Sequence specific recognition and cleavage of DNA by synthetic molecules

DNA recognition is an essential biological process responsible for the regulation of cellular functions including protein synthesis and cell division and is implicated in the mechanism of action of some anticancer drugs. Studies directed towards defining the elements responsible for sequence specific DNA recognition through the study of the interactions of synthetic organic ligands with DNA are described.

DNA recognition by poly-N-methylpyrrolecarboxamides was studied by the synthesis and characterization of a series of molecules where the number of contiguous N-methylpyrrolecarboxamide units was increased from 2 to 9. The effect of this incremental change in structure on DNA recognition has been investigated at base pair resolution using affinity cleaving and MPE•Fe(II) footprinting techniques. These studies led to a quantitative relationship between the number of amides in the molecule and the DNA binding site size. This relationship is called the n + 1 rule and it states that a poly-N methylpyrrolecarboxamide molecule with n amides will bind n + 1 base pairs of DNA. This rule is consistent with a model where the carboxamides of these compounds form three center bridging hydrogen bonds between adjacent base pairs on opposite strands of the helix. The poly-N methylpyrrolecarboxamide recognition element was found to preferentially bind poly dA•poly dT stretches; however, both binding site selection and orientation were found to be affected by flanking sequences. Cleavage of large DNA is also described.

One approach towards the design of molecules that bind large sequences of double helical DNA sequence specifically is to couple DNA binding subunits of similar or diverse base pair specificity. Bis-EDTA-distamycin-fumaramide (BEDF) is an octaamide dimer of two tri-N methylpyrrolecarboxamide subunits linked by fumaramide. DNA recognition by BEDF was compared to P7E, an octaamide molecule containing seven consecutive pyrroles. These two compounds were found to recognize the same sites on pBR322 with approximately the same affinities demonstrating that fumaramide is an effective linking element for Nmethylpyrrolecarboxamide recognition subunits. Further studies involved the synthesis and characterization of a trimer of tetra-N-methylpyrrolecarboxamide subunits linked by β-alanine ((P4)_(3)E). This trimerization produced a molecule which is capable of recognizing 16 base pairs of A•T DNA, more than a turn and a half of the DNA helix.

DNA footprinting is a powerful direct method for determining the binding sites of proteins and small molecules on heterogeneous DNA. It was found that attachment of EDTA•Fe(II) to spermine creates a molecule, SE•Fe(II), which binds and cleaves DNA sequence neutrally. This lack of specificity provides evidence that at the nucleotide level polyamines recognize heterogeneous DNA independent of sequence and allows SE•Fe(II) to be used as a footprinting reagent. SE•Fe(II) was compared with two other small molecule footprinting reagents, EDTA•Fe(II) and MPE•Fe(II).

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.

DNA-Mediated Charge Transport Devices for Protein Detection

Detection of biologically relevant targets, including small molecules, proteins, DNA, and RNA, is vital for fundamental research as well as clinical diagnostics. Sensors with biological elements provide a natural foundation for such devices because of the inherent recognition capabilities of biomolecules. Electrochemical DNA platforms are simple, sensitive, and do not require complex target labeling or expensive instrumentation. Sensitivity and specificity are added to DNA electrochemical platforms when the physical properties of DNA are harnessed. The inherent structure of DNA, with its stacked core of aromatic bases, enables DNA to act as a wire via DNA-mediated charge transport (DNA CT). DNA CT is not only robust over long molecular distances of at least 34 nm, but is also especially sensitive to anything that perturbs proper base stacking, including DNA mismatches, lesions, or DNA-binding proteins that distort the π-stack. Electrochemical sensors based on DNA CT have previously been used for single-nucleotide polymorphism detection, hybridization assays, and DNA-binding protein detection. Here, improvements to (i) the structure of DNA monolayers and (ii) the signal amplification with DNA CT platforms for improved sensitivity and detection are described.

First, improvements to the control over DNA monolayer formation are reported through the incorporation of copper-free click chemistry into DNA monolayer assembly. As opposed to conventional film formation involving the self-assembly of thiolated DNA, copper-free click chemistry enables DNA to be tethered to a pre-formed mixed alkylthiol monolayer. The total amount of DNA in the final film is directly related to the amount of azide in the underlying alkylthiol monolayer. DNA monolayers formed with this technique are significantly more homogeneous and lower density, with a larger amount of individual helices exposed to the analyte solution. With these improved monolayers, significantly more sensitive detection of the transcription factor TATA binding protein (TBP) is achieved.

