DNA polymerase activity on solid support : from diagnostics to directed enzyme evolution

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From diagnostics to directed enzyme evolution

Dissertation

zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften

(Dr. rer. nat.) an der Universität Konstanz Naturwissenschaftliche Sektion

Fachbereich Chemie

vorgelegt von Ramon Kranaster

2009

Konstanzer Online-Publikations-System (KOPS) URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-123778

URL: http://kops.ub.uni-konstanz.de/volltexte/2010/12377/

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Prüfungsvorsitzender und mündlicher Prüfer: Herr Professor Dr. Hartig

1. Referent und mündlicher Prüfer: Herr Professor Dr. Marx 2. Referent und mündlicher Prüfer: Herr Professor Dr. Wittmann 3. Referent: Herr Professor Dr. Fischer

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Biotechnol J. 2010, 5(2), 224-231. Kranaster, R., Drum, M., Engel, N., Weidmann, M., Hufert, F.T., Marx, A.

“One-step RNA pathogen detection with reverse transcriptase activity of a mutated thermostable Thermus aquaticus DNA polymerase.”

EMBO J. 2010, 29(10), 1738-1747. Obeid, S., Blatter, N., Kranaster, R., Schnur, A., Diederichs, K., Welte, W., Marx, A., “Replication through an abasic DNA lesion: structural basis for adenine selectivity.”

Angew. Chem. Int. Ed. 2009, 48(25), 4625-4628.

Kranaster R. & Marx A. “Taking fingerprints of DNA polymerases: Multiplex enzyme profiling on DNA arrays”

Nucleic Acids Symposium Series, 2008, 52, 477-478.

Kranaster, R. & Marx, A. “New Strategies for DNA Polymerase Library Screening”

ChemBioChem, 2008, 9, 694 – 697.

Kranaster, R., Ketzer, P. & Marx, A. “Mutant DNA polymerase for improved detection of single-nucleotide variations in microarrayed primer extension “

Chem. Eur. J., 2007;13, 6115-6122. Kranaster, R., Marx, A. “Increased single-nucleotide discrimination in allele-specific polymerase chain reactions through primer probes bearing nucleobase and 2'-deoxyribose modifications”

Chem Biol., 2007 14,185-194. Rudinger, N.Z., Kranaster, R., Marx, A. “Hydrophobic amino acid and single-atom substitutions increase DNA polymerase selectivity”

weitere Publikationen:

Nucleic Acids Res., 2007; e143. Fahrer, J., Kranaster, R., Altmeyer, M., Marx, A., Bürkle, A.

“Quantitative analysis of the binding affinity of poly(ADP- ribose) to specific binding proteins as a function of chain length”

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1 Introduction

Self-replication is a process in which an entity (e.g. a cell or virus) makes a copy of itself and is thus a fundamental basis of life. Living cells accomplish cell division by cooperative action of a plethora of proteins and nucleic acids. Before a cell divides, it has to replicate its own genetic information for biological inheritance. This information is stored in a polymeric macromolecule, deoxyribonucleic acid (DNA) and is used by all living organisms on earth. DNA consists of a chain of four different components, the nucleotides: adenosine, cytidine, guanosine and thymidine. Each nucleotide is a deoxyribose sugar linked with a monophosphate and a heterocyclic base and is connected to the next base via phosphodiester linkages creating an alternating phosphate-deoxyribose strand. Two antiparallel strands are coiled together to form a characteristic double-helical structure (Figure 1). The phosphate- deoxyribose backbone shields the inward pointing bases, which are interacting with the opposite DNA strand through specific hydrogen bonds forming base pairs. Adenine pairs with thymine, and cytosine pairs with guanine (see Figure 1,(B)).

Figure 1 Double-helical structure of DNA. (A)Stick –(left) and surface model (right). PDB-code:

1BNA¹. (B)Watson-Crick base pairing² of A with T and G with C.

The sequence of the four different bases in DNA is very important because they intrinsically carry the genetic code. A set of three neighboured bases is called a triplet codon and usually encodes for a specific single amino acid. Thus, each specific DNA sequence may code for at least one specific protein. The entirety of DNA in one cell constitutes the genome, which in principal dictates the production of proteins,

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1.1 DNA Synthesis - Biological role of DNA polymerases

In living cells, DNA synthesis is mainly divided into the processes of replication, repair and lesion bypass. DNA polymerases are the main actors in these processes. They are catalysing the DNA synthesis under consumption of the corresponding nucleotide triphosphates via nucleotidyl transfer³. DNA replication is the duplication of the genome in a semiconservative process in that each strand of the original double- stranded DNA serves as a template for the synthesis of the complementary strand. DNA repair is the identification and repair of certain genomic DNA lesions like DNA adducts, single and double strand breaks, photoproducts and abasic sites. DNA translesion synthesis (TLS) is the process of lesion bypass performed by specialised DNA polymerases able to bypass DNA lesions, which are typically strong obstructions for these enzymes⁴.

1.1.1 Structural model for DNA polymerase I from Thermus aquaticus

1958 Kornberg and coworkers⁵ discovered the first enzymes that catalyse the incorporation of deoxyribonucleotides into DNA extracted from Escherichia coli (E.coli) cells. These first serendipities triggered an avalanche of DNA and RNA polymerase studies about their biological function, properties and catalytic mechanisms.

Nowadays several crystal structures of polymerases are available in the presence and absence of DNA, RNA and nucleotide substrates. Kim et al.⁶ solved the structure of thermostable DNA polymerase I from Thermus aquaticus (Taq) by x-ray scattering 1995, which displays a typical DNA polymerase structure. The structure is very similar to a right hand consisting of a finger domain, a thumb – and a palm domain.

Additionally, the Taq DNA polymerase has an N-terminal attached nuclease domain (see Figure 2). Li et al.⁷ could further crystallise an N-terminal shortened form of the Taq polymerase (KlenTaq) bound to the primer template DNA in the so-called 'binary complex' and an additionally bound triphosphate constituting the 'ternary complex'.

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Figure 2 Crystal structure of thermostable DNA polymerase I from Thermus aquaticus (Taq). The structure is very similar to a right hand consisting of a finger-domain, a thumb– and a palm-domain.

