Optimizing locked nucleic acid modification in double-stranded biosensors for live single cell analysis

Double-stranded (ds) biosensors are homogeneous oligonucleotide probes for detection of nucleic acid sequences in biochemical assays and live cell imaging. Locked nucleic acid (LNA) modification can be incorporated in the biosensors to enhance the binding affinity, specificity, and resistance to nuclease degradation. However, LNA monomers in the quencher sequence can also prevent the target-fluorophore probe binding, which reduces the signal of the dsLNA biosensor. This study investigates the influence of LNA modification on dsLNA biosensors by altering the position and amount of LNA monomers present in the quencher sequence. We characterize the fluorophore-quencher interaction, target detection, and specificity of the biosensor in free solution and evaluate the performance of the dsLNA biosensor in 2D monolayers and 3D spheroids. The data indicate that a large amount of LNA monomers in the quencher sequence can enhance the specificity of the biosensor, but prevents effective target binding. Together, our results provide guidelines for improving the performance of dsLNA biosensors in nucleic acid detection and gene expression analysis in live cells.

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Metadata

Work Title Optimizing locked nucleic acid modification in double-stranded biosensors for live single cell analysis
Access
Open Access
Creators
  1. Samuel A. Vilchez Mercedes
  2. Ian Eder
  3. Mona Ahmed
  4. Ninghao Zhu
  5. Pak Kin Wong
License In Copyright (Rights Reserved)
Work Type Article
Publisher
  1. Analyst
Publication Date January 27, 2022
Publisher Identifier (DOI)
  1. https://doi.org/10.1039/d1an01802g
Deposited February 17, 2023

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Version 1
published

  • Created
  • Added Analyst-dsLNA-MS-2st-rev.docx
  • Added Creator Samuel A. Vilchez Mercedes
  • Added Creator Ian Eder
  • Added Creator Mona Ahmed
  • Added Creator Ninghao Zhu
  • Added Creator Pak Kin Wong
  • Published
  • Updated Publisher, Publication Date Show Changes
    Publisher
    • The Analyst
    • Analyst
    Publication Date
    • 2022-02-21
    • 2022-01-27