In-vitro studies on aptamer-induced FRET between _l63_1tnN_1tn2_1tn2 [lambda_1tnN_1tn2_1tn2] tagged fluorescent protein variants [Elektronische Ressource] / vorgelegt von Amrita Kuthiala

Amrita Kuthiala

Sujets

• Biologie

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• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 33
• Poids de l'ouvrage: 9 Mo

Extrait

In-vitro Studies on Aptamer - Induced
FRET Between λ Tagged Fluorescent N22
Protein Variants
Amrita Kuthiala
München 2010In-vitro Studies on Aptamer - Induced
FRET Between λ TaggedN22
Fluorescent Protein Variants
Amrita Kuthiala
Dissertation
an der Fakultät für Biologie
der Ludwig-Maximilians-Universität
München
Angefertigt am Max-Planck-Institutü r f Neurobiologie
vorgelegt von
Amrita Kuthiala
München, September 2010Erstgutachter: Prof. Dr. Alexander Borst
Zweitgutachter: Prof. Dr. Rainer Uhl
Tag der mündlichen Prüfung: 08.12.20102Contents
1 Introduction 9
1.1 Thecentraldogmaofmolecularbiology . . . . . . . . . . . . . . . . . . . . . . . . 9
1.1.1 TheRNAworld . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
1.1.1.1 miRNA(micro-RNA) . . . . . . . . . . . . . . . . . . . . . . . . 10
1.1.1.2 snRNA(smallnuclearRNA)/snoRNA(smallnucleolarRNA) . . 10
1.1.1.3 piRNA(PIWI-interactingRNA) . . . . . . . . . . . . . . . . . . . 11
1.1.1.4 rRNA(ribosomalRNA) . . . . . . . . . . . . . . . . . . . . . . . 11
1.1.1.5 tRNA(transferRNA) . . . . . . . . . . . . . . . . . . . . . . . . 11
1.1.1.6 mRNA(messengerRNA) . . . . . . . . . . . . . . . . . . . . . . 12
1.1.2 RegulatoryconformationalchangesinRNA . . . . . . . . . . . . . . . . . . 12
1.2 RNAlocalizationandtrafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
1.2.1 AdvantagesofmRNAlocalization . . . . . . . . . . . . . . . . . . . . . . 15
1.2.2 mRNAlocalizationindifferentorganisms . . . . . . . . . . . . . . . . . . 15
1.3 mRNAtransportinneurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
1.4 MethodstodetectRNAincells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1.5 UseofbacteriophageRNAbindingpeptidesformRNAtagging . . . . . . . . . . . 21
1.5.1 Bindingoftheλ peptidetoboxB . . . . . . . . . . . . . . . . . . . . . . . 23N
1.6 Tn5transposonsystem . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
1.7 Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
1.8 FÖRSTER resonanceenergytransfer(FRET) . . . . . . . . . . . . . . . . . . . . . . 30
1.8.1 PrincipleofFRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
31.8.2 MeasurementofFRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
1.8.2.1 Onlydonorisfluorescent . . . . . . . . . . . . . . . . . . . . . . 33
1.8.2.2 Donorandacceptorarefluorescent . . . . . . . . . . . . . . . . . 33
1.8.2.3 DesignrulesoffluorescentproteinsforFRET . . . . . . . . . . . 34
1.9 Fluorescentproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
1.9.1 History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
1.9.2 Structureandproperties . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
1.9.3 GFPderivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
1.9.4 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
1.10 Aimofthethesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
2 Materialsandmethods 41
2.1 Molecularbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.1.1 DNArestrictiondigestion . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
2.1.2 LigationofDNAfragments . . . . . . . . . . . . . . . . . . . . . . . . . . 42
2.1.3 Polymerasechainreaction(PCR) . . . . . . . . . . . . . . . . . . . . . . . 42
2.1.4 λ insertionsinmKO2usingtransposonTn5 . . . . . . . . . . . . . . . . 44N22
2.1.5 Transformationofchemically-competentE.coli . . . . . . . . . . . . . . . 44
2.1.6 PreparationofE.coli . . . . . . . . . . . . . . . . . . 45
2.1.7 In-vitromRNAtranscription . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.1.8 RNAisolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
2.1.9 cDNAsynthesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
2.1.10 Agarosegel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
2.2 Proteinbiochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
2.2.1 ProteinexpressionusingtheE.coliBL21strain . . . . . . . . . . . . . . . . 49
2.2.2 PurificationofHis-tagproteinsviaaffinitychormatography . . . . . . . . . 49
2.2.3 Spectrophotometer measurements of the fluorescent proteins for calculating
extinctioncoefficientandquantumyield . . . . . . . . . . . . . . . . . . . . 52
2.2.4 Gelelectrophoresis: denaturingSDS-PAGE . . . . . . . . . . . . . . . . . 53
