Oligonukleotid-modifizierte Nukleotide
Zusammenfassung der Projektergebnisse
Nucleic acid-based diagnostics span a wide field reaching from the detection of pathogenderived nucleic acids (such as viruses) to the analysis of single nucleotide variations in the entire genome such as point mutations and single nucleotide polymorphisms (SNPs). For most analytical approach are based on the polymerase chain reaction (PCR) and require sophisticated equipment. The aim of this project was to further develop methods that allows the detection of a nucleic acid target by the naked eye with single nucleotide precision without requiring PCR-based technology. Such a system would be highly useful for pointof-care testing or the detection of pathogens in the field. DNA polymerase based reactions hold great potential in this regard, since they are catalyzing nucleotide incorporation in a template-dependent fashion with high sequence selectivity. We discovered that nucleotides that are modified with large functional entities for signal generation such as DNA constructs with enzymatic activity (i.e., DNAzymes) or proteins (i.e., horse radish peroxidase, antibodies) are processed by DNA polymerases and sequence selectively incorporated into a growing DNA strand. Thereby the functional entities were connected to solid supports and resulting in sequence-selective signal generation that is detectable by naked eye. These studies were supported by structural investigations on the mechanism of how such large modifications are accepted by DNA polymerases. We also increased the sensitivity of the systems by exploiting loop-mediated isothermal amplification (LAMP) and antibody-based detection systems. Furthermore, we thoroughly investigated the incorporation of the modified nucleotides by DNA polymerases by functional and structural means. This resulted in a broader understanding of the mechanisms by which these enzymes process nucleotides that are modified with entities being up to several times larger than the diameter of the DNA polymerases itself. On the other hand, new insights into the complex mechanisms by which DNA polymerases function were obtained due to structural investigations of relevant DNA polymerases that have not been crystallized before with bound modified substrates.
Projektbezogene Publikationen (Auswahl)
- “Snapshot of a DNA polymerase while incorporating two consecutive C5-modified nucleotides”, J. Am. Chem. Soc. 2013, 135, 15667-9
S. Obeid, H. Bußkamp, W. Welte, K. Diederichs, A. Marx
(Siehe online unter https://doi.org/10.1021/ja405346s) - “Structural insights into DNA replication without hydrogen bonds“ J. Am. Chem. Soc. 2013, 135, 18637-43
K. Betz, D. A. Malyshev, T. Lavergne, W. Welte, K. Diederichs, F. E. Romesberg, A. Marx
(Siehe online unter https://doi.org/10.1021/ja409609j) - “Structures of KOD and 9°N Polymerases Complexed to Primer Template Duplex” ChemBioChem 2013, 14, 1058-62
K. Bergen, K. Betz, W. Welte, K. Diederichs, A. Marx
(Siehe online unter https://doi.org/10.1002/cbic.201300175) - “DNA polymerase-catalyzed incorporation of nucleotides modified with a G-quadruplex-derived DNAzyme” Chem. Commun. 2015, 51, 7379-81
D. Verga, M. Welter, A.L. Steck, A. Marx
(Siehe online unter https://doi.org/10.1039/c5cc01387a) - “Sequence selective naked-eye detection of DBA harnessing extension of oligonucleotide-modified nucleotides” Bioorg. Med. Chem. Lett. 2016, 26, 841-4
D. Verga, M. Welter, A. Marx
(Siehe online unter https://doi.org/10.1016/j.bmcl.2015.12.082) - “Sequence-specific Incorporation of Enzyme-Nucleotide Chimera by DNA Polymerases” Angew. Chem. Int. Ed. 2016, 55, 10131-5
M. Welter, D. Verga, A. Marx
(Siehe online unter https://doi.org/10.1002/anie.201604641) - “Structural Insights into the Processing of Nucleobase-Modified Nucleotides by DNA Polymerases” Acc. Chem. Res. 2016, 49, 418-427
A. Hottin, A. Marx
(Siehe online unter https://doi.org/10.1021/acs.accounts.5b00544) - “Crystal structures of ternary complexes of archael B-family DNA polymerases” Plos ONE 2017, 12, e0188005
H.M. Kropp, K. Betz, J. Wirth, K. Diederichs, A. Marx
(Siehe online unter https://doi.org/10.1371/journal.pone.0188005) - “Structural basis for expansion of the genetic alphabet by an artificial base pair” Angew. Chem. Int. Ed. 2017, 56, 12000-12003
K. Betz, M. Kimoto, K. Diederichs, I. Hirao, A. Marx
(Siehe online unter https://doi.org/10.1002/anie.201704190) - “Structural basis for the KlenTaq DNA Polymerase catalyzed Incorporation of Alkene- versus Alkyne-modified Nucleotides” Chem. – Eur. J. 2017, 23, 2109-18
A. Hottin, K. Betz, K. Diederichs, A. Marx
(Siehe online unter https://doi.org/10.1002/chem.201604515) - “Structural basis for the selective incorporation of an artificial nucleotide opposite a DNA adduct by a DNA polymerase” Chem. Commun. 2017, 53, 12704-7
K. Betz, A. Nilforoushan, L.A. Wyss, K. Diederichs, S.J. Sturla, A. Marx
(Siehe online unter https://doi.org/10.1039/c7cc07173f) - “Antibody–nucleotide conjugate as a substrate for DNA polymerases” Chem. Sci. 2018, 9, 7122-7125
J. Balintova, M. Welter, A. Marx
(Siehe online unter https://doi.org/10.1039/c8sc01839a) - “Preparation and Application of Enzyme-Nucleotide Conjugates” Curr. Protoc. Chem. Biol. 2018, 10, 49-71
M. Welter, A. Marx
(Siehe online unter https://doi.org/10.1002/cpch.36) - “Snapshots of a modified nucleotide “moving” through the confines of a DNA polymerase” Proc. Natl. Acad. Sci. USA 2018, 115, 9992-9997
H.M. Kropp, S.L. Dürr, C. Peter, K. Diederichs, A. Marx
(Siehe online unter https://doi.org/10.1073/pnas.1811518115) - “Structure of an archaeal B-family DNA polymerase in complex with a chemically modified nucleotide” Angew. Chem. Int. Ed. 2019, 58, 5457-61
H. M. Kropp, K. Diederichs, A. Marx
(Siehe online unter https://doi.org/10.1002/anie.201900315) - “The structural basis for processing of unnatural base pairs by DNA polymerases” Chem. Eur. J. 2020, 26, 3446-3463
A. Marx, K. Betz
(Siehe online unter https://doi.org/10.1002/chem.201903525) - „Combining the Sensitivity of LAMP and Simplicity of Primer Extension via a DNA-Modified Nucleotide” Chemistry 2020, 2, 490-8
M. Welter, A. Marx
(Siehe online unter https://doi.org/10.3390/chemistry2020029)
