules indicated that small changes in the length and nature of the linker had significant effects on DNA binding/cleaving efficiency (a measure of DNA binding affinity). DNA binding/cleaving efficiency was found to decrease with changes in the linker in the order β-alanine > succinamide > fumaramide > N-methylpyrrolecarboxamide > malonamide >glycine, γ-aminobutanamide > oxalamide. In general, the Bis(Netropsin)-EDTA:Fe compounds retained the specificity for seven contiguous A:T base pairs characteristic of P5E:Fe binding. However, Bis(Netropsin)Oxalamide- EDTA:Fe exhibited decreased specificity for A:T base pairs, and Bis(Netropsin)-Gaba-EDT A:Fe exhibited some DNA binding sites of less than seven base pairs. Bis(Netropsin)s linked with diacids have C₂-symmmetrical DNA binding subunits and exhibited little DNA binding orientation preference. Bis(Netropsin)s linked with amino acids lack C₂-symmetrical DNA binding subunits and exhibited higher orientation preferences. A model for the high DNA binding orientation preferences observed with head-to-tail DNA minor groove binding molecules is presented.

Chapter Two describes the design, synthesis, and DNA binding properties of a series of chiral molecules: Bis(Netropsin)-EDTA compounds with linkers derived from (R,R)-, (S,S)-, and (RS,SR)-tartaric acids, (R,R)-, (S,S)-, and (RS,SR)-tartaric acid acetonides, (R)- and (S)-malic acids, N ,N-dimethylaminoaspartic acid, and (R)- and (S)-alanine, as well as three constitutional isomers in which an N-methylpyrrolecarboxamide (P1) subunit and a tri(N-methylpyrrolecarboxamide)-EDTA (P3-EDTA) subunit were linked by succinic acid, (R ,R)-, and (S ,S)-tartaric acids. DNA binding/cleaving efficiencies among this series of molecules and the Bis(Netropsin)s described in Chapter One were found to decrease with changes in the linker in the order β-alanine > succinamide > P1-succinamide-P3 > fumaramide > (S)-malicamide > N-methylpyrrolecarboxamide > (R)-malicamide > malonamide > N ,N-dimethylaminoaspanamide > glycine = Gaba = (S,S)-tartaramide = P1-(S,S)-tanaramide-P3 > oxalamide > (RS,SR)-tartaramide = P1- (R,R)-tanaramide-P3 > (R,R)-tartaramide (no sequence-specific DNA binding was detected for Bis(Netropsin)s linked by (R)- or (S)-alanine or by tartaric acid acetonides). The chiral molecules retained DNA binding specificity for seven contiguous A:T base pairs. From the DNA affinity cleaving data it could be determined that: 1) Addition of one or two substituents to the linker of Bis(Netropsin)-Succinamide resulted in stepwise decreases in DNA binding affinity; 2) molecules with single hydroxyl substituents bound DNA more strongly than molecules with single dimethylamino substituents; 3) hydroxyl-substituted molecules of (S) configuration bound more strongly to DNA than molecules of (R) configuration. This stereochemical regulation of DNA binding is proposed to arise from the inherent right-handed twist of (S)-enantiomeric Bis(Netropsin)s versus the inherent lefthanded twist of (R)-enantiomeric Bis(Netropsin)s, which makes the (S)-enantiomers more complementary to the right-handed twist of B form DNA.

