Many classes of non-coding RNAs (ncRNAs; including Y RNAs, vault RNAs, RNase P RNAs, and MRP RNAs, as well as a novel class recently discovered in Dictyostelium discoideum) can be characterized by a pattern of short but well-conserved sequence elements that are separated by poorly conserved regions of sometimes highly variable lengths. Local alignment algorithms such as BLAST are therefore ill-suited for the discovery of new homologs of such ncRNAs in genomic sequences. The Fragrep tool instead implements an efficient algorithm for detecting the pattern fragments that occur in a given order. For each pattern fragment, the mismatch tolerance and bounds on the length of the intervening sequences can be specified separately. Furthermore, matches can be ranked by a statistically well-motivated scoring scheme.

In situ hybridization is used for detection of RNA expression when conservation of tissue architecture is important. Most in situ hybridization protocols are written for tissues from animals (i.e., rat) which can be harvested and preserved rapidly. In contrast, human tissue is more difficult to obtain, hence in situ hybridization experiments must frequently be performed with less than optimal tissue preservation. This procedure details hybridization of a radiolabeled single-stranded RNA probe (riboprobe) to complementary sequences of cellular RNA in human tissue sections. This method enables detection of rare mRNA species in specific cell types of human tissue, offering distinct advantages over other in situ methods due to increased sensitivity. In particular, we have found that UV cross-linking and ribonuclease treatment protocols need to be altered for human tissues to ensure successful results, making this protocol unique to those previously described. In situ hybridization experiments can be performed using either DNA or RNA probes. RNA probes are advantageous since they form stable hybrids, are single-stranded, have little or no reannealing during hybridization, and can be synthesized to high specific activity. RNA probes can be readily created utilizing SP6, T3, or T7 promoters in both sense and antisense orientations to provide non-specific (control) and specific probes. Disadvantages of RNA riboprobes include a tendency for RNA to stick non- selectively more than DNA, and degradation by RNase (hence strict adherence to RNase-free precautions is mandatory during most of the protocol). The following protocol includes: (1) preparation of human tissues (tissue fixation and sectioning are highlighted as critical for probe penetration, preservation of tissue architecture, retention of tissue RNA, and overall success); (2) generation of radiolabeled riboprobes (total incorporation of radionucleotide is important to increase sensitivity; 35S was chosen as a compromise between excellent sensitivity, cellular resolution, and required exposure times (compared with 32p or 3H); non-isotopic methods have not been tested in a side-by-side comparison with 35S in human tissues by us, but theoretically might offer faster exposure times while maintaining high resolution); (3) hybridization conditions (stringency, temperature, washes, tissue dehydration); and (4) sample visualization (application of photographic emulsion, developing, fixing, staining, and counterstaining of individual slides).

