Person: Baccaro, Anna
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Structures of KlenTaq DNA Polymerase caught while incorporating C5-Modified Pyrimidine and C7-Modified 7-Deazapurine Nucleoside Triphosphates
The capability of DNA polymerases to accept chemically modified nucleotides is of paramount importance for many biotechnological applications. Although these analogues are widely used, the structural basis for the acceptance of the unnatural nucleotide surrogates has been only sparsely explored. Here we present in total six crystal structures of modified 2′-deoxynucleoside-5′-O-triphosphates (dNTPs) carrying modifications at the C5 positions of pyrimidines or C7 positions of 7-deazapurines in complex with a DNA polymerase and a primer/template complex. The modified dNTPs are in positions poised for catalysis leading to incorporation. These structural data provide insight into the mechanism of incorporation and acceptance of modified dNTPs. Our results open the door for rational design of modified nucleotides, which should offer great opportunities for future applications.
Enzymatic Synthesis of Organic-Polymer-Grafted DNA
To create bioorganic hybrid materials, interdisciplinary work in the fields of chemistry, biology and materials science is conducted. DNA block copolymers are promising hybrid materials due to the combination of properties intrinsic to both the polymer and the nucleic acid blocks. Until now, the coupling of DNA and organic polymers has been exercised post-synthetically in solution or on solid support. Herein, we report the first enzyme-catalysed synthesis of DNA organic polymer chimeras. For this purpose, four novel 2 -deoxyuridine triphosphates carrying polymer-like moieties linked to the nucleobase were synthesised. Linear polyethylene glycol monomethyl ethers of different sizes (1) and branched polyamido dendrons with varying terminal groups (2) were chosen as building blocks. We investigated the ability of DNA polymerases to accept the copolymers in comparison to the natural substrate and showed, through primer extensions, polymerase chain reactions and rolling circle amplification, that these building blocks could serve as a surrogate for the natural thymidine. By this method, DNA hybrid materials with high molecular weight, modification density, and defined structure are accessible.
DNA Conjugation by Staudinger Ligation
Two 5 modified 2'-deoxyuridin triphosphates and a 7 modified 2'-deoxy-7-deazaadenosine were synthesized carrying a terminal azide linked to the base. For probing the sterical influence on incorporation and Staudinger ligation different sized flexible linkers were chosen. All three nucleotides can completely replace their natural counterparts in primer extension as well as polymerase chain reactions (PCR) using Pwo DNA polymerase. For azide labeled primer extension products subsequent conjugation of suitably functionalized phosphines via Staudinger ligation was achieved, e.g. for the conjugation of biotin as an affinity tag.
Barcoded Nucleotides
DNA as an information storage system is simple and at the same time complex owing to the various different arrangements of the four natural nucleotides.[1] The DNA synthesis by DNA polymerases is intriguing, since these enzymes are able to catalyze the elongation of the primer strand by recognizing the DNA template and selecting the corresponding nucleotide.[1b, 2] This feature can be exploited to diversify the four-base-code by substitution of the natural substrates with modified analogues.[3] Nucleotide analogues equipped with various marker groups (e.g. dyes, tags, or spin labels[4])can be employed in DNA polymerase catalyzed reactions to increase the application scope of DNA (e.g. sequencing,structural characterization, and immobilization[4d, 5]). The “information” embedded in the marker groups allow conclusions to be drawn from the evaluation of the resulting signals. A significant gain in information would result by embedding a marker that exhibits the properties of a barcode. Typically, the barcode label bears no descriptive data but it consists of a series of signs which code for the deposited information (the term was used in other contexts with DNA before).[6] For universal adoption the barcode should be simple, affixed easily, and allow a reliable assignment of the deposited information. Oligodeoxynucleotides (ODNs) meet the requirements of a barcode label to a great extent, since they have a simple code and can be distinguished by
characteristics such as self-assembly and hybridization specificity. For a simple introduction of these DNA barcode labels into DNA, an enzyme-mediated approach utilizing ODN-modified nucleotides would be desirable.[7] However, the acceptance of these modified nucleotides by DNA polymerases should be hampered by the steric demand of the ODN-modified nucleotides. Herein, we show that despite
the steric demand the enzymatic synthesis of barcoded DNA is feasible by using ODN-modified nucleoside triphosphates
that are about 40-times larger than the natural nucleotides and longer than the diameter of a DNA polymerase (Figure 1A).