Chapter Three describes the design and synthesis of molecules for the study of metalloregulated DNA binding phenomena. Among a series of Bis(Netropsin)-EDTA compounds linked by homologous tethers bearing four, five, or six oxygen atoms, the Bis(Netropsin) linked by a pentaether tether exhibited strongly enhanced DNA binding/cleaving in the presence of strontium or barium cations. The observed metallospecificity was consistent with the known affinities of metal cations for the cyclic hexaether 18-crown-6 in water. High-resolution DNA affinity cleaving analysis indicated that DNA binding by this molecule in the presence of strontium or barium was not only stronger but of different sequence-specificity than the (weak) binding observed in the absence of metal cations. The metalloregulated binding sites were consistent with A:T binding by the Netropsin subunits and G:C binding by a strontium or barium:pentaether complex. A model for the observed positive metalloregulation and novel sequence-specificity is presented. The effects of 44 different cations on DNA affinity cleaving by P5E:Fe were examined. A series of Bis(Netropsin)-EDTA compounds linked by tethers bearing two, three, four, or five amino groups was also synthesized. These molecules exhibited strong and specific binding to A:T rich regions of DNA. It was found that the iron complexes of these molecules bound and cleaved DNA most efficiently at pH 6.0-6.5, while P5E:Fe bound and cleaved most efficiently at pH 7.5-8.0. Incubating the Bis(Netropsin) Polyamine-EDTA:Fe molecules with K₂PdCl₄ abolished their DNA binding/cleaving activity. It is proposed that the observed negative metalloregulation arises from kinetically inert Bis(Netropsin) Polyamine:Pd(II) complexes or aggregates, which are sterically unsuitable for DNA complexation. Finally, attempts to produce a synthetic metalloregulated DNA binding protein are described. For this study, five derivatives of a synthetic 52 amino acid residue DNA binding/cleaving protein were produced. The synthetic mutant proteins carried a novel pentaether ionophoric amino acid residue at different positions within the primary sequence. The proteins did not exhibit significant DNA binding/cleaving activity, but they served to illustrate the potential for introducing novel amino acid residues within DNA binding protein sequences, and for the development of the tricyclohexyl ester of EDTA as a superior reagent for the introduction of EDT A into synthetic proteins.

Chapter Four describes the discovery and characterization of a new DNA binding/cleaving agent, [SalenMn(III)]OAc. This metal complex produces single- and double-strand cleavage of DNA, with specificity for A:T rich regions, in the presence of oxygen atom donors such as iodosyl benzene, hydrogen peroxide, or peracids. Maximal cleavage by [SalenMn(III)]OAc was produced at pH 6-7. A comparison of DNA singleand double-strand cleavage by [SalenMn(III)]⁺ and other small molecules (Methidiumpropyl-EDTA:Fe, Distamycin-EDTA:Fe, Neocarzinostatin, Bleomycin:Fe) is presented. It was found that DNA cleavage by [SalenMn(III)]⁺ did not require the presence of dioxygen, and that base treatment of DNA subsequent to cleavage by [SalenMn(III)]⁺ afforded greater cleavage and alterations in the cleavage patterns. Analysis of DNA products formed upon DNA cleavage by [SalenMn(III)] indicated that cleavage was due to oxidation of the sugar-phosphate backbone of DNA. Several mechanisms consistent with the observed products and reaction requirements are discussed.

Chapter Five describes progress on some additional studies. In one study, the DNA binding/cleaving specificities of Distamycin-EDTA derivatives bearing pyrrole N-isopropyl substituents were found to be the same as those of derivatives bearing pyrrole N-methyl substituents. In a second study, the design of and synthetic progress towards a series of nucleopeptide activators of transcription are presented. Five synthetic plasmids designed to test for activation of in vitro run-off transcription by DNA triple helix-forming oligonucleotides or nucleopeptides are described.

Chapter Six contains the experimental documentation of the thesis work.

Improving the biological activity of pyrrole-imidazole polyamides

DNA is nature’s blueprint, holding within it the genetic code that defines the structure and function of an organism. A complex network of DNA-binding proteins called transcription factors can largely control the flow of information from DNA, so modulating the function of transcription factors is a promising approach for treating many diseases. Pyrrole-imidazole (Py-Im) polyamides are a class of DNA-binding oligomers, which can be synthetically programmed to bind a target sequence of DNA. Due to their unique shape complementarity and a series of favorable hydrogen bonding interactions that occur upon DNA-binding, Py-Im polyamides can bind to the minor groove of DNA with affinities comparable to transcription factors. Previous studies have demonstrated that these cell-permeable small molecules can enter cell nuclei and disrupt the transcription factor-DNA interface, thereby repressing transcription. As the use of Py-Im polyamides has significant potential as a type of modular therapeutic platform, the need for polyamides with extremely favorable biological properties and high potency will be essential. Described herein, a variety of studies have been performed aimed at improving the biological activity of Py-Im polyamides. To improve the biological potency and cellular uptake of these compounds, we have developed a next-generation class of polyamides bearing aryl-turn moieties, a simple structural modification that allows significant improvements in cellular uptake. This strategy was also applied to a panel of high-affinity cyclic Py-Im polyamides, again demonstrating the remarkable effect minor structural changes can have on biological activity. The solubility properties of Py-Im polyamides and use of formulating reagents with their treatment have also been examined. Finally, we describe the study of Py-Im polyamides as a potential artificial transcription factor.

