Enzymatic Synthesis of Functionalized DNA

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E ^NZYMATIC S ^YNTHESIS

OF FUNCTIONALIZED DNA

Dissertation

zur Erlangung des Akademischen Grades eines Doktors der Naturwissenschaften

(Dr. rer. nat.)

an der Universität Konstanz

Mathematisch-Naturwissenschaftliche Sektion

Fachbereich Chemie

vorgelegt von

A NNA B ACCARO

aus Waldshut-Tiengen 2010

Tag der mündl. Prüfung: 15.12.2010 1. Referent: Prof. Dr. Andreas Marx 2. Referent: Prof. Dr. Jörg Hartig

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Publications:

I. Journals

1. Enzymatic Synthesis of Organic-Polymer-Grafted DNA, A. Baccaro and A. Marx, Chemistry – Eur. J. 2010, 1, 218-226.

(Highlighted by the editors as “Very Important Paper”)

2. Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase, S.Obeid, A. Baccaro, A. Marx, W.Welte, K. Diederichs, accepted.

3. DNA Conjugation by the Staudinger Ligation: New Thymidine Analogues, A. Baccaro, S. H. Weisbrod, A. Marx, Synthesis 2007, 1949- 1954.

II. Book Chapter

1. Site-specific DNA labelling by Staudinger ligation, S. H. Weisbrod, A.

Baccaro, A. Marx, accepted. (Buchkapitel)

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Danksagung

Die vorgelegte Arbeit entstand von Januar 2007 bis Oktober 2010 an der Universität Konstanz im Fachbereich Chemie am Lehrstuhl für Organische und Zelluläre Chemie von Prof. Dr. Andreas Marx.

Ich danke Prof. Dr. Andreas Marx für die Überlassung der sehr interessanten und vielseitigen Themen. Ich möchte meinen besonderen Dank aussprechen, weil er mir die größtmögliche Handlungsfreiheit beim Bearbeiten der Themen gelassen hat und für sein Vertrauen in meine Fähigkeiten auch während meiner Krankheit.

Ich möchte mich bei allen Mitgliedern der Arbeitsgruppe Marx für das gute Arbeitsklima und die schöne Zeit im Labor danken. Zeitweise hatte ich das Gefühl in der AG eine zweite Familie gefunden zu haben. Gerne denke ich an unzählige Abende zurück an denen ich mich mit meinen Kollegen wissenschaftlich ausgetauscht und gefeiert habe. Außerdem danke ich vor allem meinem ehemaligen Laborkollegen Samuel Weisbrod und Bastian Holzberger für die schöne und lustige Zeit im Labor und die vielen wissenschaftlichen Gespräche. Ich werde immer an das NamensreaktionsABC zurückdenken!

Ich danke Anke Gerull und Claudia Haas die mich bei meinem Projekt mit den Dendronen unterstützt haben.Ebenso möchte ich mich bei Anke Friemel und Ulrich Haunz bedanken für ihre Unterstützung bei der Durchführung von NMR- Experimenten. Ich möchte meiner Projektpartnerin Anna-Lena danken für Ihre tolle Mitarbeit und die vielen wissenschaftlichen Diskussionen (ich könnte mir keine bessere Partnerin wünschen). Folgenden Personen sei an dieser Stelle für Ihre Beiträge zu dieser Arbeit gedankt: Samra, Sascha, Matze, Richa, Sabrina, Norman, Anna-Lena und Bac.

Ich danke vor allem Samra und Sascha die mir während meiner Krankheit beigestanden haben, sie haben mich sowohl wissenschaftlich als auch privat immer unterstützt selbst wenn ich kurz vor der Verzweiflung war.

Außerdem danke ich meinen Freunden (insbesondere Hülya) und Bekannten die mich in dieser Zeit unterstützt haben und der Familie Schmidt die mich wie ein Familienmitglied aufgenommen hat.

Meiner Mutter möchte ich dafür danken, dass sie mich moralisch während meiner Doktorarbeit unterstützt hat.

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CONTENTS

1. I NTRODUCTION

1.1. History of DNA

In 1944, Avery, MacLeod and McCarty established that DNA is the genetic material^[1]. Nearly one decade later (1953), Watson and Crick^[2,3] with contributions from Wilkins^[4] and Franklin^[5,6] were able to postulate the three- dimensional structure of DNA. These revolutionary findings have changed the biological understanding of DNA. Today, scientists from different fields are aware of the potential of DNA in research areas as biology, chemistry, physics and material science.

1.2. Structure and characteristics of DNA

DNA (deoxyribonucleic acid) acts as long-term genetic information storage in cells.

It contains the instructions to construct proteins and RNA molecules. DNA is a polymer consisting of four monomeric units (nucleotides) connected via phosphodiester bonds. A nucleotide is composed out of a sugar (deoxyribose) connected through a N-glycosidic bond with one of four different nucleobases (Scheme 1A). It is the sequence of these four nucleobases along a sugar phosphodiester backbone that encodes the information. The stored information is copied and translated based upon the molecular recognition of Watson-Crick base pairing and the ability of DNA to form double stranded regions.

Figure 1 The DNA double helix, consisting of two antiparallel DNA strands.

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1. INTRODUCTION

Scheme 1 A) DNA building blocks B) Watson Crick base pairing

The double stranded regions of two complementary strands form a geometrically well-defined duplex structure with major and minor groove (Figure 1). The Watson- Crick base pairing (Scheme 1B) is easily predictable, specific and facilitates thereby the self-assembly of oligodeoxynucleotides (ODNs). These advantageous characteristics as self-assembly, hybridisation specificity and the formation of a geometrically well-defined duplex structure make DNA an interesting tool for various applications. For instance, the creation of well-defined DNA nanostructures (2- and 3-dimensional) ^[7-9] and the appliance as basis for ODN markers in diagnostic applications are exploiting these properties.^[10]

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1. INTRODUCTION

1.3. Modified DNA

Functionalized DNA is often used as diagnostic tool. For this purpose, DNA strands harbouring fluorescent dyes or affinity tags as biotin, to mention only a few examples, were generated by the use of DNA polymerases or solid phase synthesis.