Enzymatic Synthesis of Functionalized DNA
In conclusion, the synthesis of several triphosphates with different modifications at the C5 position of the nucleobase was developed. In enzyme catalysed reactions I was able to find conditions whereby functionalised nucleotides can act as surrogate for natural nucleotides. The triphosphates synthesised in the first part of this work were functionalised with polyethylene glycol monomethyl ether residues or with polyamido dendrons bearing different end groups. Highly modified DNA fragments were generated by primer extension and PCR employing the described modified nucleotides. These modified DNA fragments were used to investigate the influence of the modifications on DNA properties. Thus, I was able to establish a method to generate DNA fragments that are grafted with linear polyethylene glycol monomethyl ethers or branched polyamido dendrons leading to entities with high molecular weight, modification density and structural accuracy.
In the second part of this work, I was able to establish the synthesis of nucleotides harbouring a DNA strand (up to 38 nucleotides) at the nucleobase. These DNA building blocks were used successfully to introduce an additional DNA strand with a free 3’-OH in a nascent primer strand by primer extension. By this method highly branched DNA is feasible by enzyme-mediated reactions. I was able to find conditions to use the incorporated DNA strands as primer in primer extension or rolling circle amplification. This method was enhanced and transferred successful on DNA microarrays. With these findings the eligibility of these nucleotides as diagnostic tool was evidenced.
DNA Conjugation by the Staudinger Ligation : New Thymidine Analogues
Two novel modified 2′-deoxyuridine triphosphates carrying an azide functionality linked to the nucleobase were synthesized. For probing the sterical influence on enzymatic incorporation and Staudinger ligation, differently sized flexible linkers were chosen. Both nucleotides can completely replace natural thymidine in primer extension as well as polymerase chain reaction (PCR) using Pyrococcus woesei DNA polymerase. For PCR with larger gene fragments as template, however, the longer linker disturbs the DNA polymerase and yields less product. For azide-labeled primer extension products, subsequent conjugation of suitably functionalized phosphines via Staudinger ligation was achieved, for example for the conjugation of biotin as an affinity tag.
Site-specific DNA labelling by Staudinger ligation
Site-specific and chemoselective labeling of DNA is still a difficult task. The Staudinger ligation is a bioorthogonal reaction between azides and phosphines that requires no catalyst to proceed, allowing for mild reaction conditions. The reaction may be extended for site-specific labeling of DNA using azidomodified triphosphates, which can be incorporated site-specifically into DNA strands by DNA polymerases in a template-dependent manner. The azido-modified DNA, in turn, can be labeled by suitable phosphines. This protocol describes (1) the synthesis of an azido-TTP analogue; (2) the enzymatic synthesis of azido-modified DNA; (3) the synthesis of suitable phosphine labels; and (4) the labeling of azido-DNA with biotin–phosphine by Staudinger ligation with approximately 70% conversion.
Structural basis for the synthesis of nucleobase modified DNA by Thermus aquaticus DNA polymerase
Numerous 2'-deoxynucleoside triphosphates (dNTPs) that are functionalized with spacious modifications such as dyes and affinity tags like biotin are substrates for DNA polymerases. They are widely employed in many cutting-edge technologies like advanced DNA sequencing approaches, microarrays, and single molecule techniques. Modifications attached to the nucleobase are accepted by many DNA polymerases, and thus, dNTPs bearing nucleobase modifications are predominantly employed. When pyrimidines are used the modifications are almost exclusively at the C5 position to avoid disturbing of Watson Crick base pairing ability. However, the detailed molecular mechanism by which C5 modifications are processed by a DNA polymerase is poorly understood. Here, we present the first crystal structures of a DNA polymerase from Thermus aquaticus processing two C5 modified substrates that are accepted by the enzyme with different efficiencies. The structures were obtained as ternary complex of the enzyme bound to primer/template duplex with the respective modified dNTP in position poised for catalysis leading to incorporation. Thus, the study provides insights into the incorporation mechanism of the modified nucleotides elucidating how bulky modifications are accepted by the enzyme. The structures show a varied degree of perturbation of the enzyme substrate complexes depending on the nature of the modifications suggesting design principles for future developments of modified substrates for DNA polymerases.