Design, synthesis and characterization of sequence-specific DNA-cleaving metalloproteins

This thesis describes research pursued in two areas, both involving the design and synthesis of sequence specific DNA-cleaving proteins. The first involves the use of sequence-specific DNA-cleaving metalloproteins to probe the structure of a protein-DNA complex, and the second seeks to develop cleaving moieties capable of DNA cleavage through the generation of a non-diffusible oxidant under physiological conditions.

Chapter One provides a brief review of the literature concerning sequence-specific DNA-binding proteins. Chapter Two summarizes the results of affinity cleaving experiments using leucine zipper-basic region (bZip) DNA-binding proteins. Specifically, the NH_2-terminal locations of a dimer containing the DNA binding domain of the yeast transcriptional activator GCN4 were mapped on the binding sites 5'-CTGACTAAT-3' and 5'ATGACTCTT- 3' using affinity cleaving. Analysis of the DNA cleavage patterns from Fe•EDTA-GCN4(222-281) and (226-281) dimers reveals that the NH_2-termini are in the major groove nine to ten base pairs apart and symmetrically displaced four to five base pairs from the central C of the recognition site. These data are consistent with structural models put forward for this class of DNA binding proteins. The results of these experiments are evaluated in light of the recently published crystal structure for the GCN4-DNA complex. Preliminary investigations of affinity cleaving proteins based on the DNA-binding domains of the bZip proteins Jun and Fos are also described.

Chapter Three describes experiments demonstrating the simultaneous binding of GCN4(226-281) and 1-Methylimidazole-2-carboxamide-netropsin (2-ImN), a designed synthetic peptide which binds in the minor groove of DNA at 5'-TGACT-3' sites as an antiparallel, side-by-side dimer. Through the use of Fe•EDTA-GCN4(226-281) as a sequence-specific footprinting agent, it is shown that the dimeric protein GCN4(226-281) and the dimeric peptide 2- ImN can simultaneously occupy their common binding site in the major and minor grooves of DNA, respectively. The association constants for 2-ImN in the presence and in the absence of Fe•EDTA-GCN4(226-281) are found to be similar, suggesting that the binding of the two dimers is not cooperative.

Chapter Four describes the synthesis and characterization of PBA-β-OH-His- Hin(139-190), a hybrid protein containing the DNA-binding domain of Hin recombinase and the putative iron-binding and oxygen-activating domain of the antitumor antibiotic bleomycin. This 54-residue protein, comprising residues 139-190 of Hin recombinase with the dipeptide pyrimidoblamic acid-β-hydroxy-L-histidine (PBA-β-OH-His) at the NH2 terminus, was synthesized by solid phase methods. PBA-β-OH-His-Hin(139- 190) binds specifically to DNA at four distinct Hin binding sites with affinities comparable to those of the unmodified Hin(139-190). In the presence of dithiothreitol (DTT), Fe•PB-β-OH-His-Hin(139-190) cleaves DNA with specificity remarkably similar to that of Fe•EDTA-Hin(139-190), although with lower efficiency. Analysis of the cleavage pattern suggests that DNA cleavage is mediated through a diffusible species, in contrast with cleavage by bleomycin, which occurs through a non-diffusible oxidant.

Sequence specific complexation of b DNA at sites containing g,c base pairs

A series of eight related analogs of distamycin A has been synthesized. Footprinting and affinity cleaving reveal that only two of the analogs, pyridine-2- car box amide-netropsin (2-Py N) and 1-methylimidazole-2-carboxamide-netrops in (2-ImN), bind to DNA with a specificity different from that of the parent compound. A new class of sites, represented by a TGACT sequence, is a strong site for 2-PyN binding, and the major recognition site for 2-ImN on DNA. Both compounds recognize the G•C bp specifically, although A's and T's in the site may be interchanged without penalty. Additional A•T bp outside the binding site increase the binding affinity. The compounds bind in the minor groove of the DNA sequence, but protect both grooves from dimethylsulfate. The binding evidence suggests that 2-PyN or 2-ImN binding induces a DNA conformational change.

