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`LAWYERS' AND MERCHANTS' TRANSLATION BUREAU, INC.
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`Legal, Financial, Scientific, Technical and Patent Translations
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`30 BROAD STREET, 41ST FLOOR
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`Certificate of Accuracy
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`TRANSLATION
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`From German into English
`
`
`Date: August 22, 2017
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`
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`I, Charles Edward Sitch, BA, declare
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`That to the best of my knowledge and belief, the attached document, prepared by one
`of its translators competent in the art and conversant with the German and English languages,
`is a true, correct and accurate translation into the English language of the accompanying
`document.
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`Vice President
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`LAWYERS' AND MERCHANTS'
`TRANSLATION BUREAU
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`1
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`ELIX 1004
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`

`

`Isothermal In Vitro Selection and
`Amplification to Investigate
`Evolutionary Processes
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`Dissertation
`to obtain the Doctor of Philosophy degree
`from the Faculty of Chemistry
`at Ruhr-University Bochum
`
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`submitted by
`Sylvia Ehses
`from Bernkastel-Kues
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`Sankt Augustin 2005
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`This work was prepared between October 2002 and January 2005 under the supervision of Prof.
`Dr. G. von Kiedrowski and between February 2005 and April 2005 under the supervision of Prof.
`Dr. J. S. McCaskill at the Fraunhofer Society e.V. in Sankt Augustin.
`
`Parts of this work have been published: S. Ehses, J. Ackermann, J. S. McCaskill: Optimization and
`Design of oligonucleotide setup for Strand Displacement Amplification. Journal of Biochemical
`and Biophysical Methods, 63(3): 170 – 86, 2005.
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`1st Principal Investigator: Prof. Dr. J. S. McCaskill
`2nd Principal Investigator: Prof. Dr. G. von Kiedrowski
`Third examiner: Prof. Dr. W. Schuhmann
`Date of the oral examination: 07.08.2005
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`Abstract
`The development of a system is presented here that uses simple in vitro biochemical systems to
`investigate evolutionary processes. As a biochemical reaction, SDA (strand displacement
`amplification) was developed further as a DNA amplification mechanism and was adapted to meet
`the requirements of an in vitro evolution experiment. In addition to a SELEX setup based on the
`mutation and selection of nucleic acids, the system was adapted with regard to developing a self-
`contained evolvable system. Thus, it was demonstrated how the amplification mechanism can be
`controlled in microfluidic structures - a step toward complex systems that will help answer
`fundamental questions about evolution.
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`4
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` I
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` would like to take this opportunity to thank all those who accompanied me throughout the time
`this work came into being.
`
`Firstly, I owe a debt of gratitude to Prof. Dr. John S. McCaskill under whose supervision I learned
`to grasp and process the subject matter concerned.
`
` I
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` am grateful to the Fraunhofer Society for the financial and material support that enabled me to
`drive this work forward under excellent working conditions.
`
`My thanks also go to Prof. Dr. G. von Kiedrowski who, in addition to substantive discussions, also
`provided organizational assistance.
`
`Despite the structural changes and the, at times, difficult working conditions, I would like to thank
`the individual members of the former BioMIP research group for their constant willingness to help
`and their personable company.
`
` I
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` would like to thank my parents for their tireless support throughout these years. Also, I thank
`Stefan whose companionship and patience over many years made many things easier. Moreover, I
`am grateful to Robert for his sometimes sobering, but also motivating, words.
