Abstract
Background: Biomarker detection strategies have, in recent years, been moving towards nucleic acid-based detection systems in the form of aptamers, short oligonucleotide sequences which have shown promise in pre-clinical and research settings. One such aptamer is M5-15, a DNA aptamer raised against human alpha synuclein (α-syn) the causative agent in Lewy body and Parkinson's disease (PD) associated dementia. While this aptamer has shown promise, in silico methodologies have demonstrated a capacity to produce aptamers that have higher affinities for their targets than in vitro generated sequences. Methods: A Python script random generated library of DNA sequences were screened based on their thermodynamic stability with the use of DINAMelt server-QuickFold web server. The selected sequences were examined with MFold in order to generate secondary structure data that were used to produce 3D data with the use of RNA composer software. Further on, the structure was corrected and RNA was replaced with DNA and the virtual screening for α-syn aptamer took place with a series of molecular docking experiments with the use of CSD-Discovery-GOLD software. Results: Herein we propose an alternative in silico generated aptamer we call TMG-79 which demonstrates greater affinity for the target compared to M5-15 (M5-15 = -15.9 kcal/mol, TMG-79 = -17.77 kcal/mol) as well as better ChemPLP fitness scoring between the top poses (M5-15 = 32.33, TMG-79 = 53.32). Structural analysis suggests that while there are similarities, the greater potential flexibility of TMG-79 could be promoting greater affinity for the α-syn compared to M5-15. Conclusions: In silico methods of aptamer generation has the potential to revolutionise the field of aptamer design. We feel that further development of TMG-79 and validation in vitro will make it a viable candidate for diagnostic and research use in the future.
Highlights
Biomarker detection has become a critical component in the diagnosis of disease and has emerged as a critical factor in deciding courses of treatment of patients
Over 30 years ago, the first monoclonal antibody was developed for biomarker detection with the first licensed products being deployed in 1986 [1]
While monoclonal antibodies are still considered the gold standard in both clinical and research settings, being involved in a number of key molecular biology techniques like western blotting and immunofluorescence microscopy [4, 5], there are a number of drawbacks in relation to the technology
Summary
Biomarker detection has become a critical component in the diagnosis of disease and has emerged as a critical factor in deciding courses of treatment of patients. Species specific glycosylation of the antibodies can lead to batch-to-batch variations in antibody affinity [7] and the variable fragment crystallisable (fc) domain derived from the host species can cause immunogenicity problems in clinical settings [8] Despite these issues, monoclonal antibodies have seen widespread adoption, but in recent years industry and individual researchers have been looking at potential alternative strategies for biomarker detection. In recent years, been moving towards nucleic acid-based detection systems in the form of aptamers, short oligonucleotide sequences which have shown promise in pre-clinical and research settings One such aptamer is M5-15, a DNA aptamer raised against human alpha synuclein (α-syn) the causative agent in Lewy body and Parkinson's disease (PD) associated dementia. We feel that further development of TMG-79 and validation in vitro will make it a viable candidate for diagnostic and research use in the future
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