In order to understand this sequence specific complexation better, the Ackers quantitative footprinting method for measuring individual site affinity constants has been extended to small molecules. MPE•Fe(II) cleavage reactions over a 10^5 range of free ligand concentrations are analyzed by gel electrophoresis. The decrease in cleavage is calculated by densitometry of a gel autoradiogram. The apparent fraction of DNA bound is then calculated from the amount of cleavage protection. The data is fitted to a theoretical curve using non-linear least squares techniques. Affinity constants at four individual sites are determined simultaneously. The distamycin A analog binds solely at A•T rich sites. Affinities range from 10^(6)- 10^(7)M^(-1) The data for parent compound D fit closely to a monomeric binding curve. 2-PyN binds both A•T sites and the TGTCA site with an apparent affinity constant of 10^(5) M^(-1). 2-ImN binds A•T sites with affinities less than 5 x 10^(4) M^(-1). The affinity of 2-ImN for the TGTCA site does not change significantly from the 2-PyN value. At the TGTCA site, the experimental data fit a dimeric binding curve better than a monomeric curve. Both 2-PyN and 2-ImN have substantially lower DNA affinities than closely related compounds.

In order to probe the requirements of this new binding site, fourteen other derivatives have been synthesized and tested. All compounds that recognize the TGTCA site have a heterocyclic aromatic nitrogen ortho to the N or C-terminal amide of the netropsin subunit. Specificity is strongly affected by the overall length of the small molecule. Only compounds that consist of at least three aromatic rings linked by amides exhibit TGTCA site binding. Specificity is only weakly altered by substitution on the pyridine ring, which correlates best with steric factors. A model is proposed for TGTCA site binding that has as its key feature hydrogen bonding to both G's by the small molecule. The specificity is determined by the sequence dependence of the distance between G's.

One derivative of 2-PyN exhibits pH dependent sequence specificity. At low pH, 4-dimethylaminopyridine-2-carboxamide-netropsin binds tightly to A•T sites. At high pH, 4-Me_(2)NPyN binds most tightly to the TGTCA site. In aqueous solution, this compound protonates at the pyridine nitrogen at pH 6. Thus presence of the protonated form correlates with A•T specificity.

The binding site of a class of eukaryotic transcriptional activators typified by yeast protein GCN4 and the mammalian oncogene Jun contains a strong 2-ImN binding site. Specificity requirements for the protein and small molecule are similar. GCN4 and 2-lmN bind simultaneously to the same binding site. GCN4 alters the cleavage pattern of 2-ImN-EDTA derivative at only one of its binding sites. The details of the interaction suggest that GCN4 alters the conformation of an AAAAAAA sequence adjacent to its binding site. The presence of a yeast counterpart to Jun partially blocks 2-lmN binding. The differences do not appear to be caused by direct interactions between 2-lmN and the proteins, but by induced conformational changes in the DNA protein complex. It is likely that the observed differences in complexation are involved in the varying sequence specificity of these proteins.

Biological Activity of Rhodium Metalloinsertors and the Design of Bifunctional Conjugates

The Barton laboratory has established that octahedral rhodium complexes bearing the sterically expansive 5,6-chrysene diimine ligand can target thermodynamically destabilized sites, such as base pair mismatches, in DNA with high affinity and selectivity. These complexes approach DNA from the minor groove, ejecting the mismatched base pairs from the duplex in a binding mode termed metalloinsertion. In recent years, we have shown that these metalloinsertor complexes also exhibit cytotoxicity preferentially in cancer cells that are deficient in the mismatch repair (MMR) machinery.

