Development of fluorescent biosensors probing RNA function [Elektronische Ressource] / Martina Schifferer-Waritschlager. Betreuer: Oliver Griesbeck

Martina Schifferer-Waritschlager

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 35
• Langue: Deutsch
• Poids de l'ouvrage: 7 Mo

Extrait

Development of fluorescent biosensors
probing RNA function






Dissertation
der Fakultät für Biologie
der Ludwig-Maximilians-Universität
München


vorgelegt von
Martina Schifferer-Waritschlager



München, den 15.09.2011 2 Contents




























Betreuer: Dr. Oliver Griesbeck
Erstgutachter: Prof. Dr. Alexander Borst
Zweitgutachter: Prof. Dr. Charles David
Tag der mündlichen Prüfung: 08.12.2011 3 Contents











“Hier treffen wir nun auf die eigene Schwierigkeit, die nicht immer klar ins Bewusstsein tritt,
dass zwischen Idee und Erfahrung eine gewisse Kluft befestigt scheint, die zu überschreiten
unsere ganze Kraft sich vergeblich bemüht. Demohngeachtet bleibt unser ewiges Bestreben,
diesen Hiatus mit Vernunft, Verstand, Einbildungskraft, Glauben, Gefühl, Wahn und, wenn
wir sonst nichts vermögen, mit Albernheit zu überwinden.“

J.W. Goethe
Bedenken und Ergebung (1823)












4 Contents

Contents
Contents ..................................................................................................................................... 4
Table of figures .......................... 9
Abbreviations ........................................................................................................................... 12
Abstract .................................... 14
1. Introduction ......................... 16
1.1. RNA characteristics ....................................................................................................... 16
1.1.1 RNA structure .......... 16
1.1.2 RNA classification .................................................................................................... 16
1.2. mRNA fate in eukaryotic cells ....................................................................................... 17
1.2.1 mRNA transcription and processing ....................................................................... 17
1.2.2 mRNA subcellular localization ................ 19
1.2.3 mRNA turnover ....................................... 27
1.3. viral RNA-protein interactions ...................................................................................... 30
1.3.1 Arginine-rich motif (ARM) peptides ........................................................................ 30
1.3.2 HIV RRE aptamer ..................................... 31
1.3.3 Rsg1.2 –RRE interaction .......................................................... 33
1.4. Fluorescence imaging ................................................................... 34
1.4.1 Fluorescent proteins ............................................................... 34
1.4.2 Genetically encoded fluorescent biosensors .......................... 36
1.5. RNA imaging .................................................................................................................. 40
1.5.1 Chemical RNA dyes ................................................................................................. 40
1.5.2 Molecular beacons .. 42
1.5.3 Genetically encoded FP-based RNA labels ............................. 42 5 Contents

1.5.4 Recent advances in imaging technology ................................................................. 47
1.6 Project goal .................................................................................................................... 49
2. Materials and Methods ........................................................................................................ 52
2.1. DNA molecular biology . 52
2.1.1 Polymerase Chain Reaction (PCR) ........................................................................... 52
2.1.2 Error-prone PCR ...................................... 53
2.1.3 Site-directed mutagenesis via PCR ......... 54
2.1.4 DNA purification...................................................................... 56
2.1.5 Restriction digest of DNA ........................................................ 56
2.1.6 Dephosphorylation of vector DNA .......... 57
2.1.7 Ligation of DNA fragments ...................................................... 58
2.1.8 Preparation of chemically-competent E. coli .......................... 58
2.1.9 Transformation of chemically-competent E. coli .................... 59
2.1.10 Bacterial DNA purification ................................................................ 59
2.2. RNA molecular biology .................................................................................................. 59
2.2.1 In vitro transcription ............................... 60
2.2.2 Reverse transcription (RT) ...................................................................................... 61
2.2.3 Systematic Evolution of Ligands by Exponential Enrichment (SELEX) .................... 62
2.3 Protein Biochemistry...................................................................................................... 64
2.3.1 Protein expression using the E. coli BL21 strain ..................................................... 64
2.3.2 Affinity purification of recombinantly expressed proteins ..................................... 65
2.3.3 Size exclusion chromatography of recombinantly expressed proteins .................. 65
2.3.4 ATPase assay ........................................................................... 66
2.4 Spectroscopy .................................................. 66
2.4.1 Testing for in vitro ratio change of FRET RNA sensors............ 66 6 Contents

2.4.2 Spectroscopic determination of K -value ............................................................... 67 D
2.4.3 pH titration .............................................................................................................. 67
2.4.4 In vitro transcription assay ...................... 67
2.4.5 Spectroscopic REFlex FRET assay ............ 67
2.5 Bacterial screening ......................................................................................................... 68
2.5.1 Sensor library generation ....................................................................................... 68
2.5.2 Bacterial colony selection 68
2.6 Cellular biology............................................................................................................... 70
2.6.1 Preparation of dissociated hippocampal neuronal culture .................................... 70
2.6.2 Immuncytochemistry .............................................................................................. 72
2.6.3 Fluorescence in situ hybridization (FISH) 73
2.6.4 Isolation of DNA and RNA from cell lysates ............................ 74
2.6.5 Transfection methods ............................................................................................. 75
2.6.6 Cellular assays and imaging .................... 77
2.7 Materials ........................................................................................................................ 79
2.7.1 Instruments ............................................................................................................. 79
2.7.2 Consumables ........... 79
2.7.3 Buffers, solutions and media .................. 80
2.7.4 Chemicals ................................................................................................................ 82
2.7.5. Plasmids, bacterial strains, cell-lines and flies ....................... 84
3. Results .................................................................................................................................. 86
3.1 Application of genetically encodable RNA labels for imaging of neuronal RNAs .......... 86
3.1.1. Dual RNA imaging in neurons ................................................................................ 86
3.1.2. NMDAR1 splicing reporter ..................... 88
3.2. RNA imaging using an on-off chemical dye .................................................................. 90 7 Contents

3.2.1 In vitro development of the on-off dye – aptamer pair ......................................... 91
3.2.2 On-off dye performance in mammalian cell lines .................. 92
3.3. Development of a genetically encodable dynamic single fluorophore RNA sensor .... 94
3.3.1 FP emission change upon direct aptamer interaction ............................................ 94
3.3.2 Aptamer-dependent emission change in a FP-peptide hybrid ............................... 96
3.4 Engineering of the FRET-based RNA biosensor VAmPIRe ............................................. 99
3.4.1 Aptamer-peptide pair selection for the FRET biosensor ........ 99
3.4.2 Rational engineering of FR-Rsg1.2 ........................................................................ 102
3.4.3 Development of a functional screen for FRET sensor performance in bacteria .. 107
3.4.4 VAmPIRe in vitro characterization ........ 111
3.4.5 Aptamer engineering ...............................................................