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Interfering with RNA interference : Platination, kinetics and down-regulation

Alshiekh, Alak LU (2016)
Abstract (Swedish)
Cellens RNA finns i många olika storlekar, och funktioner. En viktig RNA funktion är s.k. RNA interferens (RNAi). RNAi innebär att små fraktioner av dubbelsträngat RNA kontrollerar hur mycket proteiner som skall produceras i cellen. Det finns olika typer av RNAi-molekyler, t.ex. microRNA som är producerat i cellen och s.k. siRNA som kan introduceras i cellen, t.ex. som läkemedel.
RNAi är viktigt för många olika cellulära processer och även inblandat vid utveckling av sjukdomar som cancer. Cancer är en genetiskt orsakad sjukdom, och kan beskrivas som en DNA sjukdom. En stor del av cancerbehandlingarna påverkar DNA:t direkt t.ex. cisplatin. Cisplatin är ett metall baserad läkemedel som är väldigt effektivt för cancerbehandling.... (More)
Cellens RNA finns i många olika storlekar, och funktioner. En viktig RNA funktion är s.k. RNA interferens (RNAi). RNAi innebär att små fraktioner av dubbelsträngat RNA kontrollerar hur mycket proteiner som skall produceras i cellen. Det finns olika typer av RNAi-molekyler, t.ex. microRNA som är producerat i cellen och s.k. siRNA som kan introduceras i cellen, t.ex. som läkemedel.
RNAi är viktigt för många olika cellulära processer och även inblandat vid utveckling av sjukdomar som cancer. Cancer är en genetiskt orsakad sjukdom, och kan beskrivas som en DNA sjukdom. En stor del av cancerbehandlingarna påverkar DNA:t direkt t.ex. cisplatin. Cisplatin är ett metall baserad läkemedel som är väldigt effektivt för cancerbehandling. Cisplatin reagerar med DNA men också med RNA. När Cisplatin är inbundet rekryteras specifika proteiner, vilket i sin tur startar larmsignaler i cellen som gör att cellen dör och cancern kryper ihop.
Försök är idag på gång där RNAi används som en behandling för sjukdomar genom att nedreglera specifika proteinnivåer i cellerna. I det här projektet försöker vi att ta reda på vad som händer när RNAi och platinabehandlingen arbetar tillsammans i en cancercell. Vi har undersökt hur platina påverkar RNAi, och hur snabbt cisplatin och RNA:t reagerar med varandra. En annat viktigt fråga är hur platina minskar stabiliteten av miRNA, eftersom stabiliteten är mycket viktigt för deras funktion. Vi använder en metod som utvecklats i vår forskargrupp för att kunna studera cisplatin-RNA reaktivitet som bygger på att vi kan följa effekterna av reaktionerna m.hj.a. ljus (UV/vis spektroskopi).
Vi har också designat molekyler som efterliknar cellens egna miRNA som är kemisk modifierade. Syftet är att göra en RNA molekyl som är mer stabil och starkare i nedreglering av cancerrelaterade proteiner jämfört med t.ex. de som finns naturligt i cellen. Vi har använt miRNA sekvenser som stör produktionen av BRCA1 och PDK1 undersökt hur dessa sekvenser kan modifiera produktionen av BRCA1, och PDK1.
RNA baserade modeller av miRNA kan bli är framtidens läkemedel - klinisk prövningar är redan på gång! Ny kunskap vad gäller deras kemiska natur och reaktivetet gentemot andra vanligt förekommande läkemedel bidrar till ökad förståelse kring såväl verkningsmekanismer som uppkomst av oönskade läkemedelbiverkningar.
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Abstract
Regulation of biological processes is essential for the organism’s survival. RNA interference (RNAi) is a pathway that regulates gene expression. This pathway is triggered by small double-stranded RNA molecules, such as microRNAs (miRNAs), and small interfering RNAs (siRNAs). The specific and potent regulatory functions of RNAi and its triggers caused them to be widely used research tools, and have a therapeutic application potential.
This work has probed RNAi triggers as gene silencing tools, potential drug targets, and tentative drug candidates. Mimics of endogenous miRNAs that are of importance in breast cancer context were designed, and evaluated in terms of thermal stability, and capacity to regulate expression of target genes.... (More)
Regulation of biological processes is essential for the organism’s survival. RNA interference (RNAi) is a pathway that regulates gene expression. This pathway is triggered by small double-stranded RNA molecules, such as microRNAs (miRNAs), and small interfering RNAs (siRNAs). The specific and potent regulatory functions of RNAi and its triggers caused them to be widely used research tools, and have a therapeutic application potential.