Here, we establish that a sensitive structure-activity relationship exists for rhodium metalloinsertors. We studied the relationship between the chemical structures of metalloinsertors and their effect on biological activity for ten complexes with similar DNA binding affinities, but wide variation in their lipophilicity. Drastic differences were observed in the selectivities of the complexes for MMR-deficient cells. Compounds with hydrophilic ligands were highly selective, exhibiting preferential cytotoxicity in MMR-deficient cells at low concentrations and short incubation periods, whereas complexes with lipophilic ligands displayed poor cell-selectivity. It was discovered that all of the complexes localized to the nucleus in concentrations sufficient for mismatch binding; however, highly lipophilic complexes also exhibited high mitochondrial uptake. Significantly, these results support the notion that mitochondrial DNA is not the desired target for our metalloinsertor complexes; instead, selectivity stems from targeting mismatches in genomic DNA.

We have also explored the potential for metalloinsertors to be developed into more complex structures with multiple functionalities that could either enhance their overall potency or impart mismatch selectivity onto other therapeutic cargo. We have constructed a family of bifunctional metalloinsertor conjugates incorporating cis-platinum, each unique in its chemical structure, DNA binding interactions, and biological activity. The study of these complexes in MMR-deficient cells has established that the cell-selective biological activity of rhodium metalloinsertors proceeds through a critical cellular pathway leading to necrosis.

We further explored the underlying mechanisms surrounding the biological response to mismatch recognition by metalloinsertors in the genome. Immunofluorescence assays of MMR-deficient and MMR-proficient cells revealed that a critical biomarker for DNA damage, phosphorylation of histone H2AX (γH2AX) rapidly accumulates in response to metalloinsertor treatment, signifying the induction of double strand breaks in the genome. Significantly, we have discovered that our metalloinsertor complexes selectively inhibit transcription in MMR-deficient cells, which may be a crucial checkpoint in the eventual breakdown of the cell via necrosis. Additionally, preliminary in vivo studies have revealed the capability of these compounds to traverse the complex environments of multicellular organisms and accumulate in MMR-deficient tumors. Our ever-increasing understanding of metalloinsertors, as well as the development of new generations of complexes both monofunctional and bifunctional, enables their continued progress into the clinic as promising new chemotherapeutic agents.

Investigations of Pyrrole-Imidazole Polyamide Effects on DNA Replication

Pyrrole–Imidazole polyamides are programmable, cell-permeable small molecules that bind in the minor groove of double-stranded DNA sequence-specifically. Polyamide binding has been shown to alter the local helical structure of DNA, disrupt protein-DNA interactions, and modulate endogenous gene expression. Py–Im polyamides targeted to the androgen receptor-DNA interface have been observed to decrease expression of androgen-regulated genes, upregulate p53, and induce apoptosis in a hormone-sensitive prostate cancer cell line. Here we report that androgen response element (ARE)-targeted polyamides induced DNA replication stress in a hormone-insensitive prostate cancer cell line. The ATR checkpoint kinase was activated in response to this stress, causing phosphorylation of MCM2, and FANCD2 was monoubiquitinated. Surprisingly, little single-stranded DNA was exhibited, and the ATR targets RPA2 and Chk1 were not phosphorylated. We conclude that polyamide induces relatively low level replication stress, and suggest inhibition of the replicative helicase as a putative mechanism based on in vitro assays. We also demonstrate polyamide-induced inhibition of DNA replication in cell free extracts from X. laevis oocytes. In this system, inhibition of chromatin decondensation is observed, preventing DNA replication initiation. Finally, we show that Py-Im polyamides targeted to the ARE and ETS binding sequence downregulate AR- and ERG-driven signaling in a prostate cancer cell line harboring the TMPRSS2-ERG fusion. In a mouse xenograft model, ARE-targeted polyamide treatment reduced growth of the tumor.

The application of metallointercalators in recognition of and charge transport in nucleic acids

Metal complexes that utilize the 9,10-phenanthrene quinone diimine (phi) moiety bind to DNA through the major groove. These metallointercalators can recognize DNA sites and perform reactions on DNA as a substrate. The site-specific metallointercalator Λ-1-Rh(MGP)_2phi^(5+) competitively disrupts the major groove binding of a transcription factor, yAP-1, from an oligonucleotide that contains a common binding site. The demonstration that metal complexes can prevent transcription factor binding to DNA site-specifically is an important step in using metallointercalators as therapeutics.