Additionally Taq DNA polymerase has a N-terminal attached nuclease domain. PDB-code 1TAQ⁷.

During catalysis, the polymerase is able to adopt two different conformations: an open and a closed form. Especially the finger domain has to move between these two conformations with an angle of ~46°. It is consensus that upon binding of a triphosphate the polymerase turns into the closed conformation until incorporation of deoxynucleotide takes place and a pyrophosphate is set free. The enzyme opens again and may translocate along the primer template DNA or dissociates from the extended primer template to bind to another DNA substrate.

1.1.2 Reaction pathway of DNA polymerases

The kinetic mechanism of most DNA polymerases⁸ can be described by a general, simplified scheme: In the first step, the polymerase binds to the DNA substrate. The second step involves the binding of a dNTP constituting the ternary complex. Step three is a conformational change from the open ternary to the closed ternary complex which is followed by the nucleotidyl transfer (step four, see also Section 1.1.3). In step five, the enzyme releases the extended primer template DNA substrate and relaxes to its initial conformation.

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Figure 3 Schematic representation of DNA polymerase catalysed DNA synthesis. The reaction pathway is separated into eight different kinetic steps.

During step six, the pyrophosphate is released and the polymerase may continue the synthesis on the same DNA substrate by translocation (step 7) or may dissociate from the extended primer template to bind on another DNA substrate (step 8). The number of added nucleotides by a polymerase during an association and dissociation at a single DNA substrate is defined as processivity. Replicative DNA polymerases tend to be very processive thus adding several hundred nucleotides upon binding, whereas DNA repair involved polymerases have low processivity adding only single or a few nucleotides. In E.coli for example, the DNA Polymerase III represents a processive DNA polymerase responsible for DNA replication and DNA polymerase I is a low processive representative responsible for DNA replication initiation⁹.

The question of the rate-limiting steps during enzymatic action has been explored by numerous kinetic studies and with the aid of modified substrates like α-S-dNTPs¹⁰ or 2- aminopurines¹¹ in the template strand but is still discussed. In studies of the Klenow fragment deriving from E.coli and DNA polymerase from bacteriophage T4, the conformational change from open to close state (step 3) seems to be the rate-limiting step¹¹, whereas in stopped flow fluorescence studies with human DNA polymerase β the nucleotidyl transfer step is indicated with the lowest rate constant¹². Taken together, it could be possible that DNA polymerases involved in DNA repair and/or lesion bypass have a different rate-limiting step than DNA polymerases involved in replication. In principle, all enzymatic steps shown in Figure 3 are reversible.

Pyrophosphorolysis is the reverse process and generates dNTPs under degradation of

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the primer strand. High concentrations of pyrophosphate shift the equilibrium to the pyrophosphorolysis process, which plays an important role in drug resistance of HIV RT polymerase^13,14. Furthermore, in biotechnological applications in which incorporation of artificial substrates into DNA by DNA polymerases are used, the degradation and generation of native triphosphates can easily be avoided by addition of pyrophosphatase into the reaction mix¹⁵.

1.1.3 Chemical mechanism of the catalysed nucleotidyl transfer

Although several crystal structures and numerous kinetic data have been published, the catalytic mechanism of DNA polymerases is still discussed and not fully understood. It is well established that a nucleophilic attack on the α-phosphorous atom of the nucleoside triphosphate by the primer 3´C-OH group leads to formation of the phosphodiester bond and to the release of a pyrophosphate. All known DNA and RNA polymerases require two divalent cations (usually Mg²⁺) in their active center and use a two-metal-ion mechanism for the catalysed nucleotidyl transfer. The most recent study on the mechanism of DNA polymerases (see Figure 4) proposes an extended two metal ion mechanism by a two proton transfer reaction catalysed by typical acid and basic amino acids: The protonation of the pyrophosphate leaving group by a proposed acid and the deprotonation of the 3´C-OH group by a proposed basic amino acid³.

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Figure 4 Extended two-metal-ion mechanism of nucleotidyl transfer which includes a general acid catalysis³. Shown is the active center of a polymerase with a nucleoside triphosphate (red) and two divalent metal cations (Mg²⁺). One metal ion is coordinated by the phosphates of the triphosphate and an aspartate residue located in motif A of all polymerases (blue), and probably water molecules. The other metal ion is coordinated by the 3´C-OH of the primer terminus (green), the α-phosphate of the nucleoside triphosphate and widely conserved aspartate residues of structural motifs A and C. A proposed acid (A) which protonates the pyrophosphate leaving group is indicated by four different model polymerases which where used in the study of Castro et al. to proof their concept of a general acid catalysis. A proposed basic amino acid (B) which deprotonates the 3´C-OH group stays unidentified until now. The figure was designed according to reference³.

Both metal ions stabilise the structural adjustment. Metal ion A lowers the pKa of the 3´C-OH group and thus supports the deprotonation by an unidentified base B and the subsequent nucleophilic attack at physiological pH conditions. It is believed that the α-phophate adopts during this attack a SN2 type trigonal bipyramidal transition state³. 1.2 Biotechnological role of DNA polymerases

DNA polymerases are used in a plethora of biotechnological applications¹⁶. Nowadays, they are the workhorses in numerous applications like DNA sequencing and microarrayed nucleic acid diagnostic tools for the direct diagnosis of single-nucleotide variations within genes, forensic DNA testing, pathogen detection, et cetera.

1.2.1 Polymerase chain reaction (PCR)

A key technology for the use of DNA polymerases in biotechnological applications is the polymerase chain reaction (PCR), which was development by Mullis and coworkers in 1987¹⁷. In theory, PCR allows the exponential enrichment of a particular DNA sequence by amplification of a single or few copies of template strands. For PCR applications a DNA polymerase needs specific primers (short DNA fragments) which contain sequences complementary to a target DNA region. During repeated cycles of

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heating and cooling the DNA is generated and is itself used as a template for replication. Due to the enzymatic replication under consumption of the primers and deoxynucleotide triphosphates (dNTPs) the selected DNA sequence flanked by the primers is exponentially amplified. In almost every PCR application, heat-stable DNA polymerases resistant against the thermal cycling steps necessary to physically separate the two strands of the DNA double helix (usually at high temperatures ~95°C) are employed. Nowadays, PCR methods in presence of fluorescent dyes (e.g.

