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The Study of a Hybrid-SELEX Method for the Development and Characterisation of an Aptamer to the Dendritic Cell Surface Receptor: Clec9A

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posted on 16.09.2022, 20:19 authored by Palmer, Rebecca

Current immunotherapies utilise antibodies to inhibit or aid immunological functions, or as delivery vehicles capable of targeting therapeutic drugs to the immune system. However, the limitations of antibodies compromise the growth of the field of immunotherapy and thus investigations into new avenues should be fast-tracked. One potential avenue is the use of aptamers. Aptamers are single-stranded pieces of DNA that, through the formation of unique 3D-structures specific to the sequence, have the capacity to bind virtually any molecule of interest due to their creation through systematic evolution of ligands by exponential enrichment, or SELEX. They have shown to exhibit great potential for immunotherapies and specifically, mitigate the limitations presented by antibodies. Aptamers can be developed to bind cell surface receptors that play a role in the immunological response. Furthermore, direct conjugation with immunostimulants such as antigenic peptides or adjuvants could elicit direct and tailored responses. A receptor of interest is the class V C-type lectin receptor, Clec9A. Clec9A is found on a small subset of dendritic cells and its restricted nature makes it highly desirable for targeted antigen delivery.

In this study, I hypothesised that a novel Hybrid-SELEX approach would generate aptamers with high affinity and specificity to their cognate target. This was tested by utilising the Hybrid-SELEX to develop a DNA aptamer to Clec9A. This was achieved by firstly enriching a random oligonucleotide library to recombinant Clec9A immobilized on magnetic beads and then to its natural state as cell receptors on CD8+ cDCs. A bioinformatics pipeline was applied to NGS data to identify potential aptamers that bind Clec9A which was based on a range of criteria shown to be indicative of target affinity. These included reads per million, cluster number, enrichment, and rank. Candidate aptamers were screened for their affinity to Clec9A using two established characterization techniques, a modified gold nanoparticle assay, and circular dichroism (CD) spectroscopy. CD spectroscopy results found four candidate aptamers that were suggested to exhibit an affinity to Clec9A, however, further experiments will need to be performed to confirm this. Furthermore, this study highlights the necessity for standardized and robust characterization procedures to obtain reproducible, comparable, and reliable results.

In summary, A novel hybrid approach of SELEX technology found four potential aptamer candidates that exhibited specific binding interactions towards Clec9a.

History

Copyright Date

17/09/2022

Date of Award

17/09/2022

Publisher

Te Herenga Waka—Victoria University of Wellington

Rights License

Author Retains Copyright

Degree Discipline

Biotechnology

Degree Grantor

Te Herenga Waka—Victoria University of Wellington

Degree Level

Masters

Degree Name

Master of Science

ANZSRC Socio-Economic Outcome code

209999 Other health not elsewhere classified

ANZSRC Type Of Activity code

4 Experimental research

Victoria University of Wellington Item Type

Awarded Research Masters Thesis

Language

en_NZ

Victoria University of Wellington School

School of Biological Sciences

Advisors

Pitman, Janet; Soundy, Jennifer