The distinctive photochemistry of metallointercalators can also be applied to promote long range charge transport in DNA. Experiments using duplexes with regions 4 to 10 nucleotides long containing strictly adenine and thymine sequences of varying order showed that radical migration is more dependent on the sequence of bases, and less dependent on the distance between the guanine doublets. This result suggests that mechanistic proposals of long range charge transport must involve all the bases.

RNA/DNA hybrids show charge migration to guanines from a remote site, thus demonstrating that nucleic acid stacking other than B-form can serve as a radical bridge. Double crossover DNA assemblies also provide a medium for charge transport at distances up to 100 Å from the site of radical introduction by a tethered metal complex. This radical migration was found to be robust to mismatches, and limited to individual, electronically distinct base stacks. In single DNA crossover assemblies, which have considerably greater flexibility, charge migration proceeds to both base stacks due to conformational isomers not present in the rigid and tightly annealed double crossovers.

Finally, a rapid, efficient, gel-based technique was developed to investigate thymine dimer repair. Two oligonucleotides, one radioactively labeled, are photoligated via the bases of a thymine-thymine interface; reversal of this ligation is easily visualized by gel electrophoresis. This assay was used to show that the repair of thymine dimers from a distance through DNA charge transport can be accomplished with different photooxidants.

Thus, nucleic acids that support long range charge transport have been shown to include A-track DNA, RNA/DNA hybrids, and single and double crossovers, and a method for thymine dimer repair detection using charge transport was developed. These observations underscore and extend the remarkable finding that DNA can serve a medium for charge transport via the heteroaromatic base stack.

Targeting DNA repeat sequences with Py-Im polyamides

Hairpin pyrrole-imdazole polyamides are cell-permeable, sequence-programmable oligomers that bind in the minor groove of DNA. This thesis describes studies of Py-Im polyamides targeted to biologically important DNA repeat sequences for the purpose of modulating disease states. Design of a hairpin polyamide that binds the CG dyad, a site of DNA methylation that can become dysregulated in cancer, is described. We report the synthesis of a DNA methylation antagonist, its sequence specificity and affinity informed by Bind-n-Seq and iteratively designed, which improves inhibitory activity in a cell-free assay by 1000-fold to low nanomolar IC50. Additionally, a hairpin polyamide targeted to the telomeric sequence is found to trigger a slow necrotic-type cell death with the release of inflammatory molecules in a model of B cell lymphoma. The effects of the polyamide are unique in this class of oligomers; its effects are characterized and a functional assay of phagocytosis by macrophages is described. Additionally, hairpin polyamides targeted to pathologically expanded CTG•CAG triplet repeat DNA sequences, the molecular cause of myotonic dystrophy type 1, are synthesized and assessed for toxicity. Lastly, ChIP-seq of Hypoxia-Inducible Factor is performed under hypoxia-induced conditions. The study results show that ChIP-seq can be employed to understand the genome-wide perturbation of Hypoxia-Inducible Factor occupancy by a Py-Im polyamide.

Recognition of double helical DNA by purine oligonucleotides via triple helix formation

Oligonucleotide-directed triple helix formation is one of the most versatile methods for the sequence specific recognition of double helical DNA. Chapter 2 describes affinity cleaving experiments carried out to assess the recognition potential for purine-rich oligonucleotides via the formation of triple helices. Purine-rich oligodeoxyribonucleotides were shown to bind specifically to purine tracts of double helical DNA in the major groove antiparallel to the purine strand of the duplex. Specificity was derived from the formation of reverse Hoogsteen G•GC, A•AT and T•AT triplets and binding was limited to mostly purine tracts. This triple helical structure was stabilized by multivalent cations, destabilized by high concentrations of monovalent cations and was insensitive to pH. A single mismatched base triplet was shown to destabilize a 15 mer triple helix by 1.0 kcal/mole at 25°C. In addition, stability appeared to be correlated to the number of G•GC triplets formed in the triple helix. This structure provides an additional framework as a basis for the design of new sequence specific DNA binding molecules.

