Oligonucleotide-modified nucleotides
Final Report Abstract
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.
Publications
- “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
(See online at 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
(See online at 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
(See online at 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
(See online at 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
(See online at 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
(See online at 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
(See online at 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
(See online at 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
(See online at 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
(See online at 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
(See online at 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
(See online at 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
(See online at 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
(See online at 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
(See online at 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
(See online at 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
(See online at https://doi.org/10.3390/chemistry2020029)
