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Lide Gu1, Wanli Yan1, Shu Liu1

  • 1Jiangsu Key Laboratory of Marine Bioresources and Environment, Huaihai Institute of Technology, Lianyungang, 222005, PR China; Jiangsu Marine Resources Development Research Institute, Lianyungang, 222005, PR China; College of Marine Life and Fisheries, Huaihai Institute of Technology, Lianyungang, 222005, PR China; Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Huaihai Institute of Technology, Lianyungang, 222005, PR China.

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Summary

This study introduces a novel trypsin-enhanced screening method for selecting high-affinity DNA aptamers. This method effectively identifies aptamers targeting Helicobacter pylori antigen and cancer biomarkers like CEA, CA125, and CA19-9.

Keywords:
AptamerMethodProteinTrypsin

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Biochemistry

Background:

  • Aptamers are crucial for diagnostics and therapeutics.
  • Existing protein aptamer selection methods require optimization for efficiency and specificity.
  • Helicobacter pylori antigen and common cancer biomarkers (CEA, CA125, CA19-9) are important targets.

Purpose of the Study:

  • To develop and validate a novel, efficient screening method for protein-specific aptamer selection.
  • To compare the efficacy of trypsin versus urea in protein aptamer selection.
  • To generate high-affinity and high-specificity aptamers against Helicobacter pylori antigen and cancer biomarkers.

Main Methods:

  • A trypsin-enhanced screening method was developed for aptamer selection.
  • Target proteins (HP-Ag, CEA, CA125, CA19-9) were immobilized on 96-well plates.
  • Single-stranded DNA (ssDNA) libraries were incubated with target proteins, followed by trypsin digestion to isolate specific ssDNA.
  • Aptamer enrichment and affinity were analyzed after sequencing.
  • Trypsin's efficacy was compared to urea.

Main Results:

  • The trypsin-enhanced method successfully screened aptamers with high affinity and specificity for HP-Ag, CEA, CA125, and CA19-9.
  • Trypsin was found to be more effective than urea for protein aptamer selection.
  • The concentration of shed ssDNA correlated with aptamer specificity and affinity.

Conclusions:

  • A novel and effective protocol for generating protein-specific aptamers using trypsin was established.
  • This method offers a significant advancement in aptamer selection technology.
  • The developed aptamers hold potential for diagnostic applications targeting H. pylori and cancer.