This work has probed RNAi triggers as gene silencing tools, potential drug targets, and tentative drug candidates. Mimics of endogenous miRNAs that are of importance in breast cancer context were designed, and evaluated in terms of thermal stability, and capacity to regulate expression of target genes. The effects of both trigger-target complementarity, and chemical modifications such as 2′-O-methyl and non-nucleosidic spacers, on the stability and function of RNAi triggers were investigated using UV/vis spectroscopy, luciferase assays and RT-PCR. Our design approach yielded miRNA mimics of improved stability and various degrees of gene expression regulation capacity. The designed miRNA mimics can thus be further optimized as drug candidates, and serve as research tools for studying RNAi silencing and drug interactions.
The interaction of model RNA sequences resembling RNAi triggers with cisplatin, an anticancer compound that modulates nucleic acids, was studied. The model sequences were of sizes similar to miRNAs, and contained a central tentative platination site. The kinetics of cisplatin-RNA reaction was monitored in biologically-relevant conditions by following metal induced duplex dissociation using UV/vis and fluorescence spectroscopy. The reactivity dependence on cisplatin concentration and temperature was examined. Kinetic traces of cisplatin-RNA reaction were recorded in conditions that differ in bulk ion concentration, type of counter ion, and hydrophobicity. Furthermore, the influence RNA sequence context factors such as sequence length and base-paring flanking the platination site, on reactivity with cisplatin was followed using the same methods.
In our systems, RNA-stabilizing conditions, such as increased duplex complementarity, and higher salt concentrations retarded the reactivity with cisplatin. The reactivity was slower in hydrophobic conditions. Cisplatin-RNA reaction is dependent on both temperature, and cisplatin concentration.
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Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Kjems, Jørgen, Department of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center, Aarhus University, Denmark
organization
publishing date
type
Thesis
publication status
published
subject
keywords
miRNA, chemical modifications, siRNA, kinetics, , nucleic acids, cisplatin, anticancer drugs
pages
198 pages
publisher
Lund University, Faculty of Science, Division of Biochemistry and Structural Biology
defense location
Center for chemistry and chemical engineering, lecture hall C, Naturvetarvägen 14 (former Getingevägen 60), Lund
defense date
2016-05-20 09:15
ISBN
978-91-7422-446-7
language
English
LU publication?
yes
id
bb99661d-958d-46c9-bc10-87b93bd36609
date added to LUP
2016-04-25 11:01:24
date last changed
2016-09-19 08:45:20
@misc{bb99661d-958d-46c9-bc10-87b93bd36609,
  abstract     = {Regulation of biological processes is essential for the organism’s survival. RNA interference (RNAi) is a pathway that regulates gene expression. This pathway is triggered by small double-stranded RNA molecules, such as microRNAs (miRNAs), and small interfering RNAs (siRNAs). The specific and potent regulatory functions of RNAi and its triggers caused them to be widely used research tools, and have a therapeutic application potential.<br/>This work has probed RNAi triggers as gene silencing tools, potential drug targets, and tentative drug candidates. Mimics of endogenous miRNAs that are of importance in breast cancer context were designed, and evaluated in terms of thermal stability, and capacity to regulate expression of target genes. The effects of both trigger-target complementarity, and chemical modifications such as 2′-O-methyl and non-nucleosidic spacers, on the stability and function of RNAi triggers were investigated using UV/vis spectroscopy, luciferase assays and RT-PCR. Our design approach yielded miRNA mimics of improved stability and various degrees of gene expression regulation capacity. The designed miRNA mimics can thus be further optimized as drug candidates, and serve as research tools for studying RNAi silencing and drug interactions.<br/>The interaction of model RNA sequences resembling RNAi triggers with cisplatin, an anticancer compound that modulates nucleic acids, was studied. The model sequences were of sizes similar to miRNAs, and contained a central tentative platination site. The kinetics of cisplatin-RNA reaction was monitored in biologically-relevant conditions by following metal induced duplex dissociation using UV/vis and fluorescence spectroscopy. The reactivity dependence on cisplatin concentration and temperature was examined. Kinetic traces of cisplatin-RNA reaction were recorded in conditions that differ in bulk ion concentration, type of counter ion, and hydrophobicity. Furthermore, the influence RNA sequence context factors such as sequence length and base-paring flanking the platination site, on reactivity with cisplatin was followed using the same methods. <br/>In our systems, RNA-stabilizing conditions, such as increased duplex complementarity, and higher salt concentrations retarded the reactivity with cisplatin. The reactivity was slower in hydrophobic conditions. Cisplatin-RNA reaction is dependent on both temperature, and cisplatin concentration.<br/>},
  author       = {Alshiekh, Alak},
  isbn         = {978-91-7422-446-7},
  keyword      = {miRNA,chemical modifications,siRNA, kinetics, ,nucleic acids,cisplatin,anticancer drugs},
  language     = {eng},
  pages        = {198},
  publisher    = {ARRAY(0x99571b8)},
  title        = {Interfering with RNA interference : Platination, kinetics and down-regulation},
  year         = {2016},
}