Biotechnological applications of thermophoresis

Measuring parameters such as stability and con-formation of biomolecules, especially of nucleicacids, is important in the field of biology, medicaldiagnostics and biotechnology. We present athermophoretic method to analyse the conformationand thermal stability of nucleic acids. It relies on thedirected movement of molecules in a temperaturegradient that depends on surface characteristicsof the molecule, such as size, charge and hydropho-bicity. By measuring thermophoresis of nucleicacids over temperature, we find clear melting tran-sitions and resolve intermediate conformationalstates. These intermediate states are indicated byan additional peak in the thermophoretic signalpreceding most melting transitions. We analysedsingle nucleotide polymorphisms, DNA modifica-tions, conformational states of DNA hairpins andmicroRNA duplexes. The method is validated suc-cessfully against calculated melting temperaturesand UV absorbance measurements. Interestingly,the methylation of DNA is detected by thethermophoretic amplitude even if it does not affectthe melting temperature. In the described setup,thermophoresis is measured all-optical in a simplesetup using a reproducible capillary format with only250 nl probe consumption. The thermophoreticanalysis of nucleic acids shows the technique’s ver-satility for the investigation of nucleic acids relevantin cellular processes like RNA interference or genesilencing.