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Prolonged function and optimization of actomyosin motility for up scaled network-based biocomputation
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.ORCID iD: 0000-0003-4835-0598
Lund University, Sweden.
Lund University, Sweden.
Technische Universität Chemnitz, Germany;Fraunhofer Institute for Electronic Nanosystems (ENAS), Germany.
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2021 (English)In: New Journal of Physics, E-ISSN 1367-2630, Vol. 23, article id 085005Article in journal (Refereed) Published
Abstract [en]

Significant advancements have been made towards exploitation of naturally available molecular motors and their associated cytoskeletal filaments in nanotechnological applications. For instance, myosin motors and actin filaments from muscle have been used with the aims to establish new approaches in biosensing and network-based biocomputation. The basis for these developments is a version of the in vitro motility assay (IVMA) where surface-adsorbed myosin motors propel the actin filaments along suitably derivatized nano-scale channels on nanostructured chips. These chips are generally assembled into custom-made microfluidic flow cells. For effective applications, particularly in biocomputation, it is important to appreciably prolong function of the biological system. Here, we systematically investigated potentially critical factors necessary to achieve this, such as biocompatibility of different components of the flow cell, the degree of air exposure, assay solution composition and nanofabrication methods. After optimizing these factors we prolonged the function of actin and myosin in nanodevices for biocomputation from <20 min to >60 min. In addition, we demonstrated that further optimizations could increase motility run times to >20 h. Of great importance for the latter development was a switch of glucose oxidase in the chemical oxygen scavenger system (glucose oxidase–glucose–catalase) to pyranose oxidase, combined with the use of blocking actin (non-fluorescent filaments that block dead motors). To allow effective testing of these approaches we adapted commercially available microfluidic channel slides, for the first time demonstrating their usefulness in the IVMA. As part of our study, we also demonstrate that myosin motor fragments can be stored at −80 °C for more than 10 years before use for nanotechnological purposes. This extended shelf-life is important for the sustainability of network-based biocomputation.

Place, publisher, year, edition, pages
Institute of Physics (IOP), 2021. Vol. 23, article id 085005
National Category
Nano Technology
Research subject
Natural Science, Biomedical Sciences
Identifiers
URN: urn:nbn:se:lnu:diva-106117DOI: 10.1088/1367-2630/ac1809ISI: 000685182100001Scopus ID: 2-s2.0-85112640174Local ID: 2021OAI: oai:DiVA.org:lnu-106117DiVA, id: diva2:1583925
Funder
European Commission, 732482Swedish Research Council, 2015-05290;2019-03456Available from: 2021-08-10 Created: 2021-08-10 Last updated: 2024-01-17Bibliographically approved
In thesis
1. Actomyosin in biocomputation
Open this publication in new window or tab >>Actomyosin in biocomputation
2021 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

There exist complex mathematical problems that are important in real world applications such as weather prediction, molecular modelling, network route optimization and more. In general, such problems are solved using supercomputers with higher computing efficiency but this also consumes high energy along with high production and maintenance cost. Network-based biocomputation (NBC) is an alternate computing approach, now at development stage, that can perform parallel computing in a highly energy efficient manner. Actin and myosin constitute one type of molecular motor system that has been utilized for the development of NBC. These proteins are key components in the sarcomere, the smallest functional unit of muscle and their interactions that underlie muscle contraction are powered by the cellular fuel adenosine triphosphate (ATP). To solve larger complex problems using actin-myosin based NBC, factors such as maintained biological function and longevity of operation are essential for practical relevance. In this thesis, the in vitro motility assay (IVMA) has been used as a central method to study actomyosin function and its operation within NBC devices. In the IVMA, actin filaments are propelled by myosin motors that are immobilized on functionalized surfaces in a flow cell. With the aim to improve motile fraction by reducing the interaction between actin and non-functional motor heads in the IVMA, two known methods were quantitatively compared in paper I, the affinity purification and the blocking actin method. Both approaches significantly improved the motile fraction to above 90% but affinity purification, due to the presence of ATP during incubation, induced significant reduction in sliding velocity, not seen with blocking actin. In paper III, critical parameters in the actomyosin IVMA system were investigated allowing us to extensively improve function and longevity, including: biocompatibility of flow cell components, effects of air exposure with oxygen scavenging and nanofabrication parameters such as plasma etching type and time, process of resist development, and surface silanization time. The above developments together with optimized network encoding of the problems enabled us (paper IV) to solve four instances of 3-SAT problem encoded in NBC with 99% probability of satisfiability. In parallel, (paper II) a method have been developed to recycle the surfaces with immobilized motor proteins by treatment of proteinase-K enzyme and detergent. This will allow re-cycling of advanced NBC chips. Finally, with aim to develop programmable gating for NBC, attempts have been made towards the integration of engineered light sensitive myosin XI motors with nanofabricated devices made up of Au/SiO2, SiO2/polymer and glass/polymer (paper V). In addition important factors such as standardized motor density, limiting of air exposure and longevity function have been optimized in the use of light sensitive motors.

Overall, this thesis reports critical insights for the upscaling of actomyosin based NBC. Described results, are also useful for the development of actomyosin based nanotechnological applications such as biosensing or diagnostics and other fundamental studies based on single molecule or drug testing.

Place, publisher, year, edition, pages
Linnaeus University Press, 2021. p. 79
Series
Linnaeus University Dissertations ; 420/2021
Keywords
biocomputation, molecular motors, 3-SAT, surface chemistry, actomyosin, actin, myosin II, heavy meromyosin, myosin XI, engineered myosin, in vitro motility assay, nanofabrication, alternate computing, network based biocomputation, protein-surface interaction, longevity, prolongation, surface recycling, affinity purification, blocking actin
National Category
Nano Technology Biophysics
Research subject
Chemistry, Biotechnology; Chemistry, Biochemistry
Identifiers
urn:nbn:se:lnu:diva-106118 (URN)9789189460041 (ISBN)9789189460034 (ISBN)
Public defence
2021-09-01, Room Vi2166 (Azur), Linnaeus University, building Vita, Kalmar, 11:58 (English)
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Supervisors
Available from: 2021-08-12 Created: 2021-08-10 Last updated: 2024-03-06Bibliographically approved

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Salhotra, AseemUšaj, MarkoNorrby, MarleneMånsson, Alf

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