Detection of single nucleotide substitutions (SNS) in DNA and RNA has a growing importance in biology and medicine. One traditional approach for recognition of SNS takes advantage of hybridization probes that bind target nucleic acids followed by measuring ?Tm, the difference in melting temperatures of matched and mismatched hybrids. The approach enables SNS differentiation at elevated temperatures (usually 40-65oC) often only in a narrow range of < 10oC and requires high-resolution melting devices. Here we demonstrate that a specially designed DNA probe (X sensor) can broaden ?Tm from ~10oC to ~16oC and distinguish SNS in the interval of ~5-40oC. Therefore, there is no need for heating or measuring Tm for accurate SNS differentiation. Our data indicate that this wide differentiation range is in part due to the non-equilibrium hybridization conditions. Further we explored the idea that it is possible to improve the performance of an X sensor operable in close to equilibrium conditions by shifting its operability to non-equilibrium conditions. One way to achieve this is to introduce as many as possible structured ligands in analyte's dissociated state. Here we show that by introducing the maximum possible conformational constraints in X probe it is possible to shift its operation to non-equilibrium conditions and to improve its selectivity at temperatures < 15oC. Thus, this work points towards a new strategy for the design of highly selective hybridization sensors which operate in non-equilibrium conditions at close to room temperature. The X sensors could be utilized in qPCR, microarrays, as well as RNA analysis in living cells and for ambient temperature point-of-care diagnostics. In the last part of this work, X sensors were used in real time detection of PCR products. The sensors were optimized to operate in PCR buffer with optimal Mg2+ concentration. They were able to detect the target amplicon together with nonspecific products. The results presented here suggest that X sensors might be adopted for real time PCR format.

Nukleinsäuretemplatkontrollierte Reaktionen, die häufig auf Ligations- oder Transferreaktionen basieren, unterliegen dem Effekt der Produktinhibierung. Dadurch können besonders niedrige Templatmengen nur schwer detektiert werden. Im Rahmen dieser Arbeit konnte erstmalig eine grundlegend neue Kategorie templatkontrollierter Reaktionen etabliert werden: templatkontrollierte Spaltungsreaktionen. Dazu wurde ein spaltbarer Linker auf N-Alkylpicoliniumbasis (NAP) entwickelt, der mit einfachen, orthogonalen Konjugationsmethoden (SPAAC, Maleimid-Thiol-Konjugation oder via Amidbinungsknüpfung) sowohl in PNA- als auch in DNA-Strukturen inkludiert werden kann. Die Templat-vermittelte Photoreduktion induziert eine C-O-Bindungsspaltung des Linkers. Daraus resultieren Produkte, die eine geringere Templataffinität besitzen als das Edukt, sodass die Reaktion keiner Produktinhibierung unterliegt. Dies konnte zum Beispiel mittels Triplex-bildender, spaltbarer PNA-Sonden realisiert werden, die eine rasche Spaltungsgeschwindigkeit aufweisen. Hierzu bindet die spaltbare PNA-Sonde auf dem Templat benachbart zu einer mit einem Ruthenium(II)-Komplex modifizierten Assistenzsonde, die die Photoreduktion lichtkontrolliert induzieren kann. In einem alternativen Ansatz wurde die Fluorophor-induzierte Photolyse von NAP-Derivaten näher untersucht und führte letztlich zur Entwicklung eines selbst-spaltenden Molecular Beacons (iMB). Dieser verhält sich wie ein konventioneller iMB, wodurch eine neue Klasse an Molecular Beacons vorgestellt werden konnte. Die templatkontrollierte Photolyse konnte nicht nur in wässrigem Milieu, sondern auch in komplexen Umgebungen wie Zellkulturmedium, Zelllysat und RNA-Extrakt durchgeführt werden. / Nucleic acid templated reactions, which are often based on ligation or transfer reactions, are limited by the phenomenon of product inhibition. As a result, the usage of catalytical amounts of target are up to date only applicable to a limited extend. In this work, a fundamentally new category of nucleic acid templated reactions could be established: nucleic acid templated cleavage reactions. For this purpose, a cleavable linker based on N-alkylpicolinium (NAP) was developed, which can be included in both PNA and DNA structures using simple, orthogonal conjugation methods (SPAAC, maleimide-thiol conjugation or via amide bond formation). A template-mediated photoreduction induces the C-O bond cleavage of the linker. The target affinity of the cleavage products is lower than the parental oligonucleotide prior to cleavage, hence providing a thermodynamical driving force for amplified nucleic acid detection. This could be realized, for example, using triplex-forming, cleavable PNA probes which have a fast cleavage rate. In a first approach various triplex-forming PNA probes were developed that would undergo a photo-reductive C-O-bond cleavage upon irradiation when placed on a template adjacent to an assistant probe equipped with a sensitizer (Ruthenium(II)-complex). In an alternative approach, the fluorophore-induced photolysis of NAP derivatives was investigated and ultimately led to the development of a self-immolative Molecular Beacon (iMB). The iMB behaves like a conventional MB, therefore a new class of Molecular Beacons was introduced. The template-controlled photolysis can be performed not only in aqueous environments, but also in various complex environments such as cell culture medium, cell lysate or RNA extract.

Detection, imaging and quantification of gene expression in living cells can provide essential information on basic biological issues and disease processes. To establish this technology, we need to develop molecular probes and cellular delivery methods to detect specific RNAs in live cells with potential for in vivo applications. In this thesis work, the major focus is placed on the development of molecular beacons and biochemical approaches (peptides etc.) to deliver such probes to different cellular compartments. These approaches are then employed to study the expression and localization of mRNAs, co-localization of mRNAs with cytoplasmic organelles and cytoskeleton, and co-localization of RNA molecules in the nuclei of living cells. Further along this direction, we were interested in developing a better understanding of the functional states of mRNAs and the fluorescent signal observed in optical imaging experiments. To acheive this goal, we altered the translational process and studied its effect on the detection of mRNAs in living cells. The results of these studies indicate that the translational state of mRNAs favors the hybridization of molecular beacon with its target sequence. This study has also provided the evidence that molecular beacons are reversibly bound to target mRNAs and the repression of the translational process can prevent molecular beacon from binding to its target mRNA. Further, using these approaches in combination with FRAP based biophysical analysis, the dynamics of endogenous RNA in living cells are studied. These studies revealed the possible subcellular organization of RNA molecules and their dynamics in living cells. The results also demonstrated the role of cytoskeleton and ATP in the mobility of specific mRNAs in the cytoplasm. In addition to optical probes, studies have been carried out to develop an MRI contrast agent using iron-oxide nanoparticles for deep tissue molecular imaging. Specifically, we have functionalized magnetic nanoparticles that are water-soluble, mono-dispersed, biocompatible, and easily adaptable for multifunctional bioconjugation of probes and ligands. We have successfully delivered magnetic nanoparticle bioconjugates into live cells and demonstrated their effect on relaxivity. We have further studied the role of coating thickness for optimization of contrast and further enhance the fundamental understanding of contrast mechanisms.