High-Throughput Quantification of the Impact of Different Osmolytes on the Thermal Stability of DNA

Abstract

Small molecules (osmolytes) are known to either stabilize or destabilize proteins/nucleotides depending on the concentrations and/or solvent conditions. The presence of different molecules and ions in the surrounding medium affects the stability of DNA in solution. In this work, we have developed a High-Throughput method for quantifying the energetic impact of addition of various osmolytes on short DNA duplexes. Six 19-base pair, non-self-complementary duplex DNA oligomers along with a 16-base pair control duplex DNA, having varied GC-content (ranging from 16% to 79%), nearest neighbors and end sequences were used. We sampled thirteen different osmolytes that are common in humans and throughout nature by covering different chemical classes including, sugars, polyols, amino acids, and methylamines. Varying concentrations of these osmolytes (from 0.5 M up to 3.0 M) were examined for their effects on these duplexes. Experiments were performed in 384-well plates that were prepared using a robotic device, which was calibrated for the correct dispense volume for different components of the plate. Temperature-induced melting transitions monitored by fluorescence were measured for these duplexes and the Tm values along with the m-values and melting transition enthalpies were determined. Conventional approaches, including Circular Dichroism (CD) were used to verify the thermodynamic parameters. Osmolytes had varied effects on DNA stability, and the (de)stabilizing effect does not necessarily correlate with their effects on proteins. The m-values can drastically depend on the GC-content.