SYBRGreenI) or fluorescence resonant probes (e.g. TaqMan)^18-21 report the amount of amplified DNA in real-time. Both fluorescent dyes and modified DNA polymerases have significantly shortened conventional PCR methods. Consequently, real-time PCR methods are the method of choice for the detection and quantification of DNA and RNA targets such as retroviruses and viral pathogens²¹.

Today the principle of a PCR is extended in numerous biotechnological applications:

Allele-specific PCR for the detection of single nucleotide variations^22-24, multiplex PCR for the multiple amplification of different DNA fragments in one reaction vessel²⁵, nested PCR which increases the specificity of the DNA amplification reaction²⁶, quantitative PCR to quantify and compare certain DNA strands²⁷, reverse transcription PCR for the detection of RNA targets²⁸ et cetera.

1.2.2 Modified and mutated DNA polymerases

Highly processive and accurate DNA polymerases are desired for cloning procedures in order to give shorter extension times as well as a more robust and high yield amplification. The processivity of a DNA polymerase was significantly enhanced recently by protein fusion technology²⁹: DNA polymerase processivity is definded as a value of the average number of nucleotides added by a DNA polymerase per association/disassociation with the DNA template. It is known that the Taq polymerase (see Section 1.1.1) consists of two distinct structural and functional domains, the 5´- 3´ nuclease domain and the polymerase domain. The N-terminal shortened form of Taq (KlenTaq) lacking the nuclease domain is significantly less processive than the full- length Taq²⁹, suggesting that the nuclease domain interacts with the DNA template

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specific, double-stranded DNA binding domain resulting in enzymes with increased processivities without compromising catalytic activity and enzyme stability. By monitoring the single primer extension products by sequencing gels, it was demonstrated in detail that Taq wild-type added up to 35 nucleotides whereas fused Taq produced products up to added 200 nt.

A higher DNA polymerase fidelity may increase the reliability of diagnostic application systems³⁰. Marx and coworkers³¹ demonstrated that the selectivity of Taq DNA polymerase can be increased by nonpolar substitution mutations of three amino acids QVH (Gln, Val, His) of motif C directly neighboured to the catalytic center.

Furthermore, they showed that these obtained mutants can be applied as a useful tool in genotyping assays like allele specific real-time PCR^31,32.

To enhance the efficiency of forensic DNA testing, DNA polymerases resistant to inhibitors from blood and soil would enable the PCR without prior DNA purification.

Barnes and coworkers³³ have recently evolved Taq DNA polymerase mutants with enhanced resistance to various known inhibitors of PCR reactions, including whole blood, plasma, hemoglobin, lactoferrin, serum IgG, soil extracts and humic acid, as well as high concentrations of DNA binding dyes. The mutated position of the Taq polymerase (Glu 708) is located in an alpha-helix region on the surface of the enzyme, known as “P-domain” (residues 704–717). This domain is situated about 40 residues apart from the “finger” domain, which binds the incoming dNTPs and interacts with the single stranded DNA template. Because the mutation site is at the hinge region one might speculate that it may affect the movement of the finger domains during incorporation. The described mutation in this example is not directly involved in interaction between substrate and enzyme thus indicating a so-called remote effect³⁴. The recovery of ancient DNA samples, which could be more than 40 000 years old, requires DNA polymerases with an increased substrate spectrum to efficiently amplify and overcome typical DNA lesions³⁵. In 2007 Marx and coworkers³⁶, and Holliger and coworkers³⁵ published successfully evolved DNA polymerases that are able to amplify from highly damaged DNA templates and bypass lesions found in ancient DNA such as abasic sites.

Further improvements of DNA polymerases are required, for example, to meet the requirements of next generation DNA sequencing technologies, which rely on the ability of DNA polymerases to efficiently process modified nucleotides³⁷. For example

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the sequencing technology from Illumina Inc. uses fluorescent reversible terminator deoxyribonucleotides³⁸. The triphosphates have a 3’-O-azidomethyl group, which stops the polymerase after incorporation of one nucleoside. All four 2’- deoxynucleoside triphosphates (A, C, G and T) are additionally labelled with a different removable fluorophore to determine the sequence by fluorescence readout after each incorporation step.

Figure 5 Highly modified deoxyribonucleotides are used in the sequencing technology from Illumina Inc. The triphosphate (A) has to be efficiently processed by the DNA polymerase. The next sequencing cycle can begin, after chemical removal of the fluorescent dye and 3´-OH protecting group (B).

To improve the efficient incorporation of these unnatural nucleotides they had to engineer the active site of 9°N DNA polymerase. The figure was designed according to reference 38.

After readout, the 3´-O-azidomethyl group and the fluorescent dye will be chemically removed by tris(2-carboxyethyl)phosphine (TCEP), that the next single incorporation cycle can begin. However, to improve the efficient incorporation of these unnatural nucleotides they had to engineer the active site of 9°N DNA polymerase to gain a sufficient sequencing setup.

Taken together, customized and artificially engineered DNA polymerases that lead to more robust and specific reaction systems are urgently needed.

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1.2.3 Directed evolution of DNA polymerases

Native proteins and enzymes are the natural products of several million years of evolution. Alliances of enzymes in one living organism cause it to be good or less good adapted to certain environmental conditions. Natural selection takes place in a way that the best adapted to the given conditions prevails.

This process can be artificially enhanced by modern biochemical methods in order to obtain enzymes, e.g. DNA polymerases, with new features. Alterations are mainly achieved by directed molecular evolution using genetic complementation and/or screening28,31,32,36,39-43

, phage display^44-47, or in vitro compartmentalization^48-50. In general, three steps are required for a successful directed molecular evolution of DNA polymerases: introduction of mutations by certain methods of mutagenesis, expression of different enzyme variants and screening or selection of best enzyme variants. These steps can be repeated until the desired feature is obtained (see Figure 6).

Figure 6 Scheme of directed evolution of DNA polymerases. 1. Introduction of arbitrary mutations indicated as red dots. 2 Separation of different mutants. 3. Screening/Selection of mutants by appropriate assays. These steps can be repeated until the desired feature is obtained e.g. higher mismatch discrimination.