The synthesis on solid support offers the feasibility of short highly modified DNA strands. Therefore, the label has to be available as phosphoramidite, which can be incorporated within the synthesizer cycle. In particular, steric demanding labels (e.g.

dyes) can be introduced by automated DNA synthesis. This method is limited by the chemical incompatibility with synthesis conditions and a low stepwise yield after incorporation. To overcome the synthetic restriction of solid-supported synthesis, DNA polymerase catalysed reactions hold great promise. For instance, DNA polymerases can amplify DNA fragments with several thousand base pairs, using the polymerase chain reaction (PCR).^[11]

The substitution of natural nucleotides by functionalised nucleotides in enzyme mediated reactions can influence the properties of the resulting DNA strand. The helix conformation and the melting temperature of the modified DNA duplex can be compared with the unmodified double strand. The impact of incorporated substitutes on the DNA duplex conformation can be monitored using CD measurements, while thermal denaturation measurements allow the observation of stabilizing or destabilizing effects. Famulok and coworkers ^[12] were able to replace all four natural dNTPs by analogues. During PCR under standard conditions it was not possible to melt the modified DNA double helix. The use of nucleobase alkynylated dNTPs increased the melting temperature of the resulting DNA helix.^[13] Therefore, additives as DMSO, formamide, betaine and tetramethylammonium chloride were added, for successful product formation of DNA that consists completely of nucleotide analogues. CD measurements of this product showed an inverted CD spectra compared to a standard B-type DNA double helix. The incorporated modifications have caused the transition of the right-handed B-type DNA to a left-handed form.

Interestingly, the substitution of one natural nucleotide by an alkynylated nucleotide did not change the overall B-type DNA structure. Unfortunately, the acceptance of functionalised DNA building blocks by DNA polymerases is limited and not

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1. INTRODUCTION modification of DNA is a possible pathway. Small reactive groups can be introduced by either chemical or enzymatical DNA synthesis. These groups act as anchor for the desired label (Scheme 2B). For this purpose, bioorthogonal reactions as Diels-Alder reaction^[14-16], Huisgen [2+3] cycloaddition^[17-19] and Staudinger ligation^[20-22] have already been used. Nevertheless, the reactions mentioned above imply drawbacks as cumbersome synthesis of compounds required for the reactions, undesired presence of copper ions or incomplete conversion. Therefore, the direct incorporation of the desired modification is the preferred procedure (Scheme 2A).

Scheme 2 (A) Enzyme mediated incorporation of modifications in DNA. (B) Enzyme mediated incorporation of a reactive group and post-synthetic labelling by Staudinger ligation of DNA.^[20]

For the generation of modified DNA strands, the position and character of the chemical modification at the nucleotide, the sequence of the DNA template and the choice of the right DNA polymerase are important criteria.

1.3.1. Functionalised nucleotides

The enzyme-catalysed DNA synthesis is the method of choice to generate internal modified long DNA strands. This approach employing functionalised nucleotides has the advantage, that modifications can be introduced within the template in good yields, if the DNA polymerase is able to accept the modified nucleotide analogue.

The introduction of modifications at the nucleotide can be achieved chemically at the nucleobase, the ribose unit or the backbone level.

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1. INTRODUCTION The insertion of functional molecules in between the backbone to replace one or more nucleotides was already accomplished.^[23,24] A drawback of this method is that these modified building blocks can cause perturbations of the helix conformation and are therefore incompatible with most enzymatic reactions.^[25]

Modifications at the ribose unit can be introduced at the 2’ position to label the nucleotide analogue. However, this position is not suited for the synthesis of modified DNA by DNA polymerases due to their discrimination of dNTPs and NTPs.^[26-29] The modification of the 1’, 4’ and 5’ position is often associated with a cumbersome synthesis. Furthermore, building blocks modified at these positions show often low efficiencies ^[30-34] using DNA polymerases, since these positions are near the polymerisation reaction sites and the minor groove, respectively. Nucleotide analogues bearing the modification at the nucleobase are best suited for labelling purposes, since these analogues are often accepted by DNA polymerases. A crucial rule for the acceptance of modified nucleotides by DNA polymerases is that the modification should not disturb the Watson-Crick base pairing. Thus, the 5 position of pyrimidines has proved to be best suited for this purpose ^[12,35-38], because labels at this position fit well into the major groove of the DNA duplex. In case of purine building blocks, modifications point into the major groove if they are placed at the C7 position of 7-deazapurines.^[39-41] Furthermore, modifications at the C5 position of pyrimidines or the C7 position of 7-deazapurines can be introduced via metal mediated cross coupling reactions in good yields using the corresponding iodo or bromo compounds.^[36,42] In general, the processing of functionalised nucleotides by DNA polymerases turned out to be more efficient if the modification is accommodated in the major groove and does not interfere with duplex formation.[12,43,44]

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1. INTRODUCTION

1.3.2. Enzymatic synthesis of functionalised DNA

By the use of enzyme-mediated reactions the generation of functionalised DNA is feasible. Enzymes as terminal transferase^[45,46] and DNA polymerases^[47-49] can be used for the construction of labelled DNA. These enzymes necessitate nucleotide analogues to generate functionalised DNA. DNA polymerases can be used in several approaches to amplify highly modified DNA fragments (e.g. primer extension, polymerase chain reaction (PCR) and rolling circle amplification (RCA))[20,39,42,50-53]. For the acceptance of modified nucleotides by DNA polymerases the position of the modification is relevant. It is known that the C5 position of pyrimidines and the C7 position of 7-deazapurines are best suited for the placement of modifications. Other positions can be modified, but their incorporation is often less efficient than that of C5 functionalised pyrimidine or C7 modified 7-deazapurines. The synthesis of single or multiple functionalised DNA is possible through primer extension reactions.

There is a huge variety of nucleotide analogues which can be used as substrate for DNA polymerases in primer extension.^[53-56] The amplification of modified DNA fragments by PCR is more challenging.

