Covalent Labeling of Protein and RNA Using Fluorescent Dyes Open Access

Ayele, Tewoderos (Summer 2020)

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Living systems are complex machines that are operated by tightly regulated interactions and organizations of biomolecules. Therefore, sensitive detection and tracking of these molecules are essential for understanding their complex biochemical properties. While there are various approaches for labeling and visualization of cellular biomolecules, the utility of the majority of these approaches is limited due to their non-covalent nature. To identify and image biomolecules in the complex environment, fluorescent probes must have several essential characteristics. In addition to being compatible with the cellular environment, these probes need to be cell-permeable, non-cytotoxic, selective, produce high signal-to-background ratio, and have minimal effect on the native property of the biomolecule. To date, no one technique fulfills all of these requirements, creating a unique demand for fluorescent probes that are adaptable for various experimental conditions. As a result, synergistic advances in organic chemistry, biology, physics, chemical biology, and spectroscopy are underway for improving these fluorescent probes. The work presented here aims to address this limitation by introducing a broadly applicable strategy for covalently and fluorescently labeling proteins and RNAs in complex environments. This strategy utilizes rationally designed fluorescent probes containing various reactive linkers. 

Chapter 2 describes a photoaffinity protein labeling approach using a novel malachite green analog. We show that this technique selectively and covalently labels target proteins in live mammalian cells with temporal resolution and minimal background signal. 

Chapter 3 describes the implementation of a similar photoaffinity approach to covalently label cellular RNA. In this work, we report the design of the first covalent light-up aptamer system for visualizing the spatiotemporal localization pattern of mRNA in live mammalian cells. 

Chapter 4 describes an approach for covalent chemical labeling and affinity capture of inosine-containing RNAs using acrylomidofluorescein. 

Taken together, the results demonstrated in this work highlight the advantages and utility of covalent and fluorescent labeling of biomolecules in complex environments.

Table of Contents

Chapter 1: Fluorescent labeling of RNA and proteins. 1

1.1 Introduction. 2

1.2 Protein labeling. 2

1.2.1 FP-based protein labeling. 2

1.2.2 Organic fluorescent dyes for protein labeling. 4

1.3 RNA labeling. 5

1.3.1 Single molecule fluorescent in situ hybridization for RNA labeling. 5

1.3.2 Bacteriophage coat protein-derived RNA labeling. 7

1.3.3 Light-up aptamer-based RNA labeling. 9

1.3.4 Remaining challenges in RNA labeling. 11

1.4 References. 12

Chapter 2 : Fluorogenic Photoaffinity Labeling of Proteins in Living Cells. 16

2.1 Abstract 17

2.2 Introduction. 17

2.3 Design and synthesis of fluorogenic photoaffinity labeling system.. 19

2.4 In vitro protein labeling. 22

2.5 Protein labeling in live cells. 25

2.6 Conclusion. 27

2.7 Methods and supplemental information. 28

2.7.1 Synthesis of MG-diazirine. 28

2.7.2 Preparation of FAP fusion vectors. 31

2.7.3 Preparation of mCer3-FAP plasmid vector. 32

2.7.4 HeLa cell culture and maintenance. 33

2.7.5 Plasmid Transfection. 34

2.7.6 Imaging. 35

2.7.7 Washout experiment 36

2.7.8 Kinetic experiment for protein concentration. 37

2.7.9 MG-diazirine concentration dependent kinetic experiment 39

2.7.10 Cell viability experiment 40

2.8 References. 41

Chapter 3: Imaging and tracking mRNA in live mammalian cells via fluorogenic photoaffinity labeling. 48

3.1 Abstract 49

3.2 Introduction. 50

3.3 Results. 54

3.3.1 In vitro characterization. 54

3.3.2 Fixed-cell imaging of RNA. 56

3.3.3 Comparison of fixed cell imaging of MGA/MGD2 with FISH. 59

3.3.4 Live cell imaging of mRNA. 61

3.4 Conclusion. 63

3.5 Methods and supplemental material 64

3.5.1 In vitro fluorescence enhancement 64

3.5.2 Cell culture and transfection. 65

3.5.4 Immunofluorescence and MGA/MGD2 co-imaging. 66

3.5.5 Confocal microscopy. 67

3.5.6 Live cell imaging. 68

3.5.7 MGA array plasmid construction. 68

3.5.8 Synthesis of MGD2. 70

3.5.9 Determination of MGD2 selectivity in cellular RNA. 71

3.6 References. 78

Chapter 4 : Chemical Labeling and Affinity Capture of Inosine-Containing RNAs Using Acrylamidofluorescein. 82