`
`It would be amiss of me not to also thank those who encouraged me by simply believing that this
`project was finite…
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`5
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`1.2 
`
`Table of contents
`1 
`Introduction ................................................................................................... 13 
`1.1  Molecular evolution ............................................................................ 14 
`In vitro selection and SELEX .................................................. 14 
`1.1.1 
`1.1.2  Molecular fitness landscapes ................................................... 15 
`Isothermal amplification procedures .................................................. 16 
`1.2.1  Strand displacement amplification .......................................... 17 
`Contents of the work ........................................................................... 20 
`1.3 
`2  Materials and methods ................................................................................. 23 
`2.1  Materials ............................................................................................. 23 
`2.1.1  Chemicals, enzymes, standards ............................................... 23 
`2.1.2  Apparatus ................................................................................ 25 
`2.1.3  Software .................................................................................. 26 
`2.1.4  Oligonucleotides, plasmids and bacterial strains .................... 26 
`2.1.5  Overview of oligonucleotides ................................................. 26 
`2.1.6  Bacterial strains and plasmids ................................................. 29 
`2.1.7  Commonly used buffer solutions, media and plates ............... 30 
`Basic molecular biology methods ....................................................... 31 
`2.2.1  Determination of oligonucleotide concentrations ................... 31 
`2.2.2  DNA preparation and cleaning up of nucleic acid
`solutions .............................................................................................. 31 
`2.2.3  Gel electrophoresis .................................................................. 33 
`2.2.4  Cloning of amplification products and transformation ........... 33 
`2.2.5  Polymerase chain reaction ....................................................... 35 
`Strand displacement amplification ...................................................... 36 
`2.3.1  Standard SDA .......................................................................... 36 
`2.3.2  Nicking SDA ........................................................................... 38 
`2.3.3  Elongation ............................................................................... 38 
`2.3.4  Experimental optimization ...................................................... 39 
`2.3.5  Optimization of template and primer design ........................... 39 
`2.3.6  Saturable inhibition through the use of additives .................... 40 
`
`2.2 
`
`2.3 
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`4
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`TABLE OF CONTENTS
`
`2.4 
`
`2.5 
`
`Characterization of molecular beacons ............................................... 42 
`2.4.1  Determination of the signal-to-background ratio .................... 42 
`2.4.2  Recording of the thermal denaturation profile ........................ 44 
`2.4.3  Construction of a phase diagram ............................................. 44 
`Selection-amplification cycle ............................................................. 47 
`Immobilization
`of DNA
`oligonucleotides
`on
`2.5.1 
`microparticles ...................................................................................... 47 
`2.5.2  Determination of the coupling efficiency ................................ 47 
`2.5.3  Experimental setup .................................................................. 48 
`2.5.4  Determination of the binding energy ...................................... 49 
`SDA in microstructures ...................................................................... 50 
`2.6.1  SDA module ............................................................................ 50 
`2.6.2  SDA in microfluidic structures: fan reactor ............................ 52 
`3  Results ............................................................................................................ 59 
`3.1 
`Strand displacement amplification ...................................................... 59 
`of
`the
`system
`and
`experimental
`3.1.1  Description
`optimization of the reaction conditions .............................................. 59 
`of
`an
`algorithm
`to
`optimize
`3.1.2  Development
`oligonucleotide design ........................................................................ 71 
`3.1.3  Saturable inhibition as a means of generating non-linear
`reaction kinetics .................................................................................. 72 
`3.2  Molecular beacon MB:A11 binding studies ....................................... 77 
`3.2.1  Hybridization kinetics and thermodynamics ........................... 79 
`3.2.2  Hybridization experiments in sequence pools ......................... 83 
`Selection-amplification cycle ............................................................. 89 
`3.3.1  Selection using one probe ....................................................... 90 
`3.3.2  Selection using two probes ...................................................... 93 
`SDA in microstructures .................................................................... 104 
`3.4.1  SDA module .......................................................................... 105 
`3.4.2  SDA in the fan reactor: Temporospatially resolved
`representation of the SDA reaction under flow conditions ............... 105 
`
`2.6 
`
`3.3 
`
`3.4 
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`TABLE OF CONTENTS 5
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`4  Discussion ..................................................................................................... 111 
`4.1 
`SDA as a flexible replication tool ..................................................... 112 
`4.2 
`Selection using DNA hybridization .................................................. 113 
`4.3 
`SDA in microfluidic systems ............................................................ 115 
`4.4 