Scheme 3 Nucleotide analogues used in PCR or primer extension; R: 5-substituted dUTP (1^[41]; 2^[39]; 3^[57]; 4^[58]; 5^[42]; 6^[54]; 7^[51])

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1. INTRODUCTION In PCR the nucleotide analogues were incorporated into the nascent primer strand and serve as template for the generation of modified DNA. The complete replacement of natural nucleotides through their modified analogues is not possible in every case. Therefore, the usage of mixtures of natural nucleotide and modified analogue in PCR is common.^[59,60] These DNA fragments are not completely modified but a certain percentage is substituted. Nevertheless, there are several examples of complete substitution of natural nucleotides through their functionalised analogues (Scheme 3).[19,58,61,62] It was shown that for the incorporation of functionalised DNA building blocks family B DNA polymerases (Pwo, 9°N, KOD Dash) are more convenient than family A DNA polymerases (Taq, KF). ^[63,64]

The acceptance of modified nucleotides by DNA polymerases can be enhanced by use of a linker between the nucleotide and the modification. Functionalised nucleotides, with a significant effect on the active site of the DNA polymerase (positively, negatively charged modifications or bulky groups), are only accepted if the linker is long enough to separate the modification from the base.^[41,64]

The following sections include partial excerpts from the article "Enzymatic Synthesis of Organic-Polymer-Grafted DNA" written by me.^[65]

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1. INTRODUCTION

1.4. DNA Hybrid materials

The development of hybrid materials is of high interest because it may allow the creation of new materials with extraordinary properties. To this end, materials from different areas of chemistry, biology and materials science are combined, in a way to generate new bioorganic hybrid materials with novel characteristics. DNA block copolymers consisting of oligonucleotides and organic polymers are one of the most promising hybrid materials. These hybrid materials show combined characteristics of DNA and polymers. Due to hybridization specificity and the ability of oligonucleotides to self-assemble, they were used as tools to arrange precise structures in nanometer range.^[8,66-69] Many different methods have been developed using DNA as scaffold to orientate inorganic (e.g. silica, metal complexes, gold nanoparticles)^[70-74] as well as organic compounds (e.g. sugars, dyes, lipids)^[75-77] in nanometer range. DNA block copolymers with a DNA segment and an organic polymer unit were reported recently.^[78-81] The synthesis of these DNA block copolymers was performed in solution^[82] or on solid support.^[83] For coupling of oligonucleotides and organic polymer in solution, amide^[84] bond formation and Michael addition reactions^[85] have been used (Scheme 4).

Scheme 4 Synthesis of linear DNA block copolymers in solution

These coupling strategies require modification of the employed compounds with appropriate functional groups and must ensure the solubility of both the polymer and oligonucleotide in the required solvent. The synthesis of DNA block copolymers in solution can be performed in high yields employing water soluble polymers as polyethylene glycol.^[86,87]

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1. INTRODUCTION Reactions involving hydrophobic polymers show low coupling efficiencies in solution because of the incompatibility of polymer and DNA in the solvent.^[80] Thus, amphiphilic block copolymers consisting of hydrophobic polymers and DNA were synthesized on solid support ^[78], employing a DNA synthesizer. By the usage of a DNA synthesizer, solvent mixtures with dichloromethane, dimethylformamide and acetonitrile can be used. These solvents are better suited to dissolve hydrophobic polymers for the coupling with DNA on solid support. With this approach, terminally functionalised oligonucleotides are accessible (Scheme 5A).

Scheme 5 (A) Schematic depiction of amphiphilic DNA block copolymers and aggregation in aqueous solution. (B) Schematic representation of ssDNA diblock copolymer primers in the PCR process.

While short DNA strands (50-60 nucleotides) are available in good yields through chemical synthesis on solid support, the synthesis of longer DNA strands is challenging.

To overcome the synthetic restrictions of solid support synthesis DNA polymerase- catalyzed reactions hold great promise since they allow the synthesis of long DNA fragments. For this reason the development of enzymatic transformations towards polymer modified DNA are of high interest. However, these approaches are only scarcely exploited. Along these lines, Herrmann et al. employed primer strands bearing a polymer moiety at their 5' end in PCR to synthesize DNA block copolymers (Scheme 5B) with extended DNA fragments.^[79] Numerous DNA block copolymers with polymeric block type architecture are known, while there are only a few examples of grafted DNA copolymers^[88,89] built out of an oligonucleotide

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1. INTRODUCTION DNA-polymer hybrids opens up the possibility to build a DNA backbone equipped with organic polymer side chains.

1.5. Nucleotide functionalised DNA

As mentioned before, there is a huge repertoire of modifications, accepted by DNA polymerases. The usage of labelled nucleotides (e.g. with cyanine dyes, biotin) is widespread and already a standard approach to generate modified DNA in many important biotechnological applications.^[90-93] The acceptance of nucleotides by DNA polymerases as well as the detection of the incorporated label can be improved by the usage of a linker, which increases the distance between the label and the nucleotide.

In particular, the detection of affinity tags or the signal intensity of dyes can be enhanced.^[94,95]

For instance, Kore et al. has synthesised a new nucleotide, connecting a triphosphate by a linker with 5-bromo deoxyuridine (BrdU) employing solid phase synthesis (Figure 2).^[95] This triphosphate was designed to facilitate the BrdU detection in DNA products. BrdU is used to study the cell-cycle status or the viability of cultured or harvested cells, by incorporation of BrdU into the DNA of proliferating cells.^[96]

The detection of BrdU requires the denaturation of the DNA duplex under unfavourable harsh acidic conditions. With this new BrdU labelled nucleotide the detection can be followed without denaturation step, because it is not involved in the Watson-Crick base pairing. The BrdU moiety in the DNA duplex is located in the major groove and therefore available in duplex DNA. This probe has the potential to enhance established methods in immunochemistry (e.g. labelling and detection assays).

Figure 2 Deoxyribo BrdU probe for labelling and detection of nucleic acids

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1. INTRODUCTION

1.6. DNA microarrays

DNA diagnostics was revolutionised by the introduction of DNA microarray technology in 1995. These microarrays were prepared by high-speed printing of probes on activated glass slides. Because of the small format and high density of the arrays and the usage of small volumes of probe (nanoliter range) this method is an advantageous instrument for screening approaches.^[97] This technology has vastly increased the throughput for genotyping and DNA sequencing. For the application of this methodology as diagnostic tool sensitivity, accuracy, specificity and reproducibility of microarray results are essential.

