Comparison of protein capture from a human cancer cell line by genomic G-quadruplex DNA sequences toward aptamer discovery.

A genome-inspired route to aptamer discovery that expands the sequence space beyond that available in traditional, combinatorial selection approaches is investigated for discovery of DNA-protein interactions in cancer. These interactions could then serve as the basis for new DNA aptamers to cancer-related proteins. The genome-inspired approach uses specific DNA sequences from the human genome to capture proteins from biological protein pools. The use of naturally occurring DNA sequences takes advantage of biological evolution of DNA sequences that bind to specific proteins to perform biological functions. Linking aptamer discovery to nature increa`ses the chances of uncovering protein-DNA affinity binding interactions that have biological significance as well as analytical utility. Here, the focus is on genomic, G-rich sequences that can form G-quadruplex (G4) structures. These structures are underrepresented in combinatorial libraries used for conventional aptamer selection. Additionally, G4-forming sequences are prone to inefficient PCR amplification, further biasing aptamer selection away from these structures. Nature provides a large diversity of G4-forming sequences throughout the human genome. They are prevalent in gene promoter regions, especially in oncogene promoters, and are therefore promising candidates for aptamers to regulatory proteins in cancer. The present work investigates protein capture from nuclear and cytoplasmic extracts of the breast cancer cell line MDA-MB-468 by G4-forming sequences from the CMYC, RB, and VEGF gene promoters. The studies included the effects of modifications of the VEGF sequence on the selectivity of protein capture, from which we identified promising aptamer candidates, subject to further refinement, to the proteins nucleolin and RPL19, both of which play important regulatory functions related to cancer.

In situ imidazole activation of ribonucleotides for abiotic RNA oligomerization reactions.

The hypothesis that RNA played a significant role in the origin of life requires effective and efficient abiotic pathways to produce RNA oligomers. The most successful abiotic oligomerization reactions to date have utilized high-energy, modified, or pre-activated ribonucleotides to generate strands of RNA up to 50-mers in length. In spite of their success, these modifications and pre-activation reactions significantly alter the ribonucleotides in ways that are highly unlikely to have occurred on a prebiotic Earth. This research seeks to address this problem by exploring an aqueous based method for activating the canonical ribonucleotides in situ using 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and imidazole. The reactions were run with and without a montmorillonite clay catalyst and compared to reactions that used ribonucleotides that were pre-activated with imidazole. The effects of pH and ribonucleotide concentration were also investigated. The results demonstrate the ability of in situ activation of ribonucleotides to generate linear RNA oligomers in solution, providing an alternative route to produce RNA for use in prebiotic Earth scenarios.