`Outlook ............................................................................................. 117 
`A  Sequence data for the 2-probe selection ........................................................ I 
`A.1  Round 4 ..................................................................................... I 
`A.2  Round 6 .................................................................................... II 
`A.3  Round 8 .................................................................................. IV 
`A.4  Round 10 ................................................................................ VI 
`A.5  Round 12 ............................................................................... VII 
`A.6  Round 14 ................................................................................ IX 
`A.7  Round 16 .................................................................................. X 
`B  Binding energies of the 2-probe selection ............................................... XIII 
`C  Algorithms ................................................................................................. XXI 
`C.1  Optimization of oligonucleotide design ........................................... XXI 
`C.2  Determination of binding energies ................................................ XXV 
`C.3 
`Characterization of the thermodynamics of MB:A11 .................... XXX 
`C.4  Model for the characterization of selection events ..................... XXXII 
`D  List of abbreviations ............................................................................. XXXV 
`E  Symbols used in formulas .................................................................. XXXVII 
`References .................................................................................................... XXXIX 
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`6
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`TABLE OF CONTENTS
`TABLE OF CONTENTS
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`9
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`List of figures
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`Figure 1.1: Pictorial representation of “fitness landscapes” ............................................................ 18 
`
`Figure 1.2: Basic mechanism of SDA using exo- DNA polymerase and the restriction enzyme
`BsoBI. ................................................................................................................................................. 19 
`
`Figure 2.1: Cloning vector pCR2.1-TOPO ...................................................................................... 30 
`
`Figure 2.2: SDA oligonucleotide structure. ..................................................................................... 40 
`
`Figure 2.3: Use of the partition function to screen for possible non-specific hybridization and
`elongation reactions. .......................................................................................................................... 41 
`
`Figure 2.4: Phase transitions in solutions with molecular beacons. ................................................ 43 
`
`Figure 2.5: SDA module for the real-time detection of the SDA reaction in microstructures. ...... 51 
`
`Figure 2.6: Setup of the tandem optics used to observe reactions in microstructures. ................... 53 
`
`Figure 2.7: Tandem optic components ............................................................................................. 54 
`
`Figure 2.8: Sketch of the fan reactor used to perform SDA under flow conditions. ....................... 55 
`
`Figure 2.9: Microscope setup for the detection of the SDA reaction in the fan reactor under flow
`conditions. .......................................................................................................................................... 56 
`
`Figure 3.1: Standard and nicking SDA in relation to reaction temperature. ................................... 61 
`
`Figure 3.2: SDA in relation to magnesium ion concentration. ........................................................ 62 
`
`Figure 3.3: Enzyme series: Dependency of nicking SDA on the concentrations of exo- Bst
`polymerase and the restriction enzyme N.BstNBI. ........................................................................... 63 
`
`Figure 3.4: Real-time detection of SDA on tSDAI and fitting of measurements to exponential and
`logistic growth models ....................................................................................................................... 63 
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`Figure 3.5: Asymmetric SDA. .......................................................................................................... 65 
`
`Figure 3.6: Analysis of the SDA reaction products at different template starting concentrations. 67 
`
`Figure 3.7: Elongation experiment to characterize side products due to primer-dimer formation. 68 
`
`Figure 3.8: Effect of additives (hp-DNA, heparin and polyC) on the SDA. ................................... 70 
`
`Figure 3.9: Primer dilution series. .................................................................................................... 71 
`
`Figure 3.10: Flowchart of the SDA optimization algorithm ............................................................ 73 
`
`Figure 3.11: Analysis of the SDA reaction products of the optimized oligo design with the target
`sequence tSDAmin5. .......................................................................................................................... 74 
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`8
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`LIST OF FIGURES
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`Figure 3.12: Inhibition of the SDA reaction SDAI with TOTO-1 at a constant template
`concentration. ..................................................................................................................................... 75 
`
`Figure 3.13: Inhibition of the SDA reaction with TOTO-1 at different initial template
`concentrations .................................................................................................................................... 76 
`
`Figure 3.14: Inhibition of the SDA reaction with TOTO-1 at different dye concentrations in the
`SDA module ....................................................................................................................................... 77 
`
`Figure 3.15: Inhibition of the SDA with PNA oligonucleotides in relation to concentration of
