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Hyphenating Chromatographic, Electrophoretic and On-Line Immobilized Enzymatic Strategies for Improved Oligonucleotide Analysis

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Hyphenating Chromatographic, Electrophoretic and On-Line Immobilized Enzymatic Strategies for Improved Oligonucleotide Analysis Hyphenating chromatographic, electrophoretic and on-line immobilized enzymatic strategies for improved oligonucleotide analysis Thesis submitted to the Faculty of Science in fulfilment of the requirements for the degree of Doctor in Science (Chemistry) PIOTR WIKTOR ALVAREZ POREBSKI Promotor Prof. Dr. Frederic Lynen 2016 Table of contents Table of Contents TABLE OF CONTENTS…………………………………………….………………………………………….……………………………… I I GENERAL INTRODUCTION AND SCOPE ............................................................................................. 1 INTRODUCTION .................................................................................................................................. 1 SCOPE .............................................................................................................................................. 3 REFERENCES ...................................................................................................................................... 4 II PRINCIPLES OF THE SEPARATION TECHNIQUES USED IN OLIGONUCLEOTIDE ANALYSIS ................ 7 SUMMARY ............................................................................................................................................. 7 PRINCIPLES OF HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) ...................................................... 8 Ion exchange chromatography ........................................................................................... 10 Ion pair chromatography .................................................................................................... 11 Hydrophilic interaction liquid chromatography .................................................................. 12 FUNDAMENTAL CHROMATOGRAPHIC PARAMETERS ................................................................................. 13 Retention factor .................................................................................................................. 15 Selectivity factor .................................................................................................................. 16 Efficiency ............................................................................................................................. 16 Resolution ............................................................................................................................ 16 Van Deemter equation ........................................................................................................ 17 Peak capacity ...................................................................................................................... 19 ULTRAHIGH PERFORMANCE LIQUID CHROMATOGRAPHY ........................................................................... 22 PRINCIPLES OF ELECTRODRIVEN SEPARATION TECHNIQUES USED FOR OLIGONUCLEOTIDE ANALYSIS ................... 24 Basic principles of capillary electrophoresis ....................................................................... 26 II.4.1.1 Electrophoresis ............................................................................................................. 26 II.4.1.2 Electroosmosis ............................................................................................................. 27 II.4.1.3 Apparent electrophoretic mobility and migration time ............................................... 28 Zone dispersion in capillary electrophoresis ....................................................................... 28 Resolution in CE ................................................................................................................... 29 Modes of operation in capillary electrophoresis ................................................................. 29 I Table of contents II.4.4.1 Capillary zone electrophoresis (CZE) ............................................................................ 29 II.4.4.2 Capillary gel electrophoresis (CGE) .............................................................................. 30 II.4.4.2.1 Entangled polymer solutions ............................................................................... 30 II.4.4.2.1.1 Entanglement threshold ......................................................................................... 31 II.4.4.2.1.2 Mesh size ........................................................................................................... 31 II.4.4.2.1.3 Mechanisms of electrophoretic migration in entangled polymer solutions ............................ 33 FUNDAMENTAL ASPECTS OF MULTIDIMENSIONAL SEPARATION TECHNIQUES ................................................. 34 Orthogonality, sampling frequency and peak capacity ...................................................... 37 II.5.1.1 Orthogonality ............................................................................................................... 37 II.5.1.2 Sampling frequency ...................................................................................................... 38 II.5.1.3 Peak capacity in 2D separations .................................................................................. 38 REFERENCES ................................................................................................................................... 40 III OLIGONUCLEOTIDES: CHARACTERISTICS AND CONTEMPORARY ANALYTICAL STRATEGIES ...... 43 SUMMARY ........................................................................................................................................... 43 INTRODUCTION .............................................................................................................................. 44 RELEVANT MECHANISMS OF ACTION OF ANTISENSE OLIGONUCLEOTIDES ..................................................... 49 RELEVANCE OF NUCLEASE RESISTANCE OF OLIGONUCLEOTIDES ................................................................. 51 TOXICITY ASPECTS OF ANTISENSE OLIGONUCLEOTIDES ............................................................................. 54 ANALYTICAL SEPARATION METHODS FOR THE ANALYSIS OF OLIGONUCLEOTIDES ........................................... 55 Electrophoretic methods for the analysis of oligonucleotides ........................................... 56 Liquid chromatography methods for the analysis of oligonucleotides .............................. 59 III.5.2.1 Ion-exchange chromatography for the analysis of oligonucleotides ......................... 59 III.5.2.2 Ion-pair chromatography for the analysis of oligonucleotides .................................. 61 III.5.2.3 Hydrophilic interaction liquid chromatography for the analysis of Oligonucleotides 63 III.5.2.4 Mixed mode, affinity and bidimensional separation methods for the analysis of oligonucleotides ....................................................................................................................... 65 REFERENCES .................................................................................................................................. 68 IV COMBINING LIQUID CHROMATOGRAPHY WITH MULTIPLEXED CAPILLARY GEL ELECTROPHORESIS FOR OFFLINE COMPREHENSIVE ANALYSIS OF COMPLEX OLIGONUCLEOTIDE SAMPLES ...................................................................................................................................... 75 SUMMARY ........................................................................................................................................... 75 INTRODUCTION .............................................................................................................................. 76 II Table of contents EXPERIMENTAL .............................................................................................................................. 79 Chemicals ........................................................................................................................... 79 Instrumentation ................................................................................................................. 79 Chromatographic conditions ............................................................................................. 80 Electrophoretic conditions ................................................................................................. 80 IV.2.4.1 Single capillary system ................................................................................................ 80 IV.2.4.2 Multiplexed capillary system ...................................................................................... 81 RESULTS AND DISCUSSION ................................................................................................................ 82 Offline comprehensive ON analysis by IEC x CGE .............................................................. 82 Offline comprehensive ON analysis by IPC x CGE .............................................................. 86 Peak capacity of IEC x CGE and IPC x CGE approaches ...................................................... 89 Figures of merit of the developed methodology................................................................ 91 CONCLUSIONS ............................................................................................................................... 93 REFERENCES .................................................................................................................................. 93 V POSSIBILITIES AND LIMITATIONS OF SPECTRAL DECONVOLUTION IN PHOTODIODE ARRAY DETECTION SEPARATIONS OF OLIGONUCLEOTIDES
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