A novel approach towards aptamer discovery based on ChIP detection method
DNA–protein interactions constitute essential biological processes such as replication, transcription and DNA repair. Mechanistic knowledge of these interactions offers insight into cell regulation, which holds the key to unlocking therapeutic targets for diseases such as cancer.
One common approach for identifying DNA sequence(s) that bind a particular protein-of-interest (POI) is based on a pull-down assay also called as Chromatin immunoprecipitation (ChIP) followed by sequencing. In this assay, DNA-bound proteins are sheared into shorter DNA fragments by either sonication or digestion, and the POI, along with its associated DNA, are selectively immuno- precipitated from the mixture using a protein-specific antibody. The captured DNA sequence(s) is subsequently analyzed in a high- throughput manner by either DNA microarray (ChIP-on-chip) or by next-generation sequencing (ChIP-seq).
Alternatively, there is no question that aptamers can overpower antibodies, as they are highly selective and affinity reagents. Moreover, because of their ease of synthesis and chemical modification along with high stability, increased cellular uptake, no immunogenicity and convenient storage; aptamers have become powerful therapeutic and diagnostic alternatives to antibodies. With recent developments, G-quadruplex (G4) forming sequences from oncogene promoter regions have also shown to provide a basis for aptamers to proteins associated with many cancers.
Taking advantage of G4 sequences stability, Albanese et al. introduced a new approach to aptamer discovery that used oligonucleotides with sequences drawn from the human genome to capture proteins from natural protein pools such as cell lysates and nuclear protein extracts. This way, a new, genome-inspired, reverse selection approach to aptamer discovery was described. The authors demonstrated protein capture from two human breast cancer cells (BT474 and MCF7) using G4 forming sequences from the CMYC, vascular endothelial growth factor (VEGF) and retinoblastoma (RB) human oncogene promoter regions that are over-expressed in cancer.
The oligonucleotides were attached to magnetic beads and incubated with nuclear protein extracts from cultured BT474 and MCF7 breast cancer cells. The captured proteins were extracted from the gels and analysed using LC-MS/MS. Western blot (WB) analysis followed by ChIP –seq were performed to determine whether the proteins identified to the G4- forming oligonucleotides in vitro bind to the same G-rich sequences in the chromatin of live BT474 and MCF-7 cells. The results identified nucleolin (NCL) and ribosomal protein (RPL19) as protein targets based on G4-forming sequences from CMYC, RB and VEGF gene promoter regions. The proteins detected were known for interactions with G4 structures and a significant role in cancer.
Overall, the results offered the basis for development of new aptamers based on G4 sequecnes from the oncogene promoters towards proteins including nucleolin and RPL19. These steps will refine the sequences and tune their selectivity and affinity towards the protein targets. Aptamer group has been consistently involved with custom developed aptamers against such protein targets. The bound sequences are eluted and amplified for additional rounds of screening with increasingly stringent elution as per the customer requirements. For more information regarding the benefits of aptamers in ChIP process, please get in touch.
Reference: Albanese CM, Suttapitugsakul S, Perati S, McGown LB. A genome-inspired, reverse selection approach to aptamer discovery. Talanta. 2018;177:150–156. doi:10.1016/j.talanta.2017.08.093