By the use of DNA microarrays high-throughput single-nucleotide polymorphism (SNP) discovery and genotyping has been enabled. SNPs are single base alternations occurring at a specific position in human DNA. The difference between point mutations and SNPs is their frequency in a population. They occur approximately every 1000-2000 bases in human DNA.^[98-100]If SNPs are located in coding and regulating gene regions they can cause modified structures, activities and functions of expressed proteins. Hence, specified SNPs can be utilised to identify risks of common adult diseases as breast and prostate cancer, vascular diseases or diabetes.^[101,102] DNA microarrays are attractive as a platform for high-thoughput single-nucleotide polymorphism (SNP) analysis.^[103-105]

Scheme 6 Schematic illustration of allele-specific primer extension reactions.

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1. INTRODUCTION For SNP detection Kranaster et al. has used oligonucleotide arrays with short (23- 25mer) oligodeoxynucleotide primer probes tethered on a microarray surface.^[106]

The immobilised allele-specific primer probes captured the target sequence by hybridisation of the nucleic acid strand and only the fully matched primer was elongated in contrast to the mismatched counterpart, by the use of a mutant derived from Pyrococcus furiosus (Pfu) DNA polymerase (Scheme 6). For signal generation, the incorporation of a fluorophor-labelled dUTP anlogue (F3-dUTP) was used.

Array-based analysis of genomic DNA has already entered clinical practice.^[107] But a drawback of DNA microarrays is the sensitivity. The sensitivity threshold defines the concentration by which accurate measurements can be made. Up to now, to examine human genomic DNA, PCR has to be used to amplify the required regions which are used for analysis.

Signal amplification would allow to decrease the required concentration of the target sequence, in order to perform array-based analysis of human genomic DNA without further amplification directly after extraction from blood. This would simplify the usage of microarrays in clinical diagnostics.

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1. INTRODUCTION

1.7. Concept and objective

1.7.1. DNA hybrid materials

In order to create bioorganic hybrid materials, scientists are working interdisciplinary in the fields of chemistry, biology and material science. DNA block copolymers are among the most promising hybrid materials due to combination of properties, intrinsic for the polymer and the nucleic acid blocks. Up to now, the coupling of DNA and organic polymers is exercised post-synthetically in solution or on solid support. Employing water-soluble polymers the coupling with DNA can be carried out in solution. The synthesis of amphiphilic DNA copolymers is realised by the use of solid phase synthesis. With these established methods the polymer was attached terminal at the DNA strand. For the realisation of internal functionalised DNA strands enzyme catalyzed synthesis of DNA-organic polymer chimeras is promising.

To establish the enzymatic synthesis of DNA hybrid materials build on a DNA backbone equipped with organic polymer side chains, functionalised nucleotides were envisioned. For this purpose novel 2’-deoxyuridine triphosphates carrying linear polyethylene glycol or branched dendron moieties linked to the nucleobase should be synthesized. In enzyme catalysed reactions the acceptance of these building blocks and their eligibility to generate DNA hybrid materials with high molecular weight, modification density and structural accuracy should be investigated.

1.7.2. ODN labelled DNA

The labelling of DNA is used in several diagnostic approaches. Therefore, labelled nucleotides (e.g. F3-dUTP, 11-biotin-dUTP) are used to generate modified DNA.^[106,108] For this purpose, labels with characteristics as fluorescence and affinity are utilised in established diagnostic methods. Up to now, there is no modified nucleotide available for enzyme-catalysed DNA synthesis with properties as self- assembly or hybridisation specificity. These properties are still unexploited for enzyme mediated DNA labelling.

Inspired by the work of Kore et al.^[95] it was investigated, whether the synthesis of triphosphates harbouring oligodeoxynucleotides (ODNs) at the nucleobase is

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1. INTRODUCTION possible. With the ability to self-assemble and the hybridisation specificity DNA is a versatile label. The ODN label can be varied by alternations of the sequence similar to a barcode system. The acceptance of ODN modified building blocks by DNA polymerases would make a template-dependent introduction of the ODN label feasible.

Thus, the synthesis of 2’-deoxy triphosphates harbouring an ODN at the nucleobase was envisioned. These ODN functionalised building blocks should be designed in a way that DNA polymerases would accept them in enzyme-catalysed reactions. If this could be realised the incorporated ODN labels could function as probes for their complementary nucleic acid sequences, due to the hybridisation specificity and their tendency to self assemble. By this method, additional DNA strands with a specified sequence would be introduced in a DNA duplex. The possibility to use this incorporated ODN modified building block for post-synthetic transformations could be investigated. Therefore, the ODN modified nucleotides should be designed with a ODN bearing a free 3’-OH to enable enzyme catalysed transformations after incorporation of these modified nucleotides in DNA. If the synthesis of these DNA modified building blocks would succeed their qualification as diagnostic tool should be investigated.

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2. RESULTS AND DISCUSSION

2. R ESULTS AND D ^ISCUSSION

2.1. Enzymatic synthesis of DNA hybrid materials

2.1.1. Synthesis strategy

For the enzymatic generation of DNA hybrid materials four novel modified nucleotide analogues were envisioned. The synthesis was set up starting with commercially available 5-iodo deoxyuridine. Since, it has been reported that the acceptance of nucleotide analogues by DNA polymerases heavily depends on the position and the structure of the modifications[38,41,109], the C5-position of the pyrimidine was chosen to introduce the modifications. In addition, it was shown that modified 2’-deoxyuridine triphosphates bearing a C5-substituent are accepted as substrates by DNA polymerases in several cases. Reasons here fore are that modifications at this position do not interfere with Watson–Crick base pairing or between interactions of the DNA backbone and the DNA polymerase and they do not significantly perturb the DNA duplex conformation.^[20,110] The molecules that were chosen as modifications had to meet special conditions, as biocompatibility and water-solubility. The type of modification has a significant influence on the acceptance of the nucleotide^[21], thus triphosphates should be equipped with linear as well as branched moieties. Linear polyethylene glycol monomethyl ethers with a range of lengths were chosen due to their ubiquitous use.^[111] It is also reported that polyethylene glycol can serve as an enhancer in PCR.^[112] Furthermore, branched polyamido dendrons were designed to generate thymidine analogues with varied steric demand.

