6th August 2019
Proteins can undergo post-translational modifications (PTM) of their amino acids which can lead to a change in their activity and increase their functional repertoire. The most common of these modifications include phosphorylation, glycosylation, ubiquitination, citrullination, acetylation, methylation, and hydroxylation (Figure A). PTM on proteins can therefore serve as useful biomarkers for disease. However, their discovery and detection in biological fluids is challenging. In most cases antibodies the receptors of choice; but they come with limitations. These include lack of specificity, background binding and batch-to-batch variability. Aptamers have been shown to address these challenges by discriminating against proteins with PTMs due to their facile and reproducible in vitro, along with their chemical stability as well as comparable affinity and selectivity to antibodies.
Figure A: Types of post translational modification (PTM)
At Aptamer Group (AG), we have developed our selection process tailored to customer requirements such as introducing specific buffers, matrices, and most importantly counter-selection steps to ensure that the aptamers are specific to the target of interest (Figure B). Automated parallel processing allows multiple targets and variables thus increasing throughput.
Figure B (Left): Our in vitro selection is carried out under customer defined conditions. We include negative selection to ensure the aptamers are specific to your target.
Figure C (Right): Aptamers can distinguish between closely related proteins which contain PTMs. This BLI assay shows strong binding to the target (red) and weak binding to a closely related negative target (blue). Unspecific aptamers were used with the target as a negative control (grey).
The ability to distinguish between forms of the same protein with differing post-translational modifications is an important advantage of aptamers as tools for identification and detection of biomarkers. We has successfully developed aptamers against various PTM targets and have analyzed the binding selectivity and specificity using biosensor / assay platforms such as BLI, SPR, MST, ITC etc. An example of the our data shows an aptamer generated through the in vitro selection process can distinguish between the pre and post modified forms of peptides using BLI assay (Figure C). A negative citrullinated target was introduced during the counter selection steps enabling enrichment of aptamers and high specificity to the target.
Due to the importance of PTMs in disease pathogenesis there is significant interest in studying them by cell or tissue localization studies. The small structural differences make it difficult for typical antibody reagents to discriminate. Our custom developed aptamers can provide a faster and simpler alternative and overcome the challenges in development of specific detection and purification methods of post-translationally modified proteins.
To know more about our selection process for PTM targets and how it can benefit your research, please contact us using the form below.