4.1 Abstract 83

4.2 Introduction. 83

4.3 Supporting information. 94

4.3.1 Synthesis of Acrylamidofluorescein. 94

4.3.2 Ribonucleoside Labeling and HPLC Analysis. 95

4.3.3 RNA Oligoribonucleotides. 105

4.3.4 Oligoribonucleotide labelling and PAGE analysis. 106

4.3.5 Oligoribonucleotide labelling and Immunoprecipitation pulldown. 106

4.4 References. 108

Chapter 5 : Conclusion and future direction. 112

5.1 Conclusion and future direction. 113

List of Figures

Figure 1-1 Methods for genetic tagging and fluorescent visualization of target proteins. 3

Figure 1-2 Methods for fluorescent labeling of Intracellular RNA. a, smFISH for RNA labeling. 7

Figure 2-1 a, Temporally-controlled covalent labeling of a protein of interest (POI) 20

Figure 2-2 In vitro assessment of covalent photoaffinity labeling. 23

Figure 2-3 Kinetics of labeling reaction. 24

Figure 2-4 Testing the specificity of FAP labeling in cell lysate. 25

Figure 2-5 Fluorescence imaging of HeLa cells transfected with mCerulean3-FAP. 27

Figure 2-6 Plasmid map of mCer3-FAP control expression vector highlighting important component for cloning and expression. 32

Figure 2-7 Representative fluorescence microscopy images of transfected HeLa cells incubated with MG-diazirine. 36

Figure 2-8 Washout experiment. 37

Figure 2-9 Protein concentration dependent kinetic experiment. 38

Figure 2-10 Kinetic experiment for MG-diazirine concentration. 39

Figure 2-11 Cell viability. 41

Figure 3-1 Characterization of MGA-functionalized mRNA in the presence of MGD2. 52

Figure 3-2 RNA labeling in fixed cells. 57

Figure 3-3 Live cell tracking of RNA and protein granules. 60

Figure 3-4 Selective labeling of MGA functionalized mRNA in the presence of cellular RNA extracted from HeLa cells. 72

Figure 3-5 UV dependent labeling of RNA. 76

Figure 4-1 Chemical labeling of inosine. 85

Figure 4-2 Representative HPLC traces depicting the reaction between inosine and acrylamidofluorescein over 24 hours. 86

Figure 4-3 Denaturing PAGE analysis of synthetic oligoribonucleotides labeled with acrylamidofluorescein. 89

Figure 4-4 Workflow for quantifying pulldown efficiency with acrylamidofluorescein labeling and immunoprecipitation. 91

Figure 4-5 Synthesis of acrylamidofluorescein. 94

Figure 4-6 Representative HPLC traces of ribonucleoside reactivity with acrylonitrile. 97

Figure 4-8 Percent conversion of ribonucleosides when reacted with acrylamidofluorescein. 101

Figure 4-9 ESI-MS and MS/MS spectra of isolated product fraction for the reaction of inosine and acrylonitrile. 102

Figure 4-10 ESI-MS and MS/MS spectra of isolated product fraction for the reaction of inosine and acrylamidofluorescein. 103

Figure 4-11 ESI-MS and MS/MS spectra of isolated product fraction for the reaction of pseudouridine and acrylonitrile. 104

Figure 4-12 ESI-MS and MS/MS spectra of isolated product fraction for the reaction of pseudouridine and acrylamidofluorescein. 105

List of Tables

Table 2-1 Tabular data for protein concentration dependent kinetic experiment 38

Table 2-2 Tabular data for protein concentration dependent kinetic experiment 40

Table 2-3 Tabular data for cell viability experiment 41

Table 3-1 Tabular data fluorescence output of MGA array  73

Table 3-2 Tabular data for UV dependent fluorescence enhancement of 1xMGA-mRNA compared to MGD2 in 1xPBS. 73

Table 3-3 Tabular data for UV dependent fluorescence enhancement of control mRNA compared to MGD2 in 1xPBS. 73

Table 3-4 Raw data table for fluorescence intensity of RNA foci in untrasfected Neuro-2a cells or Neuro-2a cells expressing mCDK6 functionalized with 1xMGA or 6xMGA at the 5’UTR 74

Table 3-5 Tabular data for fluorescence of RNA foci 76

Table 4-1 Preparation of RNA-I-Cy5 and RNA-A-Cy3 mixture solutions. 107

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