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Related Experiment Videos

Effect of loop sequence and size on DNA aptamer stability.

I Smirnov1, R H Shafer

  • 1Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco 94143, USA.

Biochemistry
|February 24, 2000
PubMed
Summary

This study investigated thrombin aptamer analogues, revealing that modifications to loop sequences and G-addition at the 3'-end optimize thermodynamic stability. Differential scanning calorimetry detected heat capacity changes during aptamer unfolding.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biophysical Chemistry

Background:

  • The thrombin aptamer is a 15-mer oligodeoxyribonucleotide with high affinity for thrombin.
  • It forms a unimolecular quadruplex structure with guanine quartets and loops.

Purpose of the Study:

  • To systematically examine the thermodynamic stability of thrombin aptamer analogues.
  • To investigate the impact of sequence modifications in loops and quartet number on stability.

Main Methods:

  • UV melting studies to obtain thermodynamic parameters.
  • van't Hoff analysis using a two-state model for denaturation.
  • Differential scanning calorimetry (DSC) to detect heat capacity changes.

Main Results:

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  • Central loop sequence is optimal for parent aptamer stability.
  • Loop modifications affect enthalpy (ΔH°) and entropy (ΔS°).
  • Adding a G at the 3'-end increases stability; adding at the 5'-end decreases it.
  • DSC revealed a heat capacity change during unfolding not detected by UV.

Conclusions:

  • The central loop sequence is critical for thrombin aptamer stability.
  • Specific modifications can fine-tune aptamer thermodynamic properties.
  • DSC provides complementary thermodynamic data to UV melting studies.