In work described in Chapter 3, the triplet specificities and required strand orientations of two classes of DNA triple helices were combined to target double helical sequences containing all four base pairs by alternate strand triple helix formation. This allowed for the use of oligonucleotides containing only natural 3'-5' phosphodiester linkages to simultaneously bind both strands of double helical DNA in the major groove. The stabilities and structures of these alternate strand triple helices depended on whether the binding site sequence was 5'-(purine)_m (pyrimidine)_n-3' or 5'- (pyrimidine)_m (purine)_n-3'.

In Chapter 4, the ability of oligonucleotide-cerium(III) chelates to direct the transesterfication of RNA was investigated. Procedures were developed for the modification of DNA and RNA oligonucleotides with a hexadentate Schiff-base macrocyclic cerium(III) complex. In addition, oligoribonucleotides modified by covalent attachment of the metal complex through two different linker structures were prepared. The ability of these structures to direct transesterification to specific RNA phosphodiesters was assessed by gel electrophoresis. No reproducible cleavage of the RNA strand consistent with transesterification could be detected in any of these experiments.

Insights into the mechanism of human erythrocyte hexose transport : a transferred NOE study of glucose binding to GLUT1

This study examines binding of α- and β-D-glucose in their equilibrium mixture to the glucose transporter (GLUT1) in human erythrocyte membrane preparations by an ^1H NMR method, the transferred NOE (TRNOE). This method is shown theoretically and experimentally to be a sensitive probe of weak ligand-macromolecule interactions. The TRNOEs observed are shown to arise solely from glucose binding to GLUT1. Sites at both membrane faces contribute to the TRNOEs. Binding curves obtained are consistent with a homogeneous class of sugar sites, with an apparent K_D which varies (from ~30 mM to ~70 mM for both anomers) depending on the membrane preparation examined. Preparations with a higher proportion of the cytoplasmic membrane face exposed to bulk solution yield higher apparent KK_Ds. The glucose transport inhibitor cytochalasin B essentially eliminates the TRNOE. Nonlinearity was found in the dependence on sugar concentration of the apparent inhibition constant for cytochalasin B reversal of the TRNOE observed in the α anomer (and probably the β anomer); such nonlinearity implies the existence of ternary complexes of sugar, inhibitor and transporter. The inhibition results furthermore imply the presence of a class of relatively high-affinity (K_D < 2mM) sugar sites specific for the α anomer which do not contribute to NMR-observable binding. The presence of two classes of sugar-sensitive cytochalasin B sites is also indicated. These results are compared with predictions of the alternating conformer model of glucose transport. Variation of apparent K_D in the NMR-observable sites, the formation of ternary complexes and the presence of an anomer-specific site are shown to be inconsistent with this model. An alternate model is developed which reconciles these results with the known transport behavior of GLUT1. In this model, the transporter possesses (at minimum) three classes of sugar sites: (i) transport sites, which are alternately exposed to the cytoplasmic or the extracellular compartment, but never to both simultaneously, (ii) a class of sites (probably relatively low-affinity) which are confined to one compartment, and (iii) the high-affinity α anomer-specific sites, which are confined to the cytoplasmic compartment.

Indentification and characterization of a novel CA^(2+)-binding protein in avian erythrocytes

A novel Ca^(2+)-binding protein with Mr of 23 K (designated p23) has been identified in avian erythrocytes and thrombocytes. p23 localizes to the marginal bands (MBs), centrosomes and discrete sites around the nuclear membrane in mature avian erythrocytes. p23 appears to bind Ca^(2+) directly and its interaction with subcellular organelles seems to be modulated by intracellular [Ca^(2+)]. However, its unique protein sequence lacks any known Ca^(2+)-binding motif. Developmental analysis reveals that p23 association to its target structures occurs only at very late stages of bone marrow definitive erythropoeisis. In primitive erythroid cells, p23 distributes diffusely in the cytoplasm and lacks any distinct localization. It is postulated that p23 association to subcellular structures may be induced in part by decreased intracellular [Ca^(2+)]. In vitro and in vivo experiments indicate that p23 does not appear to act as a classical microtubule-associated protein (MAP) but p23 homologues appear to be expressed in MB-containing cells of a variety of species from different vertebrate classes. It has been hypothesized that p23 may play a regulatory role in MB stabilization in a Ca^(2+)-dependent manner.

