| dc.contributor.advisor | Pena, Vladimir Dr. | |
| dc.contributor.author | Mieczkowski, Mateusz | |
| dc.date.accessioned | 2021-06-30T09:24:02Z | |
| dc.date.available | 2022-03-24T00:50:16Z | |
| dc.date.issued | 2021-06-30 | |
| dc.identifier.uri | http://hdl.handle.net/21.11130/00-1735-0000-0008-5885-F | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-8548 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 572 | de |
| dc.title | Structural investigation of functional nucleic acids | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Tittmann, Kai Prof. Dr. | |
| dc.date.examination | 2021-03-26 | |
| dc.description.abstracteng | DNA enzymes, also known as deoxyribozymes, are synthetic single-stranded DNA molecules able to catalyze chemical reactions. There are two main reasons for studying deoxyribozymes: their practical value in various applications, and the understanding of basic properties - such as folding and catalysis - of a biopolymer that is of central importance for life. Compared to ribozymes, the DNA enzymes have a potential value as tools for industrial or therapeutic applications, owing to more cost-effective synthesis and higher stability. The first crystal structure of a deoxyribozyme demonstrated that DNA possesses the intrinsic ability to adopt complex tertiary folds that support catalysis and unveiled the active site of a DNA enzyme in the post-catalytic state (Ponce-Salvatierra, Wawrzyniak-Turek et al. 2016). The second reported crystal structure of the RNA-cleaving deoxyribozyme complements observations about the folds and catalysis of DNA enzymes although the structure was derived with DNA as a substrate mimic of RNA (Liu, Yu et al. 2017). These crystal structures represent a breakthrough in the field, but they are still insufficient to derive a clear mechanistic picture of the specific features of different RNA ligating and RNA cleaving deoxyribozymes. Therefore, ongoing efforts are devoted to structurally investigating additional deoxyribozymes. The new DNA enzymes were evolved to discriminate modified and unmodified RNA substrates and provide attractive tools for studying the natural epitranscriptomic RNA modification N6-methyladenosine (Sednev, Mykhailiuk et al. 2018). In the present study, the goal is to elucidate the structural basis for recognition of the methylated nucleobase by solving the crystal structure of the m6A sensitive RNA-cleaving deoxyribozyme in complex with an uncleavable analog of the RNA substrate, containing either methylated and unmethylated adenosine. Surprisingly, the RNA substrate dissociated from the deoxyribozyme during the crystallization process. Two structures for unmethylated and one of the methylated RNA substrate analog were solved. The next goal is to elucidate the crystal structure of the RNA-ligating deoxyribozyme in the pre-catalytic state of reaction. The previously reported crystal structure of the 9DB1 in the post-catalytic state of reaction could not explain the role of magnesium cations as cofactors for accelerating RNA ligation and properly describe the ligation mechanism. The structural investigation of the 9DB1 in the pre-catalytic state resulted in the ligation of the two RNA substrates during the crystallization process. In the future, other strategies are necessary to solve the questions on substrate recognition and catalytic mechanism of the RNA-cleaving and RNA-ligating deoxyribozymes investigated in this study.
The second project deals with synthetic RNA aptamers that were identified by in vitro selection to mimic fluorescent proteins for RNA imaging and the development of biosensors. Several examples of fluorogen-activating RNA aptamers are known, and for some, the crystal structures have recently been solved e.g. of the Spinach, Mango, and Corn aptamers, that bind synthetic analogs of the GFP chromophore (Neubacher and Hennig 2019). The Chili is a new fluorogenic-RNA aptamer that mimics large Stokes shift (LSS) fluorescent proteins (FPs) by inducing highly Stokes‐shifted emission from several new green and red HBI (4-hydroxybenzylidene imidazolinone) derivatives that are non‐fluorescent when free in solution (Steinmetzger, Palanisamy et al. 2019). The new fluorophores are the first variants of fluorogenic aptamer ligands with permanently cationic sidechains that are bound by the RNA in their protonated phenol form, while emission occurs from the phenolate intermediate after excited-state proton transfer. The Chili–DMHBO+ complex is the longest wavelength-emitting (592 nm) and tightest binding (KD=12 nM) RNA fluorophore currently known in the growing family of HBI-binding aptamers. By employing X-ray crystallography, I have elucidated the three-dimensional structure of the Chili fluorophore binding site and revealed the structural basis for the large apparent Stokes shift and the promiscuity of the Chili aptamer to activate red and green-emitting chromophores | de |
| dc.contributor.coReferee | Höbartner, Claudia Prof. Dr. | |
| dc.contributor.thirdReferee | Urlaub, Henning Prof. Dr. | |
| dc.contributor.thirdReferee | Faesen, Alexis Caspar Dr. | |
| dc.contributor.thirdReferee | Bohnsack, Markus Prof. Dr. | |
| dc.subject.eng | Structural biology | de |
| dc.subject.eng | Nucleic acid structural biology | de |
| dc.subject.eng | DNA enzymes | de |
| dc.subject.eng | Fluorogenic aptamers | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-21.11130/00-1735-0000-0008-5885-F-4 | |
| dc.affiliation.institute | Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.description.embargoed | 2022-03-24 | |
| dc.identifier.ppn | 1761688154 | |