An extensible software toolbox for the in silico optimization of aptamers towards arbitrarily complex objectives Nikolaos I. Kalavros 1,2,* , George Tsekenis 1,* 1 Basic Research Center, Biomedical Research Foundation of the Academy of Athens, Athens, Soranou Ephessiou 4, 115 27 Athens, Greece 2 Department of Informatics and Telecommunications, National and Kapodistrian University of Athens, Panepistimiopolis, Ilissia, Athens 157 84, Greece * To whom correspondence should be addressed. Nikolaos I. Kalavros: Tel: +30 6931972159; Fax: +30 6931972159; Email: nkalavros@di.uoa.gr George Tsekenis: Tel: +30 6972662700; Fax: +30 6972662700; Email: gtsekenis@bioacademy.gr Abstract: Nucleic acid aptamers are a class of molecules that show great potential for a variety of applications, including biosensing, diagnostics and therapeutics. They are small molecules, with high affinity and specificity towards a particular ligand. The main method of aptamer identification is Systematic Evolution of Ligands by Exponential Enrichment (SELEX), which was first described over three decades ago. Despite the fact that SELEX and the multitude of its variants have matured significantly since then, key difficulties remain, ranging from implementation issues to cross-reactivity of the aptamer with structurally similar ligands. Due to the vast search space of possible aptamer structures, which is only enlarged when accounting for other desired properties, in silico simulations are highly desirable. Herein, we present a toolbox that aims to simplify and accelerate computational aptamer characterization utilizing a wealth of tools for every step and integrating their findings to increase accuracy and realizing a sophisticated, extensible pipeline for aptamer refinement according to arbitrarily complex designs based on user-set criteria. We compare several tools and apply our workflow in various setting, likely to be encountered by practitioners and show that our algorithm can be very accurate in the prediction of aptamer - target binding affinities, as well as able to produce refined aptamer candidates for experimental validation. Finally, we consolidate all of the programs used in a unified containerized software solution that is very modular and can be readily deployed in any environment, equipped with an API and GUI that can abstract away the complexity of using and integrating the programs and help less programmatically experienced users add computational biology workflows to their methodologies.