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2.1.2. Synthesis of polyethylene glycol monomethylether functionalised nucleotides

Due to the reasons discussed before, nucleotides harbouring a polymer moiety at the C5-position of the nucleobase were synthesized. The functionalisation of the nucleotides was performed with polydisperse polyethylene glycol monomethylethers with an average molecular weight (MW) of 350 g/mol or 750 g/mol, respectively.(Figure 3)

Figure 3 Target structures of polyethylene glycol monomethylether functionalised nucleotides

Therefore, the polyethylene glycol monomethylethers were equipped with an alkyne moiety. Thus, the alcohols 1a,b were converted into the respective tosylates 2a,b and subsequently treated with sodium hydride and propargyl alcohol to yield compounds 3a,b (Figure 8). The alkynes 3a,b were then coupled to nucleoside 4 by using a standard protocol for Sonogashira reaction^[20] to yield the nucleosides 5a,b.

Scheme 7 Reagents and conditions: a) tosyl chloride, Et3N, CH2Cl2, RT, 2a: 74 % 2b: 83 %; b) propargyl alcohol, NaH, THF, 0°C, 3a: 76 %, 3b: 67 %; c) Pd(PPh₃)₄, CuI, Et₃N, alkyne 3, DMF, RT, 5a: 31 %, 5b: - ; d) 2-Chloro-4H-1, 2, 3,-dioxaphosphorin-4-on, pyridine, dioxane, RT, (Bu3NH)2H2P2O7, nBu3N, I2, 5 % NaHSO3 aq., 33 % NH3, 6a: 31 %.

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2. RESULTS AND DISCUSSION Nucleoside 5a was then converted into the corresponding triphosphate 6a using an appropriate method for phosphorylation.^[113-116]

By mass analysis of the triphosphate 6a the polyethylene glycol monomethylether chain lengths attached at the nucleotide could be identified (n = 6-10). Compound 6a was used for first primer extension experiments to test the ability of Pwo DNA polymerase to accept this modified triphosphate as a substrate.

Building block 6a was accepted in primer extension which demonstrated the proof of principle (Figure 5). Despite these findings the usage of polydisperse polyethylene glycol monomethylethers was discarded. Main concerns, which leaded to this decision, were the possibility of inconsistent results using a triphosphate mixture and an ambiguous interpretation of obtained results. Furthermore, the purification of nucleoside 5b failed. In this case, additional to the dispersion of the polyethylene glycol monomethylether chain lengths attached at the nucleoside some impurities with an unrequested mass distribution were detected. Due to the product mixture standard purification protocols were not sufficient to purify compound 5b.

In order to simplify purification and analysis, monodisperse polyethylene glycol monomethylethers were chosen as modifications for the nucleotides with a defined chain length.

Scheme 8 Reagents and conditions: a) tosyl chloride, Et₃N, CH₂Cl₂, RT, 8a, 8b: quant.; b) propargyl alcohol, NaH, THF, 0°C, 9a: 98 %, 9b: 95 %; c) Pd(PPh3)4, CuI, Et3N, alkyne 9, DMF, RT, 10a, b: 65

%; d) protone sponge, POCl₃, PO(OMe)₃, 0°C, (Bu₃NH)₂H₂P₂O₇, nBu₃N, TEAB buffer, 33 % NH₃, 11a: 40 %, 11b: 7 %.

For the synthesis of compound 11a,b the synthetic route described above was accomplished.

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2. RESULTS AND DISCUSSION The alcohols 7a,b were converted into the respective tosylates 8a,b and subsequently treated with sodium hydride and propargyl alcohol to yield compounds 9a,b (Scheme 8). Then, Sonogashira cross coupling^[117-119] and subsequent phosphorylation^[113-116]

was performed to form the triphosphates of the corresponding nucleosides.[113,115,116]

Figure 4 Structure of nucleotides functionalised with monodisperse polyethylene glycol monomethylether

Compounds 11a,b (Figure 4) were obtained in good yields and were used in enzyme mediated reactions.

2.1.3. Synthesis of dendron functionalised nucleotides

In collaboration with the group of Prof. S. Mecking, (University of Konstanz) a nucleotide harbouring a polyamidoamine dendron at the C5-position was envisioned.

These modified nucleotides should be equipped with different generations of polyamido dendron to generate DNA polymer hybrids through enzyme catalysed synthesis. The polyamido dendron with terminal diethylamine end groups was chosen as good trade-off between water-solubility and ease of synthesis. The literature procedure used in the group of Prof. S. Mecking for generation of these polyamido dendrons was described for hydrophobic dendrons with long alkyl residues (bis(2-ethylhexyl)) as end groups.^[120] The purification of these hydrophobic dendrons was done by extraction of the product from the aqueous phase, which was sufficient to remove the excess of water-soluble reactants and by-products. For the polyamido dendron with ethyl end groups this kind of work up resulted in low yields and impure product, containing still reactants and imidazol, which is a by-product formed during the activation with CDI (Scheme 9). Thus, different purification methods were considered. Flash chromatography over silica gel seemed not to be convenient, since TLC analysis showed that the polyamido dendron is not well

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2. RESULTS AND DISCUSSION defined as a single spot and the product and by-products had similar Rf values.

Separation of polyamido dendron and by-products was only possible using reverse phase middle pressure liquid chromatography (RP-MPLC) on C-18 columns with water and acetonitrile as liquid phase. This product (Generation1 (G1) dendron 13a) was used as starting material for higher generations of dendrons or was directly treated with succinic anhydride, CDI and propargyl amine to yield the alkyne 14a (Scheme 9).

Scheme 9 Reagents and conditions: a) succinic anhydride, toluene, 70°C, CDI, then bis(3- aminopropyl)amine, 13a: 25 %; b) succinic anhydride, THF, 70°C, CDI, then propargyl amine, 14a: 63 %.

The generation of G2 dendron was accomplished by treating compound 13a with succinic anhydride, CDI and bis (3-aminopropyl) amine (Scheme 10). Initially the yield obtained for this reaction in toluene was not sufficient for the synthesis of the corresponding alkyne. Therefore, the solvent was changed from toluene to THF and compound 15a was obtained in satisfactory yield and then purified by RP-MPLC on C-18 columns with water and acetonitrile as liquid phase.

