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Monitoring the Intracellular Fate of Molecular Beacons : The Challenge of False Positive Signals

Volpati, Diogo LU ; Aoki, Pedro H.B. LU ; Johansson, Therese B. LU ; Munita, Roberto LU ; Ekstrand, Frida LU ; Ruhrmann, Sabrina LU ; Bacos, Karl LU orcid ; Ling, Charlotte LU orcid and Prinz, Christelle N. LU (2024) In Advanced NanoBiomed Research
Abstract

Molecular beacons (MBs) have been used on surfaces for detecting oligonucleotides. Attempts to use them intracellularly for monitoring mRNA content have been made, however, without any clear conclusion regarding the reliability of the method, mainly due to false positive signals. To reach an understanding of the intracellular fate of MBs, a critical question remains: how long after MB delivery and where in the cell does a false positive signal appear? To answer that question, the MB delivery method should allow for a time-stamped synchronized delivery of MBs to multiple cells, resulting in MBs being distributed in the cytosol immediately after delivery. Herein, nanostraws are used to inject MBs targeting insulin (Ins1) mRNA directly in... (More)

Molecular beacons (MBs) have been used on surfaces for detecting oligonucleotides. Attempts to use them intracellularly for monitoring mRNA content have been made, however, without any clear conclusion regarding the reliability of the method, mainly due to false positive signals. To reach an understanding of the intracellular fate of MBs, a critical question remains: how long after MB delivery and where in the cell does a false positive signal appear? To answer that question, the MB delivery method should allow for a time-stamped synchronized delivery of MBs to multiple cells, resulting in MBs being distributed in the cytosol immediately after delivery. Herein, nanostraws are used to inject MBs targeting insulin (Ins1) mRNA directly in the cytosol of clonal beta-cells, and the evolution of the MB fluorescence in time and space is monitored. The results show an MB translocation to the nucleus, where MBs are degraded or where they open nonspecifically, before the fluorophore alone is expelled back from the nucleus to the cytosol. The signal translocation to the nucleus and back to the cytosol is faster when scrambled MBs are used. The results shed light on the intracellular fate of MBs and highlight the short time scales before false positive signals become predominant.

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author
; ; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
epub
subject
keywords
beta-cells, cell interrogation, live cell imaging, molecular beacons, nanostraws
in
Advanced NanoBiomed Research
publisher
Wiley
external identifiers
  • scopus:85186901810
ISSN
2699-9307
DOI
10.1002/anbr.202300147
language
English
LU publication?
yes
id
22d0bf2d-984d-4c2f-afc0-1600c966fe29
date added to LUP
2024-04-02 15:35:23
date last changed
2024-04-02 15:35:49
@article{22d0bf2d-984d-4c2f-afc0-1600c966fe29,
  abstract     = {{<p>Molecular beacons (MBs) have been used on surfaces for detecting oligonucleotides. Attempts to use them intracellularly for monitoring mRNA content have been made, however, without any clear conclusion regarding the reliability of the method, mainly due to false positive signals. To reach an understanding of the intracellular fate of MBs, a critical question remains: how long after MB delivery and where in the cell does a false positive signal appear? To answer that question, the MB delivery method should allow for a time-stamped synchronized delivery of MBs to multiple cells, resulting in MBs being distributed in the cytosol immediately after delivery. Herein, nanostraws are used to inject MBs targeting insulin (Ins1) mRNA directly in the cytosol of clonal beta-cells, and the evolution of the MB fluorescence in time and space is monitored. The results show an MB translocation to the nucleus, where MBs are degraded or where they open nonspecifically, before the fluorophore alone is expelled back from the nucleus to the cytosol. The signal translocation to the nucleus and back to the cytosol is faster when scrambled MBs are used. The results shed light on the intracellular fate of MBs and highlight the short time scales before false positive signals become predominant.</p>}},
  author       = {{Volpati, Diogo and Aoki, Pedro H.B. and Johansson, Therese B. and Munita, Roberto and Ekstrand, Frida and Ruhrmann, Sabrina and Bacos, Karl and Ling, Charlotte and Prinz, Christelle N.}},
  issn         = {{2699-9307}},
  keywords     = {{beta-cells; cell interrogation; live cell imaging; molecular beacons; nanostraws}},
  language     = {{eng}},
  publisher    = {{Wiley}},
  series       = {{Advanced NanoBiomed Research}},
  title        = {{Monitoring the Intracellular Fate of Molecular Beacons : The Challenge of False Positive Signals}},
  url          = {{http://dx.doi.org/10.1002/anbr.202300147}},
  doi          = {{10.1002/anbr.202300147}},
  year         = {{2024}},
}