Molecular beacon nanosensors for live cell

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Molecular beacon nanosensors for live cell detection and tracking differentiation and

reprogramming

Ilieva, Mirolyuba; Dufva, Martin; Emnéus, Jenny

Pub lication date:
2013

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Citation (APA):
Ilieva, M., Dufva, M., & Emnéus, J. (2013). Molecular beacon nanosensors for live cell detection and tracking
differentiation and reprogramming.
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Molecular beacon nanosensors for live cell

detection and tracking differentiation and
reprogramming

Mirolyuba Ilieva

PhD Thesis

April 2013
Mirolyuba Simeonova Ilieva

Molecular beacon nanosensors for live cell
detection and tracking differentiation and
reprogramming

PhD Thesis
April 2013

Supervisor Assoc. Prof. Martin Dufva
Co-supervisor Professor Jenny Emnéus
Department of Micro- and Nanotechnology Technical University of Denmark
Abstract

High-sensitive and high-affinity methods to measure gene expression inside living cells have
proven to be invaluable in regards to understanding fundamental processes such as cell
differentiation, reprogramming, regeneration and cancer genesis. One tool for transcription
visualization on single cell level is molecular beacons (MBs). They are stem-loop structured
antisense oligonucleotide probes labelled with a reporter fluorophore at one end and with
quencher at the other end. Upon hybridization with complementary target, hydrogen bonds
between stem nucleotide bases brake, resulting in separation of fluorophore from quencher
and thereby emission of a fluorescent signal that can be detected.
In this project the usability and applicability of MBs for live cell detection and tracing of
gene expression was demonstrated. MBs library targeting gene markers for pluripotent stem
cells as well as markers for different stages of cell differentiation into the three germ lineages
were designed. The focus was on stem cell differentiation into neuronal lineage as well as
reprogramming into a more immature state after plasmid transfection or under the influence
of the environment and growth conditions. We have been able to detect gain of expression of
neuronal markers during differentiation of embryonic mesencephalon derived cells
(LUHMES) into dopamine neurons. Loss of expression of stem cell markers was also
observed suggesting that MBs are able to dissociate from target mRNA and regenerate from
open to closed state within living cells. Using MBs targeting pluripotent stem cell markers we
demonstrated reverse into a more immature state of LUHMES induced by neurosphere-like
growth conditions. Moreover, we have been able to trace localisation of this particular
population during differentiation and thus demonstrate the usability of MBs for monitoring
cell behaviour within 3D clusters. Finally, MBs detection of expression of human pluripotent
markers after reprograming of adult somatic cells with plasmid codding for mouse
transcription factors was demonstrated.
In conclusion, the method of using transfected MBs is an easy and rapid way to detect gene
expression. Target cells do not need to be genetically modified. Furthermore, MBs are able to
detect both up- and down regulation of gene expression on the single cell level and dynamic
tracing of specific cell populations within 3D clusters.
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Resume
Metoder med høj følsomhed og høj affinitet til at måle genekspression i levende celler har
vist sig at være uvurderlige med hensyn til at forstå fundamentale processer, såsom
celledifferentiering, -reprogrammering og -regenerering samt cancer genesis. Et redskab for
visualisering af transkription på enkelcelle niveau er molekylære beacons (MBs). Det er
antisense oligonukliotide prober med en hårnålestruktur mærket med en reporter fluorofor i
den ene ende og en quencer i den anden ende. Ved hybridisering med komplimentær target,
brydes hydrogenbindingerne mellem nukleotid baserne i de to parallelle strenge i
hårnålestukturen, resulterende i adskillelse af fluorofor fra quencer og dermed emission af et
fluorescerende signal, der kan detekteres.
I dette studie er brugbarheden og anvendeligheden af MBs for detektion og sporing af
genekspression demonstreret. Et bibliotek af MBs, målrettet genmarkører for pluripotente
stamceller såvel som markører for forskellige stadier af celledifferentiering til de tre
kimcellelinier, er designet. Focus var på stamcelledifferentiering til neuronale linier såvel
som reprogrammering til mere umodne stadier efter plasmid transfektion eller under
indflydelse af miljø og vækstbetingelser. Vi har været i stand til at detektere stigning i
ekspression af neuronale markører under differentiering af embyonale mesencephalon
udledte celler (LUHMES) til dopamin neuroner. Tab af ekspression af stamcellemarkører
blev også observeret tydende på, at MBs er i stand til at dissociere fra target mRNA og
regenerere fra åbent til lukket stadie i levende celler. Ved brug af MBs målrettet pluripotente
stamcellemarkører demonstrerede vi tilbagevending til et mere umodent stadie af LUHMES
induceret ved neurosphere-lignende vækstbetingelser. Derudover har vi været i stand til at
spore lokalisering af den bestemte population under differentiering og dermed demonstrere
brugbarheden af MBs for monitorering af celle opførelse indeni 3D klynger. Endelig blev
detektion af ekspression af humane pluripotente markører efter reprogrammering af voksen
somatiske celler med plasmid kodende for mussetranskriptionsfaktorer demonstreret ved brug
af MBs.
Afslutningsvis kan det konkluderes, at metoden med brug af MBs er en let og hurtig måde til
at bestemme genekspression. Målcellerne behøver ikke at blive genetisk modificeret.
Yderligere er MBs i stand til at detektere både op- og nedregulering af genekspression på
enkelcelle niveau samt dynamisk sporing af specifikke cellepopulationer i 3D klynger.
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Preface

The work described in this thesis has been carried out in the Fluidic Array Systems and
Technology (FAST) group at the Department of Micro- and Nanotechnology (DTU
Nanotech), Technical University of Denmark (DTU) in the time period from May 2010 to
April 2013. The work has been supervised by main supervisor assoc. prof. Martin Dufva and
co-supervisor prof. Jenny Emnéus, Bioanalytics group, DTU Nanotech. The work was
supported by FTP grant 09-070568.
The thesis consists from seven chapters. Chapter IV and Chapter VII are based on the
findings described in more details in submitted manuscripts applied as Appendix 1 and 2.

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