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Enzyme fragment complementation driven by nucleic acid hybridization sans self-labeling protein.

Zihan Xu1, Xiaoyu Zhang1, Chandan Pal1

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A novel biosensor uses enzyme fragments to detect nucleic acids in solution. This method offers sensitive and programmable detection of DNA and RNA without bulky passenger proteins.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biosensor Technology

Background:

  • Enzyme fragment complementation assays are used for detecting biomolecules.
  • Current methods for nucleic acid detection can be limited by bulky components or sequence constraints.

Purpose of the Study:

  • To develop and validate a turn-on biosensor for nucleic acid detection in dilute aqueous solutions.
  • To create a sensitive and programmable detection system free from self-labeling passenger proteins.

Main Methods:

  • Utilized fragments of NanoBiT, a split luciferase reporter enzyme.
  • Developed steramers (sterol-linked oligonucleotides) esterified to NanoBiT fragments.
  • Employed the Drosophila hedgehog autoprocessing domain (DHhC) for bioconjugation.
  • Demonstrated target sequence-agonistic detection based on NanoBiT fragment reassociation.

Main Results:

  • Achieved a 30- to 60-fold increase in NanoBiT luminescence over background in the presence of low nM concentrations of ssDNA or RNA templates.
  • The developed bioconjugate is free of self-labeling passenger protein.
  • Steramer probe sequences showed no constraints in length or composition.

Conclusions:

  • The modified NanoBiT-steramer bioconjugate functions as an effective turn-on biosensor for nucleic acid detection.
  • This approach enables programmable detection of small DNA and RNA fragments without sequence limitations or added protein bulk.
  • Opens new applications in sensitive nucleic acid analysis.