Biophysical evaluation of antiparallel triplexes for biosensing and biomedical applications.

Polypyrimidine sequences can be targeted by antiparallel clamps forming triplex structures either for biosensing or therapeutic purposes. Despite its successful implementation, their biophysical properties remain to be elusive. In this work, PAGE, circular dichroism and multivariate analysis were used to evaluate the properties of PPRHs directed to SARS-CoV-2 genome. Several PPRHs designed to target various polypyrimidine sites within the viral genome were synthesized. These PPRHs displayed varying binding affinities, influenced by factors such as the length of the PPRH and its GC content. The number and position of pyrimidine interruptions relative to the 4 T loop of the PPRH was found a critical factor, affecting the binding affinity with the corresponding target. Moreover, these factors also showed to affect in the intramolecular and intermolecular equilibria of PPRHs alone and when hybridized to their corresponding targets, highlighting the polymorphic nature of these systems. Finally, the functionality of the PPRHs was evaluated in a thermal lateral flow sensing device showing a good correspondence between their biophysical properties and detection limits. These comprehensive studies contribute to the understanding of the critical factors involved in the design of PPRHs for effective targeting of biologically relevant genomes through the formation of triplex structures under neutral conditions.

Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.

Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Ramon Eritja reports financial support was provided by Tv3 Marathon Foundation. Ramon Eritja reports financial support was provided by Ministry of Science Technology and Innovations. Anna Avino reports financial support was provided by Biomedical Research Network Centre of Bioengineering Biomaterials and Nanomedicine. Ramon Eritja and others have patent #EP21382818.9 pending to CSIC, University of Barcelona, Consorcio Centro de Investigación en red M.P. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.