Studies of G-quadruplex DNA aptamer discovery and proteomic profiling using a genome-inspired reverse selection approach

Abstract

Over the last few decades aptamers, single stranded oligonucleotides which bind with high affinity and specificity to their target molecule, have become one of the leading alternatives to antibodies as affinity reagents to a wide variety of targets including proteins. They were first discovered in 1990 using the process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). In this process, oligonucleotides are selected from a combinatorial library for high affinity, high selectivity to a specific target. SELEX has discovered many aptamers to a number of targets but does come with limitations that can hinder the process of aptamer discovery, particularly in the case of potential aptamers that form multi-tier G-quadruplexes (G4). G-quadruplex forming sequences are found throughout the human genome and merit investigation as potential aptamers. Our lab has developed a new genome-inspired reverse selection approach that allows us to explore these secondary structures. In the reserve selection approach, specific DNA sequences from the human genome are used for affinity capture of proteins from natural pools such as nuclear or cytoplasmic protein extracts or tissue lysates.

Together, these studies demonstrate the use of natural, genomic G-quadruplex forming DNA sequences as potential aptamers and the role they can play in cancer diagnosis and therapy.

The last selection of this dissertation focuses on (1) investigating the selectivity of the SELEX-selected aptamers Thrombin binding aptamer (TBA) and the VEGF binding aptamer (VBA) for capture from nuclear extracts and (2) investigating the effects of single nucleotide base mutations in the loop region on the specificity of the VEGF promoter sequence used in the above mentioned studies. From the first study we were able to determine that VBA binds to proteins other than its target VEGF165 while TBA captures only thrombin (Thr). This is an important observation, since protein-captured by SELEX-selected aptamers has not generally been described for real biological samples. In the second study we determined that replacing a pyrimidine nucleotide with a purine nucleotide and mutating two bases in the four base loop regions of the VEGF promoter sequence can reduce the number of proteins it binds to and improve its specificity. These studies demonstrate that SELEX-selected aptamers are not necessarily more selective than the sequences we explored in our genome-inspired reverse selection approach and that nucleotide base mutations in the loop regions of the VEGF promoter can improve its specificity.

The second focus of this dissertation is on profiling proteins using genomic G-quadruplex DNA. Using the genome-inspired reverse selection approach we profiled proteins from matched primary tumor and adjacent tissue from different cancer tissue types and different breast cancers. The genomic G-quadruplex DNA used was the G-quadruplex forming promoter sequences c-myc, Rb, and VEGF. From this study we were able to identify proteins only present in the primary tumor tissue, determine similarities in proteins in the match primary tumor and adjacent tissue of the different tissue types and determine similarities in proteins in the matched primary tumor and adjacent tissue of the different breast cancer types. These results provide a basis for the development of a G-quadruplex oligonucleotide-based cancer profiling array platform.

This dissertation first focuses on the G-quadruplex forming promoter sequences from the oncogenes c-myc, Rb, and VEGF as potential aptamers using the genome-inspired reverse selection approach. The in vitro interaction of the promoter sequences with nuclear and cytoplasmic proteins from the triple negative breast cancer cell line MDA-MB-468 were studied and a number of proteins were identified that bind to the G-quadruplex forming sequences. Chromatin Immunoprecipitation (ChIP) was used to study the binding interaction of the promoter sequences with these proteins in live cells. These new DNA-protein interactions not only lead to new aptamers but also could identify new biomarkers not previously known in breast cancer and could play an important role in cancer therapy.