Protein capture by G-quadruplex DNA for breast cancer profiling

Abstract

G-quadruplexes (G4s) are guanine-rich DNA sequences that fold both intermolecularly and intramolecularly to form four-stranded DNA structures. These structures come from self-stacking of two or more guanine quartets formed through hydrogen bonding. G4s can be further classified by their orientation such as parallel and antiparallel. Guanine quadruplex (G-quadruplex) structures are amongst the various structures found in aptamers. Aptamers have become an important alternative to antibodies as protein affinity reagents due to their comparable and sometimes even higher specificities than antibodies, their low to no immunogenicity, and their unlimited shelf life. However, aptamer discovery has had only limited success despite intensive efforts since their discovery in 1990. Recently, the McGown group introduced a new, human genome inspired "reverse" selection process to overcome some of these obstacles involved in aptamer discovery and enable greater representation of G4 structures due to the diversity found in the human genome. Instead of choosing an aptamer from a combinatorial library of random oligonucleotides for high affinity to a target protein as seen in the process SELEX, this approach uses G4-forming DNA sequences from the human genome to fish out specific-binding proteins from natural protein pools. We are particularly interested in G4-forming sequences in human oncogene promoter regions, where there is a tendency for G4 sequences to occur. The work described in this thesis focuses on protein capture from ductal carcinoma (breast cancer) tissue lysates of different stages by G4 sequences from the Rb, VEGF, and c-myc promoter regions. With these studies, we hope to eventually be able to profile cancers based on their stage and type.

This approach offers a rapid way of profiling breast cancer that requires no large equipment and is fairly inexpensive. Additionally, it provides the opportunity to learn more about protein expression as it relates to cancer to foster diagnosis and therapeutic treatment.

The three G4-forming sequences employed are biotinylated at the 5ꞌ end to attach to streptavidinated magnetic beads. Once attached, the oligonucleotide-modified beads were incubated with the breast cancer lysates. Any weakly bound and unbound proteins were rinsed off while captured proteins were eluted with hot water and collected. Captured proteins were then separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Bands that appeared were mainly around the 50 kDa mark and nearly all sequences showed bands around the 10-15 kDa mark. The bands are indicative of binding by the G4-forming sequences. Protein identity was then interrogated via Western blotting and the results indicate that the Rb and VEGF G4 oligonucleotides are potential candidates for aptamers to human prostatic acid phosphatase (PAP) protein. The two breast cancer lysates that contained PAP and were captured by Rb and VEGF were of the same cancer stage (IIB).