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Enzyme Fragment Complementation Driven by Nucleic Acid Hybridization.

Zihan Xu1, Xiaoyu Zhang1, Chandan Pal1

  • 1Department of Chemistry, Binghamton University, The State University of New York, 4400 Vestal Parkway East Binghamton, New York, 13902, USA.

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A novel biosensor detects nucleic acid interactions using split NanoBiT luciferase fragments. This assay offers sensitive detection of DNA and RNA, enabling broader applications in molecular biology.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biotechnology

Background:

  • Accurate detection of nucleic acid interactions is crucial for understanding biological processes.
  • Existing methods for detecting nucleic acid:nucleic acid interactions can be limited in sensitivity or range.
  • Development of sensitive and versatile biosensors is needed for advanced molecular diagnostics.

Approach:

  • A modified protein fragment complementation assay was developed using NanoBiT split luciferase fragments.
  • Fragments were esterified to sterol-modified oligodeoxynucleotides (steramers) and conjugated using the DHhC self-cleaving catalyst.
  • The assay functions as a gain-of-signal biosensor, reconstituting NanoBiT activity upon complementary nucleic acid binding.

Key Points:

  • The biosensor detects nucleic acid:nucleic acid interactions, including single-stranded DNA (ssDNA) and RNA.
  • NanoBiT luminescence signal increased 30- to 120-fold in the presence of target nucleic acids.
  • The absence of a self-labeling domain in NanoBiT bioconjugates broadens detection range from short oligos to full-length mRNA.

Conclusions:

  • The developed NanoBiT-based assay is a validated, sensitive gain-of-signal biosensor for nucleic acid interactions.
  • This system offers a versatile platform for detecting diverse nucleic acid targets with high sensitivity.
  • The elimination of extraneous domains enhances the assay's applicability for various molecular targets.