42.2.5 BCAassayforproteinconcentration . . . . . . . . . . . . . . . . . . . . . . 54
2.2.6 In-vitro FRET measurements to study aptamer induced FRET upon binding
oftheλNtaggedfluorescentproteins . . . . . . . . . . . . . . . . . . . . . 55
2.3 Cellculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
2.3.1 PreparationofHeLacellsculture . . . . . . . . . . . . . . . . . . . . . . . 55
2.3.2 TransfectionofHeLacellscultureusinglipofectamine . . . . . . . . . . . . 56
2.3.3 ConfocalimagingoftransfectedHeLacells . . . . . . . . . . . . . . . . . . 56
2.4 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
2.4.1 Primerslist . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
2.4.2 Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
2.4.3 Consumables . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
2.4.4 Buffers,solutionsandmedia . . . . . . . . . . . . . . . . . . . . . . . . . . 62
2.4.5 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3 Results 65
3.1 GenerationofmKO2variantswithλ insertions . . . . . . . . . . . . . . . . . . 65N22
3.2 ComparingthefluorescentpropertiesofthemKO2-λ variants . . . . . . . . . . . 69N22
3.3 GenerationofFRETdonorsproteinstaggedwithλ . . . . . . . . . . . . . . . . 72N22
3.4 StudyingaptamerinducedFRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
4 Discussion 83
A ExperimentResults 93
A.1 Resultgraphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
A.2 Discussiongraphs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
B VarioustolerantinsertionsitesinmKO2 111
Bibliography 125
5Abbreviations
APS AmmoniumPersulfate MOPS 3-(N-morpholino)propanesulfonicacid
bp Basepair mTFP monomericTealFluorescentProtein
BSA BovineSerumAlbumine MW Molecularweight
CMV cytomegalovirus N Lambda-Nprotein
Da Dalton NLS Nuclearlocalizationsignal
DMEM Dulbecco’sModifiedEagleMedium NTA nitriloaceticacid
DMSO Dimethylsulfoxid ODx Opticaldensityatxnm
DNA DeoxyribonucleicAcid PAGE PolyacrylamideGelElectrophoresis
dNTP Deoxyribonucleotid-triphosphate PBS PhosphateBufferedSaline
EDTA Ethylenediaminetetraaceticacid PCR PolymeraseChainReaction
EGFP Enhancedgreenfluorescentprotein pH reverselogarithmicrepresentation
EGTA ethyleneglycol-bis[β-amino-ethyl ofrelativeH+concentration
ether]N,N,N’,N’-tetraaceticAcid PMSF phenylmethylsulfonylfluoride
eppi EPPENDORF 1.5/2mlvial R ratio
FP fluorescentprotein ΔR differenceofratio
FRET fluorescenceresonanceenergytransfer RBP RNABindingPeptide
HBSS Hanks’balancedsaltsolution RNA Ribonucleicacid
IPTG isopropyl-β-D-thiogalactopyranoside RT RoomTemperature
L Lambda-N22(λ )protein SDS sodiumdodecylsulfateN22
LB LysogenybrothLuriabroth TAE tris-acetate-EDTAelectrophoresisbuffer
orLuria-Bertanibroth TEMED N,N,N´,N´-Tetramethyl-ethylen-
M Molar diamine
mKO2 monomericKusabiraOrange2 WT wildtype
6Abstract
LocalizationofmRNAinthecellplaysavitalroleinfunctioningofthecell,especiallyindevelopment
andneuronalplasticity. TheaimofthisthesiswastogenerateanewRNAlabelingsystemusingFRET
betweenfluorescentproteins. ShortspecificRNAsequences,calledaptamers,wereusedtobringthe
fluorescentproteinsincloseproximitytoobserveFRET.Toachievethis,theaptamerbindingpeptide,
λ ,wasinsertedatvarioussitesinthedonor(EGFPandmTFP1)andacceptor(mKO2andmKO3)N22
fluorescentproteins. Thisprovidedtheadvantageoftetheringtheλ taggedfluorescentproteinsatN22
differentanglesonthisspecificaptamer,theboxBRNA.TheideawastoinvestigatewhichFRETpair
wouldofferthebestFRETefficiencywhenthepeptidesbindtotheRNA.Dependingonthestructural
conformation the RNA adopts in solution, different distances and orientation between the donor and
acceptorswouldform,whichwouldhelptocomparetheFRETefficiencyofdifferentFRETpairs.
The first step towards this approach was generation of mKO2 acceptor proteins which have theλN22
peptide inserted in different tolerant sites in the middle of mKO2 protein. Using the Tn5 transposon
system acceptor proteins were generated offering a range of orientational specificity on binding the
boxB RNA. Generation of acceptors tagged with λ either at N- or C-terminal was done usingN22
a mutant form of mKO2 generated in the lab, termed as mKO3. mKO3 is a modified version of
mKO2, generated in the lab, where the C-terminus of mKO2 is mutated to introduce last 9 amino
acids from Aequorea GFP C-terminus thus making mKO3 more tolerant to C-terminus labelling in
comparisontomKO2. Whentheseacceptorproteinswerebroughtincloseproximitywiththedonors
(also tagged with λ ) on binding a 5boxB sequence in vitro, a small and unspecifi