Abstract
The characterization and development of highly specific aptamers requires the analysis of the interaction strength between aptamer and target. MicroScale Thermophoresis (MST) is a rapid and precise method to quantify biomolecular interactions in solution at microliter scale. The basis of this technology is a physical effect referred to as thermophoresis, which describes the directed movement of molecules through temperature gradients. The thermophoretic properties of a molecule depend on its size, charge, and hydration shell. Since at least one of these parameters is altered upon binding of a ligand, this method can be used to analyze virtually any biomolecular interaction in any buffer or complex bioliquid. This section provides a detailed protocol describing how MST is used to obtain quantitative binding parameters for aptamer–target interactions. The two DNA-aptamers HD1 and HD22, which are targeted against human thrombin, are used as model systems to demonstrate a rapid and straightforward screening approach to determine optimal buffer conditions.
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References
Siller-Matula JM, Schwameis M, Blann A, Mannhalter C, Jilma B (2011) Thrombin as a multi-functional enzyme. Focus on in vitro and in vivo effects. Thromb Haemost 106(6):1020–1033
Bock LC, Griffin LC, Latham JA, Vermaas EH, Toole JJ (1992) Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature 355(6360):564–566
Macaya RF, Schultze P, Smith FW, Roe JA, Feigon J (1993) Thrombin-binding DNA aptamer forms a unimolecular quadruplex structure in solution. Proc Natl Acad Sci U S A 90(8):3745–3749
Tasset DM, Kubik MF, Steiner W (1997) Oligonucleotide inhibitors of human thrombin that bind distinct epitopes. J Mol Biol 272(5):688–698
Muller J, Wulffen B, Potzsch B, Mayer G (2007) Multidomain targeting generates a high-affinity thrombin-inhibiting bivalent aptamer. Chembiochem 8(18):2223–2226
Becker RC, Povsic T, Cohen MG, Rusconi CP, Sullenger B (2010) Nucleic acid aptamers as antithrombotic agents: opportunities in extracellular therapeutics. Thromb Haemost 103(3):586–595
Baaske P, Wienken CJ, Reineck P, Duhr S, Braun D (2010) Optical thermophoresis for quantifying the buffer dependence of aptamer binding. Angew Chem Int Ed Engl 49(12):2238–2241
Jerabek-Willemsen M, Wienken CJ, Braun D, Baaske P, Duhr S (2011) Molecular interaction studies using microscale thermophoresis. Assay Drug Dev Technol 9(4):342–353
Seidel SA, Dijkman PM, Lea WA, van den Bogaart G, Jerabek-Willemsen M, Lazic A, Joseph JS, Srinivasan P, Baaske P, Simeonov A, Katritch I, Melo FA, Ladbury JE, Schreiber G, Watts A, Braun D, Duhr S (2013) Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods 59(3):301–315
Zillner K, Jerabek-Willemsen M, Duhr S, Braun D, Langst G, Baaske P (2012) Microscale thermophoresis as a sensitive method to quantify protein: nucleic acid interactions in solution. Methods Mol Biol 815:241–252
Ludwig C (1856) Diffusion zwischen ungleich erwärmten Orten gleich zusammengesetzter Lösungen. Sitzungber Bayer Akad Wiss Wien Math-Naturwiss Kl 20:539
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Breitsprecher, D., Schlinck, N., Witte, D., Duhr, S., Baaske, P., Schubert, T. (2016). Aptamer Binding Studies Using MicroScale Thermophoresis. In: Mayer, G. (eds) Nucleic Acid Aptamers. Methods in Molecular Biology, vol 1380. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3197-2_8
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DOI: https://doi.org/10.1007/978-1-4939-3197-2_8
Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-3197-2
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