Using low-density DNA monolayers, two-electrode DNA arrays were designed and fabricated to enable the placement of multiple DNA sequences onto a single underlying electrode. To pattern DNA onto the primary electrode surface of these arrays, a copper precatalyst for click chemistry was electrochemically activated at the secondary electrode. The location of the secondary electrode relative to the primary electrode enabled the patterning of up to four sequences of DNA onto a single electrode surface. As opposed to conventional electrochemical readout from the primary, DNA-modified electrode, a secondary microelectrode, coupled with electrocatalytic signal amplification, enables more sensitive detection with spatial resolution on the DNA array electrode surface. Using this two-electrode platform, arrays have been formed that facilitate differentiation between well-matched and mismatched sequences, detection of transcription factors, and sequence-selective DNA hybridization, all with the incorporation of internal controls.

For effective clinical detection, the two working electrode platform was multiplexed to contain two complementary arrays, each with fifteen electrodes. This platform, coupled with low density DNA monolayers and electrocatalysis with readout from a secondary electrode, enabled even more sensitive detection from especially small volumes (4 μL per well). This multiplexed platform has enabled the simultaneous detection of two transcription factors, TBP and CopG, with surface dissociation constants comparable to their solution dissociation constants.

With the sensitivity and selectivity obtained from the multiplexed, two working electrode array, an electrochemical signal-on assay for activity of the human methyltransferase DNMT1 was incorporated. DNMT1 is the most abundant human methyltransferase, and its aberrant methylation has been linked to the development of cancer. However, current methods to monitor methyltransferase activity are either ineffective with crude samples or are impractical to develop for clinical applications due to a reliance on radioactivity. Electrochemical detection of methyltransferase activity, in contrast, circumvents these issues. The signal-on detection assay translates methylation events into electrochemical signals via a methylation-specific restriction enzyme. Using the two working electrode platform combined with this assay, DNMT1 activity from tumor and healthy adjacent tissue lysate were evaluated. Our electrochemical measurements revealed significant differences in methyltransferase activity between tumor tissue and healthy adjacent tissue.

As differential activity was observed between colorectal tumor tissue and healthy adjacent tissue, ten tumor sets were subsequently analyzed for DNMT1 activity both electrochemically and by tritium incorporation. These results were compared to expression levels of DNMT1, measured by qPCR, and total DNMT1 protein content, measured by Western blot. The only trend detected was that hyperactivity was observed in the tumor samples as compared to the healthy adjacent tissue when measured electrochemically. These advances in DNA CT-based platforms have propelled this class of sensors from the purely academic realm into the realm of clinically relevant detection.

Studies on 4S and 5S RNA of HeLa cells

SECTION I

Section I is concerned with a partial sequence analysis conducted on 5S RNA from HeLa cells. Analysis of the oligonucleotide pattern after pancreatic ribonuclease digestion of a highly-purified preparation of 5S RNA gave results which were in general agreement with those published for KB cells, both with respect to the identity and the frequency of the partial sequences. However, the presence of a trinucleotide not found in the KB 5S pattern, together with the reproducibly much lower than expected molar yield of the larger oligonucleotides strongly suggested the occurrence of alternate sequences at various sites in the 5S molecules of human cells. The presence of ppGp and pppGp at the 5'-terminus of HeLa 5S RNA was clearly demonstrated. The implications of this finding with regard to the origin of 5S RNA are discussed.

SECTION II

In Section II the proportion of the HeLa cell genome complementary to tRNA was investigated by using RNA- DNA hybridization. The value for saturation of the HeLa DNA by tRNA was found to be 1.1 x 10^-5, which corresponds to about 4900 sites for tRNA per HeLa cell in an exponentially growing culture. Analysis of the nucleotide composition of the hybridized tRNA revealed significant differences from the nucleotide composition of the input tRNA, with the purine to pyrimidine ratio indicating, however, that these differences were not produced by excessive RNase attack of the hybrid. The size of the hybridized tRNA was only moderately smaller than that of the input RNA; the average S value in formaldehyde was 2.7 (corresponding to a length of about 65 nucleotides), suggesting that a relatively small portion near the ends of the hybridized 4S chains had been removed by RNase.

SECTION III

The proportion of the HeLa cell genome complementary to 5S RNA was investigated by using RNA-DNA hybridization. The value for saturation of the HeLa DNA by 5S RNA was found to be 2.3 x 10^-5, which corresponds to about 7,000 sites for 5S RNA per HeLa cell in an exponentially growing culture. Analysis of the nucleotide composition of the hybridized 5S RNA revealed no significant difference from the nucleotide composition of the input RNA. At the RNA to DNA input ratio of 1:1000, the average S value in formaldehyde of the hybridized 5S RNA corresponded to a polynucleotide chain about two-thirds the size of the input RNA.