After creation of a mutant library, a mutant separation process is needed which also ensures a linkage between a specific enzyme genotype and the respective phenotype.

Selection or screening approaches are described in the literature for the directed

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evolution of DNA polymerases. Common selection methods are phage-display , compartmentalised self replication³⁵ (CSR) or reporter plasmid assays⁴³.

For example, Vichier-Guerre et al.⁴⁷ employed phage display to select DNA polymerase mutants with about two orders of magnitude higher catalytic efficiency for reverse transcription when compared with the natural enzyme. In phage-display, the DNA polymerases are expressed and displayed together with a substrate on the surface of a phage. The polymerase mutant displayed onto the phage particle has to convert a linked substrate into desired product, which is then selected for example by affinity chromatography. One disadvantage might be the high degree of cross-reactions between a polymerase on one phage and a substrate attached to another phage.

Holliger and coworkers⁴⁹ employed CSR to evolve polymerases that can extend mismatches and common lesions found in ancient DNA. They demonstrated that these engineered polymerases could expand the recovery of genetic information from Pleistocene specimens³⁵. For a CSR method, each polymerase gene is encapsulated in a compartment formed by a heat-stable water-in-oil emulsion. Each polymerase has to replicate its own encoding gene and therefore results in a very high adaptive burden depending on the specific selection system.

Loeb and coworkers⁴³ employed a reporter plasmid assay for the selection of DNA polymerase I mutants from E.coli with increased fidelity. In a reporter plasmid assay, a plasmid is used which contains a reporter gene for example containing an antibiotic resistance gene but with an opal codon. Selection of mutants is possible by comparing the reversion frequencies of the wild-type with the mutants.

In standard screening methods, the mutants must be separately expressed in multi- well plates so that the phenotype is directly connected to the corresponding mutant in each well. Subsequent screening reactions of mutants can be processed by either primer extension reactions or PCR. Real-time PCR screening yields a very high sensitivity due to the exponential enrichment of the product³². Barnes and coworkers for example employed a radioactive labelled nucleotide incorporation assay to screen successfully for DNA polymerase mutants, which are more resistant against common inhibitors present in blood and soil samples³³.

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screen in a multiplexed and parallel manner for several different reactions and new functions. This approach was followed in Section 2.3.

1.2.4 Methods of mutagenesis

Numerous strategies and methods of mutagenesis can be found in the literature:

Site directed mutagenesis is a good strategy when proper information about structural and substrate-enzyme interactions is available. The mutation sites are rationally designed and are introduced by site directed mutagenesis protocols using mutagenesis primers carrying the respective nucleic acid sequence for the desired mutation⁵¹. Special enzyme features may change simply by introducing these point mutations. This strategy could be a good starting point for further directed evolution.

Saturated mutagenesis offers another option to test one amino acid position with all native possible amino acid substitutions. The amino acid position can be randomised using degenerated mutagenesis primers. When a single codon is randomised, the library size can be small (3-4 hundreds mutants) and leads to 99.9% probability of having all possible mutations included⁵².

Arbitrary gene mutations can be introduced by error-prone PCR^28,53 (epPCR). In epPCR high magnesium or manganese concentrations and/or imbalanced mixtures of deoxynucleotide triphosphates during the PCR are used, causing the DNA polymerase to produce incorporation errors. Unfortunately this encompass a few disadvantages:

Due to the nature of template amplification, an early occurring mutation in the first cycles of PCR might be enriched during amplification and thus overrepresented in the resulting protein library⁵⁴. Additionally, DNA polymerases used in epPCR preferentially produce transition than transversions errors due to the steric demands of similar bases (purines A, G and pyrimidines T, C). At least certain amino acid exchanges do occur very infrequently, because exchange of one nucleotide is not enough to change a whole amino acid codon and mutation of two neighboured nucleotides is not occurring very often⁵⁵. Nevertheless, error-prone PCR creates a good initially library for directed evolution methods especially when few information on enzyme structure and important amino acid residues exists.

Furthermore, arbitrary gene mutations can be introduced by other techniques such as sequence saturation mutagenesis⁵⁶ (SeSaM). This method uses gene fragmentation by iodine cleavage of previously introduced phosphorothioate groups randomly

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distributed in the gene. After fragmentation, these single stranded DNA fragments are used as primers for the following full-length gene synthesis. During this step, artificial bases with universally binding properties are used to ensure arbitrary randomisation.

Disadvantages of this method are that DNA fragments smaller than ~70 nt are not mutagenized due to the employed DNA extraction and purification procedures⁵⁶. The independence from the mutational bias of DNA polymerases using epPCR is exchanged for the different base-pairing preferences of universal or degenerated bases.

Homologous recombination methods enable the shuffling of different mutations and additionally introduce new mutations as well. In the DNA shuffling method, homologue genes are fragmented by DNase digestion and afterwards reassembled to the full-length gene by PCR⁵⁷. An alternative method is the staggered extension process (StEP) which also allows the combination of different homologous genes⁵⁸. It uses highly abbreviated annealing and extension steps during PCR to generate staggered DNA fragments. This procedure promotes crossover events along the full length of the template sequences resulting in a library of chimeric polynucleotide sequences.

1.3 Accuracy of enzymatic DNA synthesis

Due to its complementary structure, DNA can be copied using the respective DNA strand as a template. The energy differences based on hydrogen bonding between a correct Watson-Crick base pair and an incorrect one are not very high (~ 1-3 kcal/mol) and result theoretically in a high error-rate of one per 100 incorporated nucleotides^59,60. In living cells during replication, DNA polymerases have to copy the whole genome, in human cells these are more than 6 billion nucleotides⁶¹. The genomic sequence would rapidly undergo changes after each cell division. Fortunately, nature has evolved polymerases with error-rates much lower than one would expect from thermodynamic considerations^62,63. In bacteria for example, the overall accuracy of DNA synthesis reaches one error of 10⁸-10¹⁰ incorporated nucleotides^62,64. Even in eukaryotic cells error-rates >10¹⁰ are reached⁸. The impact of incorrectly incorporated nucleotides can vary: On the one hand, mutations in the genetic sequence may be a

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Misincorporation rates by DNA polymerases in E.coli are determined by 10 – 10, depending of course on the misincorporated base, indicating enzymatic processes that increase the accuracy beyond thermodynamic limitations⁶⁵. Factors like proofreading and mismatch repair by base and nucleotide excision repair (BER and NER) improve these rates further to the overall error-rates mentioned above (10⁸-10¹⁰).

