UV Optical Melting Analysis of Sequence-Dependent A–G Mismatch in DNA Hairpins
Skills:
Wet Lab Techniques / Structural Imaging
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UV optical melting experiments
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DNA hairpin oligonucleotide handling and sample preparation
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Buffer and solution preparation for nucleic acid thermodynamic assays
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UV-Vis spectrophotometry and melting curve acquisition
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Nuclear magnetic resonance (NMR) spectroscopy
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Experimental design across multiple trinucleotide sequence contexts
Computational & Data Analysis
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Python (data processing, analysis, and visualization)
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Jupyter Notebook for iterative data exploration and workflow documentation
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Two-state model fitting for thermodynamic parameter extraction (ΔH, ΔS, ΔG, Tm)
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Nonlinear curve fitting and melting-curve analysis
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Comparative error analysis between calculated free energies and measured melting temperatures
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Data visualization for cross-context stability comparisons
Comparison of 1D ¹H imino-proton NMR spectra for hpAG-GAG: reference spectrum from Gu et al (4). (red) and current sample (blue).
Heat map of A·G mismatch duplex thermodynamic stability
Sample raw UV melting curves for A·G hairpin constructs
Comparison of 1D ¹H imino-proton NMR spectra for hpAG-GAG: reference spectrum from Gu et al (4). (red) and current sample (blue).
Summary of Findings
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Investigated how local sequence context affects the thermodynamic stability of A–G mismatches, which can evade polymerase fidelity through transient conformations
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Used UV optical melting to analyze hairpin constructs spanning all 16 trinucleotide contexts, extracting melting temperatures and thermodynamic parameters
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Found that mismatch stability varies significantly by sequence context, with differences of up to ~3 kcal/mol
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Identified limitations in the two-state fitting model: calculated free energies were inconsistent with directly measured melting temperatures, particularly for high-stability sequences where incomplete melting compromised baseline accuracy
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Determined melting temperature to be the most reliable, model-independent stability measure, producing a consistent ranking across all contexts
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Showed that mismatches with a 3′ G neighbor are more stable than those with a 3′ T neighbor — consistent with nearest-neighbor effects from stronger hydrogen bonding, base stacking, and GC-rich environments
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Laid the groundwork for future duplex-based studies to build a more accurate thermodynamic model of mismatch behavior and mutagenesis