To What Extent Does GC Content Determine Oligonucleotide Hybridization Thermodynamics?

Overview

Oligonucleotide hybridization is essential for DNA repair, amplification, sequencing, and editing, and relates to the development of antisense drugs used to treat various diseases. Increasing our understanding of factors besides GC content that influence oligonucleotide hybridization could aid in the development of antisense drugs. I hypothesized oligonucleotides with identical GC content, but varying GC distribution would exhibit different duplex stabilities. I obtained nine 12-base oligonucleotide sequences and their complements and prepared them using two buffers, one with sodium ions and one with potassium ions. The solutions were gradually heated, and UV absorbance was measured using a UV spectrometer. Melting temperatures of each mixture were determined from melting curve derivatives. I found that oligonucleotides with different GC distributions exhibited melting temperatures varying up to 7°C, with oligonucleotides containing centrally clustered GC content demonstrating the greatest duplex stability across both buffers. Oligonucleotides with the lowest melting temperature were observed to have long, uninterrupted runs of A/T bases. The experimental melting temperatures were compared against two nearest-neighbor calculators. They were effective in predicting general trends of duplex stability, however varied in accuracy when predicting precise thermodynamic behavior of oligonucleotides. The results support my hypothesis, indicating arrangement of GC content needs to be taken into consideration when discussing oligonucleotide hybridization thermodynamics. My results could be useful in the design of antisense drugs. Future research may include the impact of overhanging nucleotides on duplex stability.

Competition history

• CSEF 2026 Chemistry (Senior Division) · Entry S-05-07

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