Kool and coworkers showed that the efficient and selective enzymatic DNA synthesis is not dependent on the hydrogen bonding between Watson-Crick base pairs alone⁶⁶. In detail, they studied base analogues that lack the ability for hydrogen bonds, but still have similar size and shape compared to the natural bases⁶⁶ (see Figure 7, A). DNA polymerases were still able to use these analogues as substrates in a selective manner.

The base analogues were selectively incorporated opposite of the respective correct template base and furthermore used as a template base selectively addressing for the correct triphosphate.

Figure 7 (A) Representative isosteric base analogue N in comparison with nature base cytosin C.

(B) Pyrene base opposite an stable abasic site analogue.

Along these lines, they constructed a pyrene instead of a natural base bearing triphosphate, which has similar size and shape as a full base pair. This artificial triphosphate was preferentially incorporated opposite of an abasic site instead of a nucleobase⁶⁷ (see Figure 7, B). All these findings lead to the assumption that size and shape of the incoming dNTP play an important role for DNA polymerase selectivity.

Further experiments concerning structural studies^68,69 have led to the common understanding that the geometry of the DNA base pair is regulated by a close fit in the polymerase active site⁷⁰.

1.3.1 Chemical approach for increasing the selectivity of DNA polymerases Incorporation of an incorrect nucleotide results in the forming of a mismatch and leads to an altered geometry within the DNA duplex. DNA polymerases are able to sense

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these mismatches and display significantly reduced extension rates compared to a matched base pair situation^22,71,72 (see Figure 8).

Figure 8 Matched and mismatched primer template situation. DNA polymerases are able to differentiate between these cases resulting in reduced mismatch extension rates compared to matched base pair situations. Black bars represent primer/template duplexes.

One approach to enhance DNA polymerase ability in discriminating between matched and mismatched situations is to use 3´ chemically modified primer probes.

Latorra et al. introduced locked nucleic acid (LNA) modification at the 3'end of primer probes and demonstrated that this modification leads to an increased single nucleotide discrimination in allele-specific PCRs⁷³. Along this line, Gaster et al.^24,74 introduced several 4'-C-modifications at the 3´primer end and could show that these modifications in combination with Vent (exo-) reveal highly increased single nucleotide discrimination properties. Especially a polar 4´-C-methoxymethylen group showed superior discrimination properties between matched and mismatched primer template situations. Presumably one might hold thermodynamic reasons responsible for this effect, but thermal denaturation studies and CD spectra revealed that differences between stabilities of canonical over non canonical duplexes at the 3´terminal primer end are negligible^22,24. Thus, it is unlikely that this effect derives from differential duplex stabilities of 4'C-modified versus unmodified duplexes. It is more likely that increased steric constraints and slightly disturbed geometries in the active center especially in the case of mismatched substrates contribute to the selectivity of the outcome of this process. Marx and coworkers^75,76 could show that altering the steric demand of the DNA substrate results in a more selective polymerase regarding the discrimination of single nucleotide variations. Additionally, these beneficial properties of modified primer probes were independent of buffer conditions and applicable in different sequence contexts²⁴. Taken together the employment of primer probes bearing modifications at the 3’ end allowed increasing the selectivity of

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between matched and mismatched cases in the asPCR as well as in primer extension and arrayed primer extension systems^19,74,77.

1.3.2 Genetic approach for increasing the selectivity of DNA polymerases

Intensive crystal structure explorations revealed that DNA polymerases have subtle hydrogen-bonding networks, especially with the minor groove of the primer/template substrates^78,79. It is believed that they have direct impact on the DNA polymerase selectivity. On the one hand, it could be possible to alter the acceptor potential of the DNA substrate. This approach was partially followed during my diploma thesis by substituting the carbonyl functionalities in thymidine for thiocarbonyl groups.

Thiolated thymidines have decreased H-bonding abilities compared to the native carbonyl groups and could have a significant effect on the selectivity of DNA polymerases which is described and analysed in Section 2.1. In addition, other modifications like electron withdrawing groups are imaginable but are accompanied all with cost-intensive consumption of chemically modified primer probes.

On the other hand, it might be more effective to have DNA polymerases with improved single nucleotide discrimination properties in combination with unmodified primer strands and thus obviate the need for chemical modifications of the primer probes.

Loeb and coworkers selected DNA polymerase I mutants from E.coli showing enhanced accuracy in DNA synthesis⁴³. Mutants were selected by using a reporter plasmid harbouring an antibiotic resistance gene. One double mutant (K601I, A726V) exhibited a ten-fold enhanced accuracy without a significant reduction in activity⁴³. The mutation A726V is located in the M-helix, which is at the juncture of the fingers and palm subdomains. It lacks any direct contacts with the substrates but may disrupt the function of the hinge and may give more time for kinetic proofreading. The K601I mutation is located near motif 1 which is involved in the binding and positioning of the DNA substrate⁸⁰.

Summerer et al.³¹ focused on three residues within the motif C of proofreading- deficient DNA polymerases I from Thermus Aquaticus. motif C is well-known for having hydrogen bonds with the DNA substrate via the minor groove and is directly neighboured to the catalytic center of the DNA polymerase. They found that mainly unpolar amino acid substitutions lead to increased single nucleotide discrimination properties. Along these lines, Rudinger et al.⁷² could transfer this concept to DNA

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polymerases from the family B and constructed a double mutant of the DNA polymerase from Pyrococcus furiosus (Pfu). This mutant is ten fold more selective for the discrimination of unmodified mismatched primer templates and may build the basis for further enhancements regarding methods for the detection of single nucleotide variations (see also Section 2.22.2). Until now, fidelity mechanisms of DNA polymerases are not fully understood. Taken together mutations, which decrease or delete distinct polar interaction with the DNA substrates, may generally result in enzymes with higher selectivity. Unfortunately, it seems that mutations with very high impact on DNA polymerase selectivity appear in tandem with a significant reduction of enzymatic activity.