Scheme 10 Reagents and conditions: a) succinic anhydride, THF, 70°C, CDI, then bis(3- aminopropyl)amine, 15a: 8 %; b) succinic anhydride, THF, 70°C, CDI, then propargyl amine,

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2. RESULTS AND DISCUSSION For purification the same C-18 column was used for G1 and for G2 dendrons but ¹H and HSQC NMR studies revealed that small amounts of the injected dendron were bound on the column and were eluted during the next purification runs. Thus, each compound was purified over a separate C-18 column to avoid dendron mixtures.

Scheme 11 a) Pd(PPh3)4, CuI, Et3N, alkyne 14a or 16a, DMF, RT, 17a: 89 %, 19: 11 %; b) 2-Chloro- 4H-1, 2, 3,-dioxaphosphorin-4-on, pyridine, dioxane, RT, (Bu₃NH)₂H₂P₂O₇, nBu₃N, I₂, 5% NaHSO₃ aq., 18a: 27 %, 20: -; c) protone sponge, POCl₃, PO(OMe)₃, 0°C, (Bu₃NH)₂H₂P₂O₇, nBu₃N, TEAB buffer, 33 % NH3, 18a: 6 %, 20: -.

The G1 (14a) and G2 (16a) dendron alkynes were then coupled to nucleoside 4 by using a standard protocol for Sonogashira reaction.^[20] (Scheme 11)

The purification of both compounds was done with flash chromatography followed by RP-MPLC. These nucleosides were then used for phosphorylation. ^[113-116]

At first the phosphorylation of the G1 dendron functionalised nucleoside was accomplished with 2-chloro-4H-1, 2, 3-dioxaphosphorin-4-on^[121] but the ¹H-NMR showed impurities in the aromatic range, which could not be removed neither by RP- MPLC nor by RP-HPLC. Therefore, the phosphorylation method employing

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2. RESULTS AND DISCUSSION phosphoroxychloride was chosen and G1 dendron functionalised nucleotide was obtained.

For the G2 dendron nucleoside both reaction pathways were used to generate a triphosphate but in several approaches the product was not obtained in sufficient amount. After purification mass spectra were recorded which showed a pattern typical for G2 dendron modified molecules (measured MS (ESI): m/z: 1781.4 [M]^- calc. mass for nucleotide 20: m/z: 1779.81[M]^-) but the amounts were not sufficient to get ¹H-NMR or ³¹P-NMR.

Because of the troublesome synthesis of the G2 dendron functionalised nucleoside and low yield for the phosphorylation this part of the project was not persecuted anymore.

Hence another type of water soluble G1 dendron was envisioned with varied end groups. Saglam et al.^[122] reported that thiazolidine equipped poly(hydroxyethylmethacrylate) (PHEMA) microbeads were able to adsorb heavy- metal ions from aqueous solutions. Therefore, thiazolidine as end group was chosen to test whether the introduction of such a thiazolidine functionalised building block would enable modified DNA to bind Cd(II) or Pb(II) ions and to use end groups with varied steric demand.

Scheme 12 Reagents and conditions: a) succinic anhydride, toluene, 70°C, CDI, then bis(3- aminopropyl)amine, 13b: 16 %; b) succinic anhydride, THF, 70°C, CDI, then propargyl amine, 14b: 31 %.

The synthesis of compound 13b started with the thiazolidine heterocycle and was prepared according to the procedure mentioned above. Compound 13b was then treated with succinic anhydride, CDI and propargyl amine to yield the corresponding alkyne (14b). Compound 14b was then coupled to nucleoside 4 by using a standard protocol for Sonogashira reaction.^[20] The nucleoside (17b) was phosphorylated using 2-chloro-4H-1, 2, 3-dioxaphosphorin-4-on^[121] in good yield.

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Scheme 13 Reagents and conditions: a) Pd(PPh₃)₄, CuI, Et₃N, alkyne 14b, DMF, RT, 17b: 63 %; b) 2- Chloro-4H-1, 2, 3,-dioxaphosphorin-4-on, pyridine, dioxane, RT, (Bu₃NH)₂H₂P₂O₇, nBu₃N, I₂, 5 % NaHSO3 aq., 33 % NH3, 18b: 18 %.

The nucleotides 11a,b and 18a,b obtained, were used in enzyme mediated reactions (e.g. primer extension, PCR, rolling circle amplification) to test their ability to substitute natural thymidine.

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2.1.4. Enzymatic incorporation of functionalised building blocks

• Primer extension

To test the ability of DNA polymerases to accept the modified triphosphate and to incorporate template specific the respective nucleotide into a nascent DNA strand, a primer extension reaction was set up. A 5'end ³²P-labelled 23-nucleotide primer and diverse templates were used. The reactions were analysed by denaturing polyacrylamide gel electrophoresis (PAGE) followed by autoradiography.

Figure 5 Incorporation experiments using Pwo DNA polymerase: (A) Partial sequence of the primer template complex; (B) Structure of compound 6a used in primer extension experiments; (C) Lane 0:

5’-³²P-labeled 23-nucleotide primer strand; Lane 1: primer template complex including dATP, dCTP, dGTP; Lane 2: same as in lane 1 including TTP; Lane 3 same as in lane 1 including 6a.

First the primer and 35-nucleotide template complex with a single A residue, coding for insertion of a TTP after extending the 23-nucleotide primer strand by three residues, was used. Reactions lacking TTP predominantly abort before the template A, while reactions including dATP, dCTP, dGTP and TTP gave full-length products (Figure 5). Pyrococcus woesei (Pwo) DNA polymerase was able to extend the nascent primer strand to full-length product, if compound 6a was used instead of natural TTP (Figure 5). For compounds 11a,b and 18a,b Pyrococcus woesei (Pwo) and 9°Nm DNA polymerases were tested since these enzymes have shown promising results with other modified nucleotides [39,47,57,123]. Both enzymes were able to extend

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2. RESULTS AND DISCUSSION the nascent DNA strand to full length, when natural TTP was replaced by one of the modified triphosphates (Figure 6).

Figure 6 Incorporation experiments using Pwo DNA polymerase: (A) Partial sequence of the primer template complex; (B) Structure of compounds used in primer extension experiments; (C) Lane 0: 5’-

32P-labeled 23-nucleotide primer strand; Lane 1: primer template complex including dATP, dCTP, dGTP; Lane 2: same as in lane 1 including TTP; Lane 3 same as in lane 1 including 11a; Lane 4:

same as in lane 1 including 11b; Lane 5: same as in lane 1 including 18a; Lane 6: same as in lane 1 including 18b.