`template tSDAI. .................................................................................................................................. 78 
`
`Figure 3.16: Effect of the PNA oligonucleotides during the course of the SDA reaction. ............. 79 
`
`Figure 3.17: Representation of the molecular beacon MB:A11. ..................................................... 80 
`
`Figure 3.18: Determination of the melting temperatures of target sequences with known base
`sequence using the partition function. ............................................................................................... 83 
`
`Figure 3.19: Denaturation profile of the molecular beacon MB:A11 in equilibrium with the
`complementary target sequence and in relation to the concentration of the target sequence. ......... 84 
`
`Figure 3.20: Determination of the thermodynamic parameters ....................................................... 85 
`
`Figure 3.21: Free energy of the three phases of a solution of the molecular beacon MB:A11 in
`equilibrium with the complementary sequence complA11. .............................................................. 86 
`
`Figure 3.22: Titration of target sequences with different mispairings with the molecular beacon
`MB:A11. ............................................................................................................................................. 87 
`
`Figure 3.23: Representation of the distribution of the melting temperatures of the sequence pool in
`relation to the number of mispairings. ............................................................................................... 88 
`
`Figure 3.24: Melting curve of the molecular beacon MB:A11 in the presence of different target
`sequences with mispairings. .............................................................................................................. 89 
`
`Figure 3.25: Comparison of the measured melting curve of the molecular beacon MB:A11 with
`the sequence pool Y=T/C=91/1 as a target sequence. ....................................................................... 90 
`
`Figure 3.26: Determination of the melting temperature from the partition function. ..................... 91 
`
`Figure 3.27: Comparison of the computed and experimentally determined melting points of the
`molecular beacon MB:A11 by hybridization with different target sequences. ................................ 93 
`
`Figure 3.28: Calibration series with pSDAw2(6.0) in order to quantify the hybridization capacity
`of micromer-M particles coupled with I1R(SDA)TIVTV-NH2. ...................................................... 95 
`
`Figure 3.29: Alignment of the sequenced products from the 1-probe selection after five (V1 - V9)
`and six (VI1 - VI10) rounds with tSDAII as the start pool ............................................................... 96 
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`LIST OF FIGURES 9
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`Figure 3.30: Representation of the probe binding regions P1 and P2 on tSDAI. ........................... 98 
`
`Figure 3.31: Calibration series with V5-0rR6G and V4-1R6G for quantification of the
`hybridization capacity of M-PVA microparticles coupled to Im5-0r and Im4-1r. ........................... 99 
`
`Figure 3.32: Alignment of the sequenced selection products after 16 rounds for tSDAI. ............ 101 
`
`Figure 3.33: Alignment of the sequenced products of the serial transfer after five and eight rounds.
` .......................................................................................................................................................... 102 
`
`Figure 3.34: Alignment of the sequenced selection/amplification products after 16 rounds with
`amplification of the selection products using PCR instead of SDA. .............................................. 102 
`
`Figure 3.35: Alignment of the sequenced selection products after 16 rounds for tSDAII. ........... 104 
`
`Figure 3.36: Model to characterize the proportion of sequences with no deletion in relation to
`selective advantage. ......................................................................................................................... 105 
`
`Figure 3.37: Representation of the molecular beacons for characterization of the selection
`products. ........................................................................................................................................... 106 
`
`Figure 3.38: Use of the molecular beacons to observe changes in binding affinity in SELEX
`experiments. ..................................................................................................................................... 106 
`
`Figure 3.39: Characterization of the SDA reaction in the SDA module. ...................................... 108 
`
`Figure 3.40: Product control of the SDA reaction in the fan reactor. ............................................ 108 
`
`Figure 3.41: SDA in the fan reactor in relation to flow velocity. .................................................. 109 
`
`Figure 3.42: Detection of the SDA on tSDAI in real-time and fitting of the measurements to
`logistic and exponential growth models. ......................................................................................... 110 
`
`Figure A.1: Sequencing of the selection/amplification products after 4 rounds starting with the
`template tSDAI. ..................................................................................................................................... I 
`
`Figure A.2: Sequencing of the selection/amplification products after 4 rounds starting with the
`sequence pool tSDAII. ........................................................................................................................ II 
`
`Figure A.3: Sequencing of the selection/amplification products after 6 rounds starting with the
`template tSDAI. ................................................................................................................................... II 
`
`Figure A.4: Sequencing of the selection/amplification products after 6 rounds starting with the
`sequence pool tSDAII. ....................................................................................................................... III 
`
`Figure A.5: Sequencing of the selection/amplification products after 8 rounds starting with the
`template tSDAI. .................................................................................................................................. IV 
`
