Functional Modulation of Activated Protein C using DNA-Aptamers

...................................................................................................................................... I Chapter 1: General introduction and outline..............................................................................1 1.1. Blood coagulation system ............................................................................................... 2 1.2. Regulation of the coagulation system ............................................................................. 3 1.3. Activated protein C .......................................................................................................... 4 1.3.1. APC structure ........................................................................................................... 4 1.3.2. APC anticoagulant activity ....................................................................................... 5 1.3.3. APC cytoprotective activity ..................................................................................... 6 1.4. Aptamers ......................................................................................................................... 7 1.4.1. Aptamer selection procedure .................................................................................. 8 1.4.2. Capillary electrophoresis (CE) .................................................................................. 9 1.4.3. Capillary Electrophoresis-based SELEX (CE-SELEX) ................................................ 11 1.5. Single-stranded DNA production .................................................................................. 12 1.6. Aptamers against coagulation factors .......................................................................... 14 1.6.1. Thrombin binding aptamers .................................................................................. 16 1.6.2. Factor IXa binding aptamer .................................................................................... 16 1.6.3. Anti-vWF aptamers ................................................................................................ 18 1.6.4. Anti-APC aptamers ................................................................................................. 18 1.7. Aims and outlines of the thesis ..................................................................................... 19 Chapter 2: Chapillary electrophoresis for the selection of DNA aptamers recognizing activated protein C............................................................................................................20 2.1. Abstract ......................................................................................................................... 22 2.2. Introduction ................................................................................................................... 22 2.3. Materials........................................................................................................................ 23 2.3.1. Capillary electrophoresis ....................................................................................... 23 2.3.2. Polymerase chain reaction (PCR) ........................................................................... 23 2.3.3. Agarose gel ............................................................................................................. 24 2.3.4. ssDNA production .................................................................................................. 24 2.3.5. Filter retention analysis ......................................................................................... 25 2.4. Methods ........................................................................................................................ 25 III 2.4.1. Installation and conditioning of a new capillary .................................................... 26 2.4.2. CE-based isolation of target-binding ssDNA-molecules ........................................ 27 2.4.3. PCR-based amplification of selected ssDNA .......................................................... 29 2.4.4. Asymmetric PCR and isolation of ssDNA ............................................................... 29 2.4.5. Filter retention experiment ................................................................................... 30 2.5. Notes ............................................................................................................................. 31 Chapter 3: Capture and Release (CaR): A simplified procedure for one-tube isolation and concentration of single-stranded DNA during SELEX..........................................................35 3.1. Abstract ......................................................................................................................... 37 3.2. Main manuscript ........................................................................................................... 37 3.3. Electronic supplementary information ( ESI†) .............................................................. 43 3.3.1. Chemicals and reagents ......................................................................................... 43 3.3.2. Prediction of DNA hybridization profiles and design of capture-molecules ......... 43 3.3.3. Binding of capture-molecules to streptavidin-coated magnetic beads (SMB) ...... 45 3.3.4. Assessment of binding and adverse release of capture molecules to /from SMB using fluorescence measurements ...................................................................................... 45 3.3.5. Exponential amplification and asymmetric PCR .................................................... 46 3.3.6. Production of asymmetrically amplified IHT1-library for evaluation purposes .... 47 3.3.7. Assessment of quality and purity of ssDNA after asymmetric PCR/ CaR during basic assay evaluation .......................................................................................................... 47 3.3.8. Quantification of streptavidin released from the SMB ......................................... 49 3.3.9. CE-SELEX against APC and FXIIIAa .......................................................................... 49 3.3.10. Yield and purity of ssDNA as produced by asymmetric PCR/ CaR during SELEX 50 3.3.11. Filter retention assay .......................................................................................... 51 3.3.12. Cloning and sequencing ..................................................................................... 52 3.3.13. Production of identified individual aptamers by asymmetric PCR/ CaR and determination of binding affinity ......................................................................................... 52 3.3.14. In silico folding predictions ................................................................................ 55 3.3.15. Determination of the reusability of loaded SMB ............................................... 55 Chapter 4: Modifying substrate specificity of the serine protease activated protein C using exocite-modulating aptamers.......................................................................................................55 4.1. Abstract ......................................................................................................................... 58 4.2. Main manuscript ........................................................................................................... 58 IV 4.3. Supplementary information .......................................................................................... 64 4.3.1. Chemicals and materials ........................................................................................ 64 4.3.2. Capillary electrophoresis-(CE)-SELEX ..................................................................... 65 4.3.3. Next generation sequencing and data analysis ..................................................... 65 4.3.4. In silico secondary structure predictions ............................................................... 65 4.3.5. Detection of G-quadruplex formation by Thioflavin T-staining ............................. 66 4.3.6. Determination of dissociation constants and binding competition experiments . 66 4.3.7. OECA-based binding competition experiments ..................................................... 67 4.3.8. APC amidolytic assay .............................................................................................. 67 4.3.9. FVa and FVIIIa inactivation assays ......................................................................... 67 4.3.10. Thrombin generation assay ................................................................................ 68 4.3.11. APC anticoagulant activity in whole blood ........................................................ 68 4.3.12. APC-APC-inhibitor complex formation testing ................................................... 69 4.4. Supplementary tables and figures ................................................................................ 69 Abbreviations ........................................................................................................................... 79 Bibliography.............................................................................................................................. 82 Acknowledgement ................................................................................................................... 91 Curriculum Vitae ....................................................................................................................... 92