Thermophoresis in nanoliter droplets to quantify aptamer binding.

Biomolecule interactions are central to pharmacology and diagnostics. These interactions can be quantified by thermophoresis, the directed molecule movement along a temperature gradient. It is sensitive to binding induced changes in size, charge, or conformation. Established capillary measurements require at least 0.5 μL per sample. We cut down sample consumption by a factor of 50, using 10 nL droplets produced with acoustic droplet robotics (Labcyte). Droplets were stabilized in an oil-surfactant mix and locally heated with an IR laser. Temperature increase, Marangoni flow, and concentration distribution were analyzed by fluorescence microscopy and numerical simulation. In 10 nL droplets, we quantified AMP-aptamer affinity, cooperativity, and buffer dependence. Miniaturization and the 1536-well plate format make the method high-throughput and automation friendly. This promotes innovative applications for diagnostic assays in human serum or label-free drug discovery screening.

[1]  Dieter Braun,et al.  Protein-binding assays in biological liquids using microscale thermophoresis. , 2010, Nature communications.

[2]  Dan S. Tawfik,et al.  Man-made cell-like compartments for molecular evolution , 1998, Nature Biotechnology.

[3]  Richard N. Ellson,et al.  Transfer of low nanoliter volumes between microplates using focused acoustics-automation considerations , 2003 .

[4]  J F Brandts,et al.  Rapid measurement of binding constants and heats of binding using a new titration calorimeter. , 1989, Analytical biochemistry.

[5]  Soyoun Kim,et al.  Patents on SELEX and therapeutic aptamers. , 2008, Recent patents on DNA & gene sequences.

[6]  J. Szostak,et al.  A DNA aptamer that binds adenosine and ATP. , 1995, Biochemistry.

[7]  J. Szostak,et al.  In vitro selection of RNA molecules that bind specific ligands , 1990, Nature.

[8]  Dieter Braun,et al.  Why molecules move along a temperature gradient , 2006, Proceedings of the National Academy of Sciences.

[9]  Aniela Wochner,et al.  Characterisation of aptamers for therapeutic studies , 2007, Expert opinion on drug discovery.

[10]  R. Kennedy,et al.  Retention and separation of adenosine and analogues by affinity chromatography with an aptamer stationary phase. , 2001, Analytical chemistry.

[11]  Eun Jeong Cho,et al.  Applications of aptamers as sensors. , 2009, Annual review of analytical chemistry.

[12]  D. Braun,et al.  Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. , 2013, Methods.

[13]  D. Braun,et al.  A Monoclonal Antibody (MCPR3-7) Interfering with the Activity of Proteinase 3 by an Allosteric Mechanism* , 2013, The Journal of Biological Chemistry.

[14]  Anthony D. Keefe,et al.  Aptamers as therapeutics , 2010, Nature Reviews Drug Discovery.

[15]  Dieter Braun,et al.  Label-Free Microscale Thermophoresis Discriminates Sites and Affinity of Protein–Ligand Binding , 2012, Angewandte Chemie.

[16]  Dieter Braun,et al.  Direct detection of antibody concentration and affinity in human serum using microscale thermophoresis. , 2012, Analytical chemistry.

[17]  E. Gragoudas,et al.  Pegaptanib for neovascular age-related macular degeneration. , 2004, The New England journal of medicine.

[18]  A. Adamis,et al.  Pegaptanib 1-year systemic safety results from a safety-pharmacokinetic trial in patients with neovascular age-related macular degeneration. , 2007, Ophthalmology.

[19]  Helmut Sigel,et al.  Interactions of metal ions with nucleotides and nucleic acids and their constituents , 1993 .

[20]  D. Patel,et al.  Structural basis of DNA folding and recognition in an AMP-DNA aptamer complex: distinct architectures but common recognition motifs for DNA and RNA aptamers complexed to AMP. , 1997, Chemistry & biology.

[21]  D. Braun,et al.  Quantifizierung der Puffer‐Abhängigkeit von Aptamer‐Bindungsreaktionen mit optischer Thermophorese , 2010 .

[22]  Dirk Trauner,et al.  Markierungsfreie “Microscale Thermophoresis” zur Bestimmung von Bindestellen und Affinitäten bei Protein-Liganden-Wechselwirkungen† , 2012 .

[23]  D. Braun,et al.  Optical thermophoresis for quantifying the buffer dependence of aptamer binding. , 2010, Angewandte Chemie.

[24]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.