Next, two different templates (69 nucleotides) were designed in a way that they code for a modified nucleotide every fourth and every second position (Figure 7A, B).

Figure 7 Multiple incorporation experiments using 9°Nm DNA polymerase: Lane 0: 5’-³²P-labeled 23- nucleotide primer strand; Lane 1: primer template complex including dATP, dCTP, dGTP; Lane 2:

same as in lane 1 including TTP; Lane 3 same as in lane 1 including 11a; Lane 4: same as in lane 1 including 11b; Lane 5: same as in lane 1 including 18a; Lane 6: same as in lane 1 including 18b.

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2. RESULTS AND DISCUSSION These templates with a more challenging sequence should shed light on the ability of the enzymes to promote multiple incorporations of functionalized nucleotides. For these templates it was found that 9°Nm DNA polymerase is more proficient in extending both templates (Figure 7A, B), while Pwo DNA polymerase was not able to incorporate modified nucleotide 11b or 18a on every second position (Figure 8B).

Therefore, 9°Nm DNA polymerase was used for further experiments. In case of substitution of natural TTP by 11a, 11b, 18a or 18b the full-length products showed lower mobility on the gel in comparison to the unmodified full-length product (Figure 7). It was supposed that this property is based on the increased steric demand and molecular weight of the modified entities. Similar effects have been reported by Famulok et al. ^[12] In order to form one entire modified DNA helix turn in the nascent DNA strand, a template with eleven adjacent A residues was used next.

Figure 8 Multiple incorporation experiments using Pwo DNA polymerase: Lane 0: 5’-³²P-labeled 23- nucleotide primer strand; Lane 1: primer template complex including dATP, dCTP, dGTP; Lane 2:

same as in lane 1 including TTP; Lane 3 same as in lane 1 including 11a; Lane 4: same as in lane 1 including 11b; Lane 5: same as in lane 1 including 18a.

For all four thymidine analogues full-length product formation was observed (Figure 9A).

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Figure 9 Multiple incorporation experiments using 9°N_m DNA polymerase: Lane 0: 5’-³²P-labeled 23- nucleotide primer strand; Lane 1: primer template complex including dATP, dCTP, dGTP; Lane 2:

same as in lane 1 including TTP; Lane 3 same as in lane 1 including 11a; Lane 4: same as in lane 1 including 11b; Lane 5: same as in lane 1 including 18a; Lane 6: same as in lane 1 including 18b.

Interestingly, comparing the mobility shifts of the full-length products, the fragment of 11b is migrating slower than the fragment of polyamido dendron 18b (Figure 9A), though building block 18b has a slightly higher molecular weight than 11b assuming that the incorporation of a linear polymer causes a lower mobility in the polyacrylamide gel than a branched one with higher molecular weight. This shows that the DNA polymerase is able to incorporate several modified nucleotides in a row and to elongate the nascent strand to full-length in spite of the modifications.

Another template with 46 A residues in a row was designed to investigate the limits of enzymatic incorporation of the modified nucleotides. In this experiment full- length product was formed with 11a (Figure 9B, lanes 3), whereas for all other modifications shorter fragments were obtained (Figure 9B, lanes 4-6). The length of the fragments obtained in these experiments for compounds 11b and 18b was not determined but the DNA polymerase was able to incorporate clearly more than 11 adjacent modified building blocks. This shows that these modified nucleotides can act as surrogate for natural thymidine in primer extension using 9°Nm DNA polymerase.

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• Polymerase chain reaction

Next, the suitability of building blocks 11a, b and 18a, b for substituting TTP in PCR was investigated. In this case 9°Nm DNA polymerase was tested with a template of 304 nucleotides in size (Figure 10A, B).

Figure 10 2.5 % agarose gel stained with ethidium bromide showing PCR products of 304 base pairs (bp) template. (A) Lane 0: Marker; Lane 1: PCR with all natural dNTPs; Lane 2: PCR with dATP, dCTP, dGTP; Lane 3: PCR with 11a instead of TTP; Lane 4: PCR with 11b instead of TTP. (B) Lane 0: Marker; Lane 1: PCR with all natural dNTPs; Lane 2: PCR with dATP, dCTP, dGTP; Lane 3: PCR with 18a instead of TTP; Lane 4: PCR with 18b instead of TTP.

As with the primer extension tests one control reaction with dATP, dCTP, dGTP and TTP was conducted leading to PCR product with the desired length (Figure 10A, B;

lanes 1). In another control the same reaction was performed in the absence of TTP leading to no observable PCR product formation (Figure 10A, B; lanes 2). The reactions were analyzed by agarose gel electrophoresis and stained with ethidium bromide. Using standard PCR conditions the DNA polymerase was able to substitute compounds 11a (Figure 10A; lane 3), 18a and 18b for TTP and amplify the modified DNA in similar fashion as with all four natural dNTPs (Figure 10B; lanes 3, 4).

Again a mobility shift of the product bands to lower mobility was observed for the modified DNA. Interestingly, the usage of compound 18a resulted in less amplification product than the usage of compound 18b. A possible reason is the different steric demand of the different end groups. Using 11b instead of TTP in PCR resulted in no amplification product. Even by changing the reaction conditions for compound 11b no product band was observed. Presumably, the larger modification of 11b compared to 11a interfered with the DNA polymerase. One reason for the reduced amplification efficiency when using 11b might be the polyethylene glycol moiety itself. Control reactions were performed and polyethylene glycol monomethyl ether with the respective molecular weight was added to a reaction with all natural dNTPs. With this experiment it was shown that

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2. RESULTS AND DISCUSSION with an average molecular weight (MW) of 550 g·mol^-1than for 350 g·mol^-1(Figure 11).

Figure 11 PCR reactions with increasing polyethylene glycol monomethyl ether concentration.

(A) PCR in presence of polyethylene glycol monomethyl ether with an average MW of 350 g·mol^-1. (B) PCR in presence of polyethylene glycol monomethyl ether with an average MW of 550 g·mol^-1

It was noted that in presence of polyethylene glycol monomethyl ether at a concentration of 500 mM for 350 g·mol^-1 and 175 mM for 550 g·mol^-1 no more PCR product was formed. Since it has been shown that 9°Nm DNA polymerase is able to accept thymidine derivatives and uses them as a template (11a, 18a, 18b), we switched to a genomic DNA fragment (1062 bp) as template for the PCR (Figure 12A, B).