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1.4 Aim of this work

The aim of this work was the functional analysis and recruitment of mutated DNA polymerases for improved biotechnological applications.

Strerath et al. ²³ demonstrated that DNA polymerases have increased mismatch discrimination features under the presence of 4´C modified triphosphates or primers.

It is believed that a subtle hydrogen-bonding network of the enzyme with minor groove of the primer/template substrate contributes to selectivity of a DNA polymerase^78,79. To proof this concept, small thiomodifications at the 2- and 4-position on thymidine were introduced and tested regarding their effects on DNA polymerase selectivity.

In this context a new diagnostic application for the detection of single nucleotide variations, under recruitment of DNA polymerases by arrayed primer extensions, should be established. It was previously demonstrated by Summerer et al.³¹ that certain mutations in DNA polymerases lead to increased single nucleotide discrimination properties. Along these lines, Rudinger et al.⁷² showed that a double mutant of the DNA polymerase from Pyrococcus furiosus (Pfu) is ten fold more selective for the discrimination of unmodified mismatched primer templates. These features of the double mutant should be exploited in establishing a reliable arrayed method for the detection of single nucleotide variations.

Besides investigating DNA polymerase selectivity, a new arrayed approach for the functional profiling of DNA modifying enzymes should be established enabling multiplexed profiling of several enzyme features in high-throughput. The system should be based on the spatial separation of different covalently attached DNA substrates on a glass slide and their selective addressing by template oligonucleotide hybridisation.

Besides screening of DNA polymerase features on solid support, other functional mutations and changes at DNA polymerases should be tested to study their effect on enzyme activity and behaviour. In detail, Sauter et al.²⁸ evolved an N-terminal shortened form of the DNA polymerase from Thermus aquaticus (Taq) with highly increased reverse transcriptase activity. The attachment of a 5´-3´ nuclease domain constituting the full-length Taq enzyme should be performed and the functionality of

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both combined activities should be tested. The resulting enzyme should be further evaluated regarding its usefulness for one-step RNA detection systems.

At least single mutations in the active center of a DNA polymerase should be tested to obtain more insights concerning non-templated DNA synthesis catalysed by some DNA polymerases following the A-rule. In this context dNTP incorporation opposite abasic DNA lesions and at blunt-end primer template complexes should be tested and further evaluated in kinetic incorporation studies.

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2 Results and discussion

2.1 SNP detection by allele-specific real-time PCR employing chemically modified DNA primers

2.1.1 Introduction

Within the human genome comprising approximately 3 billion nucleobase pairs, individuals differ in approximately 0.1 percent of the nucleotide sequence^61,81. The most frequent among these sequence variations are single-nucleotide polymorphisms (SNPs) which are changes in a single base at a specific position in the genome^82-84.

Figure 9 Representative single nucleotide variation at a specific position in the genome

SNPs are defined as sites in which the less common variant has a frequency of at least 1% in a population. A direct linkage exists between some of these dissimilarities and certain diseases. Additionally, different effects of drugs on different patients can also be linked to SNPs^82,84,85. Moreover, SNPs can be used to determine someone’s ancestry.

Recently, it has been shown that SNPs mirror the European landscape in a way that regional provenance may be estimated with an accuracy of 100km^86,87. Thus, considerable efforts have focussed on finding new SNPs and elucidating connections between them and certain phenotypes. On average, each human carries three to four million SNPs which can be detected with a variety of methods, at least by whole genome sequencing technologies³⁸. In a first step, high-throughput sequencing methods that identify unknown nucleotide variations are needed. This is followed by an investigation of the medicinal relevance of these variations. After assessment of the exact sequence context other methods are needed for high-throughput screening of populations in the search for known SNPs or to analyse individuals for SNP patterns.

Obviously in the latter case methods are essential that allow for time- and cost- effective verification of distinct nucleotide variations in daily laboratory practice^19,88-

95. Many methods rely on amplification of the target gene before analysis can take

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place. Thus, methods that enable amplification and analysis in a single step are desirable.

In principle, allele-specific PCR (asPCR) comprises these features^96-103. The concept of asPCR is based on the principle that a DNA polymerase catalyses DNA synthesis from a matched 3’-primer terminus, while a mismatch due to hybridisation of the primer probe to a different sequence variant should obviate DNA synthesis and, therefore DNA amplification should be abolished (see Figure 10).

Figure 10 In principle, the DNA polymerase catalyses the DNA-synthesis in the match case (upper half); due to incorrect hybridisation at the primer end the DNA synthesis should be oppressed in the mismatch case (lower half) (Black bars represent primer/template duplexes).

This conceptually simple and straightforward procedure is hampered somehow by the sequence dependency of the selectivity^99-103. Thus, identification of the appropriate reaction conditions requires tedious optimisation. In earlier publications, it has been shown that an essential improvement may be gained in this method by employing chemically modified primer probes23,24,73,76,104,105

. The employment of primer probes bearing a 4’-C-modification at the 3’ end allowed the selectivity of asPCR to be increased significantly. These primer probes showed increased discrimination properties between matched and mismatched cases in the asPCR, as well as in primer extension and arrayed primer extension systems⁷⁴. Additionally, these beneficial properties of modified primer probes were independent of buffer conditions and applicable in different sequence contexts23,24,73,76,104,105

. Especially the combination of primer probes bearing 4’-C-methoxymethylene residues at the 3’ end together with the commercially available DNA polymerase from Thermococcus litoralis (Vent (exo^-) DNA polymerase) was found to exhibit superior performance in allele discrimination²⁴ (see Figure 11).

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Figure 11 4´-C-methoxymethylen residues at the 3´terminal position of primer probes lead to increased allele specific discrimination using Vent (exo^-) DNA polymerase. (Black bars represent primer/template duplexes, red T represents a 4´-C-methoxymethylen residue at the 3´end of a primer)

Encouraged by these findings the effects of some nucleobase modifications in conjunction with the previously described 4’-C-methoxymethylenated 2’-deoxyribose residues were studied with regard to discrimination in the asPCR. In order to modify the size and hydrogen-bonding ability of the substrates, the carbonyl functionalities in thymidine were substituted for thiocarbonyl groups. Thiolated thymidines have an enlarged steric demand, due to the 0.45 Å longer double bond length¹⁰⁶ and additionally decreased hydrogen-bonding abilities compared to the native carbonyl groups (see Figure 12).

