(T1030-08-55) Biomolecule Characterization Using UV-Vis Spectroscopy: A Focus on Thermal Stability of Nucleic Acids and Proteins

Purpose: Drug discovery is a multifaceted process in which potential new medicines and their therapeutic effects must be demonstrated. Proper identification and characterization of drug targets are important steps in the process of drug discovery that can facilitate lead-therapeutic identification.¹ Biochemical characterization of biomolecules includes molecular weight, homogeneity and heterogeneity, solubility, thermal stability (Tm), amino acid sequence composition, secondary structure, and three-dimensional structure.^{2, 3} Tm is often used as a secondary identification test (QC check) for nucleic acids. It is also used to provide an insight into protein structure, and understanding of ligand-binding, and drug stability. Two studies are presented here using a UV-Vis spectrophotometer. Study 1 assessed the impact of increasing the temperature ramp rate on the calculated Tm of herring sperm DNA.⁴ Study 2 demonstrated the fast and simple characterization of protein thermal stability and melting temperature of a protein.⁵

Methods: Study 1* A solution of ~15 μg /mL of the herring sperm DNA was prepared in phosphate buffered saline. Neat phosphate buffered saline was used as the blank solution. Measurements were performed using method parameters shown in Figure 1.Study 2* A sample of Escherichia coli disulfide bond isomerase A (EcDsbA) was provided by La Trobe Institute for Molecular Science, Australia. A solution of ≈0.6 mg/mL of EcDsbA was prepared in a phosphate buffer solution. Neat phosphate buffer was used as the blank solution. Parameters used are shown in Figure 1 * The Agilent Cary 3500 Multizone UV-Vis spectrophotometer and Cary UV Workstation were used to conduct both studies.

Results: Study 1 The absorbance data collected at the six temperature ramp rates (1, 5, 10, 20, 30, and 40 C/min) is shown in Figure 2A. The first derivative of each scan was calculated and is shown in Figure 2B. The peak maximum of each first derivative plot identifies the midpoint of the melting curve, and, therefore, the Tm value. As shown in Figure 2, the measured Tm values of the herring sperm DNA samples was within 0.2 C for all six temperature ramp rates used in the experiments. The results show the high-level reproducibility: laboratories can use faster temperature ramp rates than the standard protocol of 0.5 °C/min, significantly reducing experimental times without compromising the quality of the results. These results have highlighted the associated time saving. An experiment that previously took 2.5 hours could be measured in approximately 10 minutes. These experiments can be done in a regulated environment with secure data control. The software used meets data integrity standards for laboratories that must follow the compliance guidelines of FDA 21 CFR Part 11, EU Annex 11, GAMP5, as well as and ISO/IEC 17025 and EPA’s 40 CFR Part 160. QC practices in oligonucleotides ID testing shows a trend towards using melting temperature (Tm) in combination with intact mass measurement, combining sequence and mass to achieve unequivocal identification. ⁶ Study 2 For the thermal stability analysis of the EcDsbA protein, the absorbance at 280 nm was monitored. As the temperature increased, a peak was observed, indicating a change in the absorbance of the protein caused by a change in the protein structure. The thermal melt plot (Figure 3A) shows a transition occurring at high temperature (above 65 °C) for the EcDsbA protein. The Tm of EcDsbA was found to be 69.86 °C with a standard deviation of 0.16 °C (n = 6; Figure 3B). The calculated Tm for the EcDsbA protein obtained using the Cary 3500 UV Vis spectrophotometer agreed with the previously reported results obtained using circular dichroism with higher sample throughput.⁷

Conclusion: Both studies showed the capabilities of UV-Vis spectroscopy in biomolecule characterization. The results demonstrate the accuracy, precision, and speed of the spectrophotometer for both DNA and protein thermal stability analyses. They also demonstrate the ability to measure all eight cuvette positions simultaneously offers further productivity improvements. Finally, they show the effectiveness of using UV-Vis spectroscopy for laboratories interested in studying the response of liquid samples to temperature changes without other experimental variables.

References: 1. Mohs, R. C.; Greig, N. H. Drug Discovery and Development: Role of Basic Biological Research, Alzheimers Dement (NY) 2017, 11, 3(4), 651 657. doi: 10.1016/j.trci.2017.10.005
2. Schenone, M. et al. Target Identification and Mechanism of Action in Chemical Biology and Drug Discovery, Nat. Chem. Biol. 2013, 9(4), 232 40. doi: 10.1038/nchembio.1199
3. Gashaw, I. et al. What Makes a Good Drug Target? Drug Discovery Today 2012, 17, Supplement, S24 S30. doi.org/10.1016/j.drudis.2011.12.008
4. Fast Determination of Thermal Melt Temperature of Double Stranded Nucleic Acids by UV Vis Spectroscopy. Agilent application note. Aug 2022. 5994 0384EN
5. Characterization of Protein Thermal Stability Using UV Vis Spectroscopy. Agilent application note. Aug 2022. 5994 5086EN
6. Evaluating an Innovative Analytical ID Testing Strategy for Oligonucleotides. Agilent case study. Sep 2022. 5994 5144EN.
7. Heras, B. et al. Staphylococcus aureus DsbA Does Not Have a Destabilizing Disulfide, a New Paradigm for Bacterial Oxidative Folding. J. Biol. Chem. 2008, 15, 283(7), 4261 71. doi:10.1074/jbc.M707838200

UV-Vis spectrophotometer method parameters used in study 1 and 2

(A) Herring sperm DNA absorbance versus temperature (B) the corresponding first derivative versus temperature and (C) The measured Tm values for the herring sperm DNA sample at each temperature ramp rate.

(A) EcDsbA protein absorbance versus temperature and (B) the corresponding first derivative plots of six separate samples.