Figure 12 0.8% agarose gel stained with ethidium bromide showing PCR products of 1062 base pairs (bp) template. (A) Lane 0: Marker; Lane 1: PCR with all natural dNTPs; Lane 2: PCR with dATP, dCTP, dGTP; Lane 3: PCR with 11a instead of TTP; Lane 4: PCR with 1b instead of TTP. (B) Lane 0: Marker; Lane 1: PCR with all natural dNTPs; Lane 2: PCR with dATP, dCTP, dGTP; Lane 3: PCR with 18a instead of TTP; Lane 4: PCR with 18b instead of TTP.

Here, compound usage of 11a resulted in higher yield of PCR product compared to the PCR including 18a and 18b, which produced less but observable product consistent with previous observations.

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• Rolling circle amplification^[124-129]

A method to generate long single DNA strands is rolling circle amplification.

Therefore the acceptance of the thymidine analogues 11a, b and 18a, b in rolling circle amplification was tested. For this reaction, a template with 42 nucleotides containing an A residue on every fourteenth position and a restriction site for restriction endonuclease from Haemophilus haemolyticus (HhaI) was designed. This linear precursor was circularized enzymatically using T4 DNA ligase and a “splint”

oligonucleotide with 16 nucleotides that aligns both ends of the precursor by hybridisation. ^[129] This DNA circle serves as a virtually never-ending template for a primer extension reaction.

Radioactive labelled RCA product was obtained by incorporation of radioactive labelled nucleotides. Therefore, alpha-³²P-dATP was added to the reaction mixture.

9°Nm DNA polymerase was tested to determine the ability to extend a short primer to a long ssDNA strand. The reactions were analysed by denaturing polyacrylamide gel electrophoresis (PAGE).

Figure 13 (A) Rolling circle amplification; Lane 0: Marker; Lane 1: primer template complex including alpha-dATP, dATP, dCTP, dGTP; Lane 2: same as in lane 1 including TTP; Lane 3: same as in lane 1 including 11a; Lane 4: same as in lane 1 including 11b; Lane 5: same as in lane 1 including 18a; Lane 6: same as in lane 1 including 18b. (B) Restriction enzyme digestion; Lane 0:

Marker; Lane 1: digestion of RCA product lacking dTTP; Lane 2: digest RCA product with all nat.

dNTPs; Lane 3: digest RCA product with 11a; Lane 4: digest RCA product with 11b; Lane 5: digest RCA product with 18a; Lane 6: digest RCA product with 18b.

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2. RESULTS AND DISCUSSION First, the control reactions with dATP, dCTP, dGTP, TTP and another one lacking TTP were performed. Then all four nucleotide analogues were tested, respectively, as surrogate for natural TTP. With the thymidine analogues 11a, b and 18a, b the nascent DNA strand was elongated indicating that all modified nucleotides were accepted (Figure 13A).

The radioactive labelled RCA products were purified with G 25-columns to remove excess alpha-³²P-dATP and salts. Afterwards the RCA products were hybridised with restriction helpers (13-nucleotide DNA strand to enhance the digestion) and digestion with restriction endonuclease HhaI was carried out. These reactions were analysed by PAGE. The resulting gel is depicted in Figure 13B. The whole RCA products were digested by the enzyme to 42-nucleotide fragments. The shifts observed in reaction products employing the modified RCA products (Figure 13B;

lane 3-6), is caused by higher molecular weight and steric demand, as mentioned before. By means of this experiment it was shown, that the restriction enzyme was not significantly inhibited by the DNA modifications.

In first experiments a 5'end ³²P-labelled 16-nucleotide primer was used for RCA. The reaction product was obtained and then digested with restriction endonuclease HhaI.

The resulting gel showed not the expected band at 42-nucleotide but a shorter one.

Starting from the labelled elongated primer till the first restriction site a shorter fragment was obtained by digestion. Thus, the reaction was labelled by the use of alpha-³²P-dATP to get the expected 42-nucleotide fragments.

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2.1.5. Attempts to immobilise modified DNA on IDA agarose beads

Saglam et al.^[122] showed that thiazolidine equipped poly(hydroxyethylmethacrylate) (PHEMA) microbeads were able to adsorb heavy-metal ions from aqueous solutions.

In order to test whether thiazolidine functionalised DNA has similar ability to bind to Pb (II) or Cd (II) ions immobilized-metal affinity chromatography was used.

Therefore, the tridentate chelating agent, iminodiacetic acid (IDA) which is covalently coupled to 4% cross-linked agarose beads was charged with metal ions (Cd²⁺, Pb²⁺). The loaded beads were filled into a microspin column and washed with water and equilibrated with buffer (100 mM MOPS, 200 mM NaCl, pH=6.5).

PCR with a 5´-radioactive labelled primer was carried out employing building block 18b instead of natural thymidine. As control natural thymidine was used. The PCR products were purified by G25 columns to remove salts and remaining nucleotides.

The radioactive labelled PCR products were applied on microspin columns containing the IDA agarose beads with Cd²⁺or Pb²⁺ and incubated 1h at rt. By centrifugation the solution was removed and the beads were washed with water.

Elution of bound DNA was carried out by the use of elution buffer containing thiazolidine. The beads were washed several times with elution buffer with increasing concentration of thiazolidine. The flow through of each washing step was collected separately and analyzed by agarose gel electrophoresis followed by autoradiography. The resulting gel of both radioactive labelled PCR products (with and without building block 18b) showed no bands. In a further experiment radioactive intensity of the beads after incubation with labelled DNA and washing with water was measured, expecting a high level of radioactivity for bound, labelled DNA. For both microspin columns containing unmodified DNA or modified DNA only a low rate of radioactivity was measured implicating that there is no interaction between the beads and the thiazolidine modified DNA.

Probably, the thiazolidine residue is sterically encumbered and the interaction between the heavy metal and the thiazolidine modified DNA was hindered.

This experiment was repeated using different buffer systems with different pH values without significant improvement.

Enzymatic Synthesis of Functionalized DNA