`Figure A.6: Sequencing of the selection/amplification products after 8 rounds starting with the
`sequence pool tSDAII. ........................................................................................................................ V 
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`12
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`10
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`LIST OF FIGURES
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`Figure A.7: Sequencing of the selection/amplification products after 10 rounds starting with the
`template tSDAI. .................................................................................................................................. VI 
`
`Figure A.8: Sequencing of the selection/amplification products after 10 rounds starting with the
`sequence pool tSDAII. ....................................................................................................................... VI 
`
`Figure A.9: Sequencing of the selection/amplification products after 12 rounds starting with the
`template tSDAI. ................................................................................................................................ VII 
`
`Figure A.10: Sequencing of the selection/amplification products after 12 rounds starting with the
`sequence pool tSDAII. .................................................................................................................... VIII 
`
`Figure A.11: Sequencing of the selection/amplification products after 14 rounds starting with the
`template tSDAI. .................................................................................................................................. IX 
`
`Figure A.12: Sequencing of the selection/amplification products after 14 rounds starting with the
`sequence pool tSDAII. ....................................................................................................................... IX 
`
`Figure A.13: Sequencing of the selection/amplification products after 16 rounds starting with the
`template tSDAI. ................................................................................................................................... X 
`
`Figure A.14: Sequencing of the selection/amplification products after 16 rounds starting with the
`sequence pool tSDAII. ....................................................................................................................... XI 
`
`Figure A.15: Sequencing of the selection/amplification products after 16 rounds starting with the
`template tSDAI or the sequence pool tSDAII, however, with amplification of the selection
`products after selection round 16 by PCR instead of SDA. ............................................................ XII 
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`
`
`List of tables
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`Table 2.6: Real-time detection settings ............................................................................................ 37 
`
`Table 2.7: Enzymes used. ................................................................................................................. 39 
`
`Table 2.8: Pipetting scheme for SDA under flow conditions in the fan reactor. ............................. 56 
`
`Table 3.1: Comparison of the binding of amplification product to tSDAI after asymmetric and
`symmetric SDA and after heat-denaturation ..................................................................................... 64 
`
`Table 3.2: Oligo designs. .................................................................................................................. 66 
`
`Table 3.3: Sequenced DNA ladder products. ................................................................................... 69 
`
`Table 3.4: Oligonucleotide sequence of the molecular beacon MB:A11 and the used target
`sequences with different mispairings. ............................................................................................... 81 
`
`Table 3.5: Presentation of sequence pools using binomial distribution .......................................... 82 
`
`Table 3.6: Comparison of computed and measured melting temperatures of the molecular beacon
`using different target sequences ......................................................................................................... 92 
`
`Table 3.7: Determination of the hybridization capacity of the micromer-M particles coupled to the
`oligonucleotide I1R(SDA)TIVTV-NH2. ........................................................................................... 94 
`
`Table 3.8: Selection steps for the 1-probe selection ......................................................................... 95 
`
`Table 3.9: Description of the selected sequences from the 1-probe experiments after rounds V and
`VI. ....................................................................................................................................................... 97 
`
`Table 3.10: Determination of the hybridization capacity of M-PVA particles coupled to the
`oligonucleotide Im4-1r or Im5-0r. ..................................................................................................... 99 
`
`Table 3.11: Selection steps for the 2-probe selection ..................................................................... 100 
`
`Table B.1: Computation of the energy of binding to the probes P1 and P2 after the
`selection/amplification experiments with the start sequence tSDAI ............................................. XIII 
`
`Table B.2: Computation of the energy of binding to the probes P1 and P2 after the
`selection/amplification experiments with the start pool tSDAII .................................................... XVI 
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`14
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`LIST OF TABLES
`LIST OF TABLES
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`12
`12
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`15
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`Chapter 1
`1 Introduction
`
`[1]
`
`[2]
`
`[3]
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`[4]
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`[5]
`
`The importance of evolution was first described by Charles Darwin in 1848 in his book “On the
`origin of species by natural selection” [27]. The success and efficiency of the Darwinian theory of
`evolution is based on the distinction between genotype and phenotype, where the former is subject
`to variation and the latter to selection.