Binucleated (bnbn) turkey erythrocytes were found to express a truncated p23 variant (designated p21) with identical subcellular localization as p23 except immunostaining reveals the presence of multi-centrosomes in bnbn cells. The p21 sequence has a 62 amino acid deletion at the C-terminus and must therefore have an additional ~40 amino acids at the N-terminus. In addition, p21 seems to have lost the ability to bind Ca^(2+) and its supramolecular interactions are not modulated by intracellular [Ca^(2+)]. These apparent differences between p23 and p21 raised the possibility that the p23/p21 allelism could be the Bn/bn genotype. However, genetic analysis suggested that p23/p21 allelism had no absolute correlation with the Bn/bn genotype.

Identification of fucose-α(1-2)-galactose binding proteins in the mammalian brain

Fucose-α(1-2)-galactose (Fucα(1-2)Gal) carbohydrates have been implicated in cognitive functions. However, the underlying molecular mechanisms that govern these processes are not well understood. While significant progress has been made toward identifying glycoconjugates bearing this carbohydrate epitope, a major challenge remains the discovery of interactions mediated by these sugars. Here, we employ the use of multivalent glycopolymers to enable the proteomic identification of weak affinity, low abundant Fucα(1-2)Gal-binding proteins (i.e. lectins) from the brain. End-biotinylated glycopolymers containing photoactivatable crosslinkers were used to capture and enrich potential Fucα(1-2)Gal-specific lectins from rat brain lysates. Candidate lectins were tested for their ability to bind Fucα(1-2)Gal, and the functional significance of the interaction was investigated for one such candidate, SV2a, using a knock-out mouse system. Our results suggest an important role for this glycan-lectin interaction in facilitating synaptic changes necessary for neuronal communication. This study highlights the use of glycopolymer mimetics to discover novel lectins and identify functional interactions between fucosyl carbohydrates and lectins in the brain.

Structurally engineered cytochromes c with novel ligand-binding properties

Semisynthesis of horse heart cytochrome c and site-directed mutagenesis of Saccharomyces cerevisiae (S. c.) iso-1-cytochrome c have been utilized to substitute Ala for the cytochrome c heme axial ligand Met80 to yield ligand-binding proteins (horse heart Ala80cyt c and S.c. Ala80cyt c) with spectroscopic properties remarkably similar to those of myoglobin. Both species of Fe(II)Ala80cyt c form exceptionally stable dioxygen complexes with autoxidation rates 10-30x smaller and O₂ binding constants ~ 3x greater than those of myoglobin. The resistance of O₂-Fe(II)Ala80cyt c to autoxidation is attributed in part to protection of the heme site from solvent as exhibited by the exceptionally slow rate of CO binding to the heme as well as the low quantum yield of CO photodissociation.

UV/vis, EPR, and paramagnetic NMR spectroscopy indicate that at pH 7 the Fe(III)Ala80cyt c heme is low-spin with axial His-OH^- coordination and that below pH ~6.5, Fe(III)Ala80cyt cis high-spin with His-H₂O heme ligation. Significant differences in the pH dependence of the ¹H NMR spectra of S.c. Fe(III)Ala80cyt c compared to wild-type demonstrate that the axial ligands influence the conformational energetics of cytochrome c.

¹H NMR spectroscopy has been utilized to determine the solution structure of the cyanide derivative of S.c. Fe(III)Ala80cyt c. 82% of the resonances in the ¹H NMR spectrum of S.c. CN-Fe(III)Ala80cyt c have been assigned through 1D and 2D experiments. The RMSD values after restrained energy minimization of the family of 17 structures obtained from distance geometry calculations are 0.68 ± 0.11 Å for the backbone and 1.32 ± 0.14 Å for all heavy atoms. The solution structure indicates that a tyrosine in the "distal" pocket of CN-Fe(III)Ala80cyt c forms a hydrogen bond with the Fe(III)-CN unit, suggesting that it may play a role analogous to that of the distal histidine in myoglobin in stabilizing the dioxygen adduct.