Figure 12 (A) Exchange of Oxygen O with Sulphur S at position 2 (^2ST) and position 4 (^4ST); (B) Connolly surface with electrostatic charge distribution. Substitution of the carbonylgroup with thiocarbonylgroup leads to an enlarged steric demand, due to the 0.45 Â longer double bond-length¹⁰⁶ and to a decreased H-bonding ability compared to the native carbonyl group.

To explore the effects of 2-S and 4-S substitutions in conjunction with 4’-C modification on the allele-discrimination in PCRs, the respective modified oligonucleotides were successfully synthesised during my diploma thesis in 2005/2006¹⁰⁷. In that work, the 2-S and 4-S thiolated commercially available nucleosides and the synthesised (4-S/2-S)-4’-C-methoxymethylene thymidines were coupled to a solid long-chain amino alkyl modified controlled pore glass (LCAA-CPG) support according to established protocols¹⁰⁸. The solid supports were subsequently

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transferred into suitable cartridges and employed in standard automated DNA synthesis to yield the desired oligonucleotides ^4ST, ^2ST, T^OMe, ^2STO^Me, ^4ST^OMe.

Thereupon, modified oligonucleotides were used in allele-specific real-time PCRs. For proof of principle experiments, it was decided to use a PCR template (90nt long) within the human acid ceramidase and the Farber disease¹⁰⁹ sequence context. Two reactions were conducted at a time in parallel: One PCR was conducted by employing a template bearing a deoxyadenosine (dA) residue opposite the respective 3’-terminal thymidine in the primer probe. In the other experiment the same primer probe was combined with a template strand that had the same sequence apart from a dA to deoxyguanosine (dG) mutation opposite the 3’-terminal thymidine moiety (see Figure 10). An unmodified reverse primer was used for both setups. PCRs were analysed by employing real-time double-stranded DNA detection through SybrGreen I fluorescence by using appropriate thermocycler equipment¹¹⁰. First results during my diploma thesis indicated that thiolated thymidines, especially ^2ST, ^2ST^OMe and ^4ST^OMe, may increase single-nucleotide discrimination by DNA polymerase.

2.1.2 Results

After successful synthesis of the base and/or 4Ć modified oligonucleotides (^4ST, ^2ST, TÔMe, ^2STO^Me, ^4STÔMe) and first preliminary asPCRs during my diploma thesis¹⁰⁷, the previous results obtained were repeated and further analysed in more detail:

The threshold-crossing point (Ct) as a measure for amplification efficiency was determined for each primer probe respectively. This parameter is defined as the point in which the reporter’s fluorescence (SybrGreen I) exceeds the background fluorescence significantly and crosses a threshold.

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Figure 13 Results of real-time asPCR experiments obtained by using primer probes bearing 4’-C-methoxymethylene residues at the 3’ end in combination with nucleobase thiomodifications. Results are shown with primer probes ^2ST, ^4ST, TÔMe, ^2STÔMe, ^4STÔMe or the unmodified primer T as indicated. PCR amplification in the presence of target template A (solid line) or template G (dashed line) is shown. All experiments were conducted under the same conditions (see the Experimental Section).

The difference in the threshold-crossing points (ΔCt) of canonical (A-T/T*) versus non- canonical (G-T/T*) primer–template amplification is a measure for single-nucleotide discrimination. Amplification efficiency and the ability to discriminate against single- nucleotide mismatches varied with the modification employed (see Figure 13, Table 1).

Table 1 Analyses of realtime asPCRs. Comparison of ΔCt-values obtained from base and 4´-C- methoxymethylene modified primer probes.

5´-...AGGA T/T*

(primer probe, 20nt) 3´-...T CC T N TCCA.. (template, 90nt)

Ct (N=A) ΔCt

T 5 0-0.5

2ST 5 3

4ST 5 1

T^OMe 6 9

2ST^OMe 6 12

4ST^OMe 20 19

T*=modified thymidine ^2ST, ^4ST, TÔMe, ^2STÔMe or ^4STÔMe N= A or G, respectively

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Usage of the ^2ST probe introduced some degree of selectivity (2.5-3 PCR cycles) as compared to use of the unmodified probe. Strikingly, these effects were significantly enhanced when a 4’-C-methoxymethylene modification was present in conjunction with a 2-thiolation. ^2STÔMe had superior properties when compared with the unmodified T, ^2ST and TÔMe probes. Different results were obtained when the effects of 4-thiolation on asPCRs were investigated. The ^4ST probe had no significant effect on the amplification selectivity or the efficiency. When ^4STÔMe was used, the amplification efficiency decreased significantly, as indicated by the Ct value of 20. Interestingly, this was combined with an unprecedented high ΔCt value of 19 cycles. Agarose gel analysis of the resulting reaction products revealed that, when mismatched primer–template complexes containing the ^4STÔMe probe were used, thermocycling resulted in amplification of non-specific PCR products.

To gain further insights into the origin of the observed effects, thermal-denaturing studies were conducted and CD spectra were recorded. Duplexes between the respective primer strands (T, ^2ST, ^4ST, TÔMe, ^2STÔMe and ^4STÔMe) and 33-mer templates corresponding to matched (A–T/T*) and mismatched cases (G–T/T*) resulted in nearly superimposable CD spectra. This result indicated little, if any, dependence of the overall helix conformation on the presence of a matched or mismatched case and modifications at the nucleobase and/or 2’-deoxyribose (see Figure 14).

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Figure 14 Resulting Circular Dichroism spectra from respective primer strands (T, ^2ST, ^4ST, TÔMe, ^2STÔMe and ^4STÔMe, 20nt) hybridised to templates (33nt) corresponding to matched (A–T/T*) and mismatched cases (G–T/T*). (A) T match, T mismatch, ^2ST match, ^2ST mismatch, ^4ST match and ^4ST mismatch; (B) TÔMematch, TÔMe mismatch, ^2STÔMe match, ^2STÔMe mismatch, ^4STÔMe match and ^4STÔMe mismatch.

T*= modified thymidines ^2ST, ^4ST, TÔMe, ^2STÔMe or ^4STÔMeas described above.