`In population genetics, the dynamics of evolution are represented as a process in the genotype
`sequence space. To describe the phenotype, empirical parameters are used. Optimization is a
`process within the genotype sequence space. However, without taking the phenotype into
`consideration, the description of evolutionary processes remains incomplete.
`Evolutionary optimization in asexually reproducing populations follows the Darwinian theory of
`evolution and is determined by the interaction of two processes with opposite effects on genetic
`heterogeneity: on the one hand, mutations increase the diversity of the genotypes; on the other
`hand, the diversity of phenotypes is reduced through selection. At first glance, the uncoupling of
`the goal of a mutation from selection appears to be disadvantageous. An advantageous mutation
`does not occur more frequently because it has a better chance of being selected. Nevertheless, in
`nature, the genotype-phenotype dichotomy ensures the random walk on the fitness landscape and
`results in optimization.
`Genotype and phenotype are different “entities” and, with few exceptions, it is impossible to map
`changes in phenotypic characteristics to known changes in the DNA sequence of the genotype.
`Metabolism is too complex in order to be able to construe it from the DNA sequence.
`Different strategies have been followed to find sufficiently simple systems that will facilitate the
`study of evolutionary processes. Thus, LENSKI and co-workers performed experiments under
`
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`13
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`16
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`14 CHAPTER 1. INTRODUCTION
`
`[6]
`
`[7]
`
`[8]
`
`controlled growth conditions with bacterial cultures in chemostats [66, 67]. However, bacteria are
`too complex in order to be able to interpret the progress of evolution experiments at a molecular
`level.
`The simplest case of an evolutionary process in vitro, the optimization of ribozymes (RNA
`molecules that exhibit a genotype (RNA structure) as well as phenotype (RNA structure)), has
`been described successfully. JOYCE and co-workers develop a system that enables the continuous
`characterization of such an evolutionary process [80]. The system combines newly acquired
`functionality with selection and amplification.
`
`1.1 Molecular evolution
`Biochemical reactions such as nucleic acid amplification permit the investigation of macro
`evolutionary processes on a “laboratory timescale”. At the end of the 1960s, SPIEGELMANN and
`co-workers first demonstrated biochemically controlled evolution using the bacteriophage Qβ
`replicase [84]. Here, RNA molecules could be replicated in a test tube and, after sufficient
`generations, evolutionary phenomena in the Darwinian sense, such as selection and evolutionary
`adaptation to the environment, could be observed. This proved that the principles of evolution are
`not confined to cellular life. Rather, molecules that are capable of replicating and mutating suffice.
`The in vitro evolution of the RNA molecules mirrored evolution at an accelerated rate.
`Experiments into the mechanisms of evolution with regard to chemical kinetics have been
`conducted by BIEBRICHER et al. [12].
`
`1.1.1 In vitro selection and SELEX
`Molecular evolution makes it possible to isolate nucleic acids with catalytic activity or specific
`binding properties. In vitro selection methods such as SELEX (systematic evolution of ligands by
`exponential amplification) use large populations of random sequences of RNA or DNA as starting
`material to select specific functional molecules [36, 56]. Since the isolation of the reverse
`transcriptase and the invention of the polymerase chain reaction in the 1990s, it has been possible
`to amplify almost any nucleic acid in vitro and to thus combine in vitro selection and directed
`evolution in order to search the sequence space for highly-specialized RNA sequences [36, 121].
`Ligands resulting from in vitro selection are described as aptamers. In general, aptamers are
`created according to the foll