Next, thermal-denaturing studies were conducted in order to investigate the impact of a chemical modification at the 3’-terminus on duplex stability. Only small variations in melting behaviour were found (see Table 2).

Table 2 Melting temperatures T_m [°C] derived from thermal denaturing experiments of primer probes in matched (A-T/T*) and mismatched (G-T/T*) complexes with respective template strand. All experiments were conducted with same DNA and buffer concentrations.

5´-...AGGA T/T* (primer probe, 20 nt) 3´-...T CC T N TCCA.. (template, 33nt)

matched case (A-T/T*) mismatched case (G-T/T*)

T 65.5 64.5

2ST 66.1 66.3

4ST 67.5 67.4

T^OMe 65.5 66.0

2ST^OMe 66.3 65.0

4ST^OMe 67.0 66.1

T*=modified thymidine ^2ST, ^4ST, TÔMe, ^2STÔMe or ^4STÔMe N=A or G, respectively

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Interestingly, all duplexes containing modified strands had slightly increased melting points when compared to the native duplexes, in both matched and mismatched cases.

Thus, the influence of a single nucleotide at the primer terminus that has been modified at the 4’-C-deoxyribose and/or the nucleobase on the intrinsic formation of aberrant conformations or duplex stability is small.

Steady-state extension efficiencies were measured to quantify the influence of the depicted chemical modifications at the primer probe 3´end on the enzyme action.

Following kinetic constants were determined using single-nucleotide incorporation experiments under single-completed-hit and steady-state conditions, as previously described¹¹¹: kcat= first order rate of catalysis; KM= Michaelis constant and kcat/KM= incorporation efficiency (see Table 3).

Table 3 Kinetic analyses of single nucleotide insertions of matched (A-T/T*) and mismatched (G- T/T*) primer termini performed by Vent (exo^-) DNA polymerase. Incorporation of dATP was observed using the modified primer probes ^2ST, ^4ST, TÔMe, ^2STÔMe and ^4STÔMe in comparison with unmodified T primer pro be.

5´-...AGGA T/T*

(primer probe, 20 nt) 3´-...T CC T N TCCA.. (template, 33nt)

matched case (A-T/T*) mismatched case (G-T/T*) primer KM

[μM]

kcat [min^-1]

kcat/KM

[min^-1μM^-1] KM

[μM]

kcat

[min^-1]

kcat/KM

[min^-1μM^-1] T 0.23±0.02 1.11±0.1 4.8 51.8±6.0 0.48±0.05 0.009

2ST 0.26±0.06 0.85±0.04 3.3 89.7±30 0.18±0.01 0.002

4ST 0.26±0.08 1.09±0.21 4.2 33.7±9.2 0.48±0.03 0.014 T^OMe 33.6±4.2 0.14±0.02 0.004 n.a. n.a. n.a.

2ST^OMe 30.2±1.3 0.21±0.02 0.007 n.a. n.a. n.a.

4ST^OMe 26.5±2.4 0.10±0.01 0.004 n.a. n.a. n.a.

T*=modified thymidine ^2ST, ^4ST, TÔMe, ^2STÔMe or ^4STÔMe N=A or G, respectively

n.a.=not accessible; no nucleotide insertions were observed when up to 8nM of DNA Polymerase, up to 1h incubation time and up to 600 μM of dATP (higher dATP concentrations caused inhibition of the reaction) were applied.

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The best discrimination properties of the non-sugar-modified primer probes were shown by ^2ST, which is coherent with the results obtained in real-time asPCR experiments (described above). Recently, similar results were obtained for the incorporation of the respective thiolated thymidine triphosphates (TTPs)¹¹². The increased selectivity of the system comprising ^2ST is mainly achieved by a decreased steady-state kcat value in the mismatched case, in comparison to that of the unmodified system. One can envision that DNA amplification from matched versus mismatched DNA complexes under PCR conditions (that is, 200 μM deoxynucleoside triphosphates (dNTPs)) very much depends on the kcat value of the proceeding nucleotide incorporation. In real-time PCR, the signal generation is dependent on the formation of double stranded DNA. Thus, the reduced steady-state kcat value of the ^2ST- comprising system in the mismatched case might well be the cause of the above- observed single-nucleotide discrimination ability in allele-specific PCR. 4’-C- modification has significant effects on the extension efficiency. The efficiency is greatly diminished due to a significantly increased KM value (about 100-fold) and an approximately 5- to 10-fold decreased kcat value. This is in line with earlier findings, although different enzymes and modifications were used⁷⁵. However, when single- completed hit conditions were used (>10-fold excess primer/template over DNA polymerase concentration), no extension of mismatched primer termini was observed.

As demonstrated by our results, these limitations can be overcome in PCRs in which standard dNTP concentrations are employed that are higher than the measured KM

values, thereby resulting in an efficient PCR when matched primer/templates are employed.

2.1.3 Conclusion

Taken together, it was shown that primer probes that bear thiolated thymidines are able to increase single-nucleotide discrimination in allele-specific PCRs. These modifications, either at the 4’-C-deoxyribose or on the nucleobase of a single- modified nucleotide at the primer terminus, do not have any significant effect on duplex stability and the conformation of the respective primer–template complex.

Therefore, the characterised discrimination properties must result from the specific interaction between the DNA polymerase, the template–primer probe duplex and the

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incoming dNTPs. Nucleobase thiolation in conjunction with 4’-C-methoxymethylene modification at the 2’-deoxyribose exhibits the most pronounced effects. These compounds are readily available and can be incorporated into DNA strands by using standard oligonucleotide chemistry. This real-time PCR system supersedes recently discovered approaches^24,32 that use unmodified nucleobases. The described system with the ^2ST^OMe primer probe can be useful for the direct diagnosis of single-nucleotide variations within genes, such as single-nucleotide polymorphisms or point mutations, directly without the need of further time- and cost-intensive post-PCR analysis.

These results were published in R. Kranaster and A. Marx, Chem. Eur. J., 2007, 13, 6115-6122.

DNA polymerase activity on solid support : from diagnostics to directed enzyme evolution

From diagnostics to directed enzyme evolution

Table of contents

1 Introduction

2 Results and discussion