Wir haben DNA-basierte Hybridisierungssonden zur erzwungenen Interkalation (sogenannte FIT-Sonden) entwickelt. Diese beruhen auf dem Ersatz einer natürlichen Nukleobase durch einen umgebungssensitiven Farbstoff. Die einzelsträngigen Sonden zeigen ein geringes Fluoreszenzsignal, da die Rotation um die zentrale Methinbrücke den angeregten Zustand effizient entvölkert. Durch Hybridisierung mit komplementären Zielsequenzen wird der Farbstoff in den Basenstapel gezwungen, wodurch die Rotation eingeschränkt wird und verstärkt Fluoreszenz auftritt. Im Rahmen dieser Arbeit wurden die Sonden optimiert, um maximale Fluoreszenzanstiege und maximale Helligkeit zu erzielen. Wir konnten zeigen, dass der Einbau von Locked Nucleic Acid (LNA) gleichzeitigt die Helligkeit und die Stabilität gegenüber Nukleaseverdau vergrößert. Solche LNA-verstärkten FIT-Sonden konnten in Zusammenarbeit mit der Arbeitsgruppe von Anne Ephrussi (EMBL-Heidelberg) wurden solche Sonden erfolgreich in der Lebendzell-Bildgebung von Oskar mRNA in Oozyten von Drosophila Melanogaster eingesetzt. Die kombinierte Verwendung von Thiazolorange und einem zweiten, terminal angebrachten Cy7-Reporter ermöglichte durch Ratiometrische Messungen die Quantifizierung von Sonden und Ziel RNA durch waschfreien Fluoreszenz in-situ Hybridisierung. Wir konnten die Farbauswahl für FIT-Sonden erweitern und etablierten Chinolinblau als den höchst-responsiven Farbstoff in DNA-FIT-Sonden, welcher bis zu 195-fache Fluoreszenzintensivierung zeigte. Die Verwendung von cyan- (Thiazolgelb), grün- (Thiazolorange) und rot-emittierende (Chinolinblau) FIT-Sonden ermöglichte die simultane Detektion drei verschiedener RNA-Sequenzen. Dieselben Farbkanäle wurden ebenfalls zur Entwicklung von Wiederholungseinheiten für FIT-Sonden verwendet. Durch die Expression transgener RNA und die benachbarter Hybridisierung zahlreicher Sonden sollten die Mehrfarbdetektion in lebenden Zellen mit sehr geringer Nachweisgrenze gelingen. / We developed oligonucleotide hybridization probes based on forced intercalation (FIT). FIT probes contain asymmetric cyanine dyes of the thiazole orange family as nucleobase surrogates as an internal label. The thiazole orange is dark in the unbound, single stranded state, due to rapid depletion of the chromophores excited state by torsional twisting. Upon hybridization with target nucleic acids, the reporter is forced into the base stack, resulting in restriction of intramolecular rotation which is accompanied by a dramatic increase in quantum yield. The probes have been optimized for highest fluorescence response and maximum brightness upon hybridization with complementary nucleic acids. We discovered that locked nucleic acids (LNA) increase both, the nuclease resistance in living cells and the brightness. FIT-probes were applied in real-time PCR and RNA localization studies in fixed tissue and in living cells. Together with our collaborator Anne Ephrussi we demonstrated that carefully chosen probe sequences disrupted RNA secondary structure and thereby altered the motility of oskar ribonucleotide-particles in living oocytes of Drosophila melanogaster. The combined use of fluorogenic base surrogates and a second, terminally attached, independent reporter allowed the rapid intracellular RNA-quantification by means of wash-free fluorescence in-situ hybridization. We expanded the color repertoire of FIT probes by screening multiple chromophores and discovered quinoline blue as the most responsive chromophore with up to 195-fold fluorescence enhancement. The combined use of cyan- (thiazole yellow), green- (thiazole orange) and red-emissive (quinoline blue) FIT probes allowed the simultaneous detection of multiple RNAs of interest. The same color channels could be used for the development of artificial repeat tags for FIT-probes that will enable live-cell multiplexing by FIT-probes with transgenic RNAs.

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.

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.

Die Fähigkeit Nukleinsäuren in lebenden Organismen nachzuweisen und zu visualisieren, ist entscheidend für das Verständnis zellulärer Prozesse. Die Forschungsgruppe von Prof. Dr. Oliver Seitz hat zu diesem Zweck fluorogene FIT-Hybridisierungssonden entwickelt, die die besondere Eigenschaft der Cyaninfarbstoffe Thiazolorange und Chinolinblau nutzen, stärker zu fluoreszieren, wenn sie in die beengte Umgebung eines Nukleinsäureduplex aus Sonde und spezifischer Zielsequenz eingebracht werden. Obwohl FIT-Sonden eine gute Fluoreszenzverstärkung und Spezifität aufweisen, wäre eine weitere Verbesserung ihrer Helligkeit und des Signal-Hintergrund-Verhältnisses wünschenswert. Um dies zu erreichen, wurde in dieser Arbeit ein Ansatz untersucht, bei dem FIT-Sonden mit zwei Fluorophoren desselben Typs ausgestattet werden (FIT2-Strategie). Dies sollte sowohl die Helligkeit der Sonde erhöhen, als auch die Fluoreszenz im Einzelstrang und bei Hybridisierung mit fehlgepaarter RNA durch eine Mischung aus kontaktvermittelter Fluo-reszenzlöschung und strahlungsfreiem Energietransfer verringern. Verschiedene Sonden-längen, Farbstoffabstände und -positionen wurden untersucht und es konnte bestätigt werden, dass FIT2-Sonden eine höhere Extinktionskoeffizienten, größere Fluoreszenzver-stärkung und eine bessere Selektivität aufweisen als einfach markierte Sonden. Außerdem behalten sie ihre Fähigkeit zur Unterscheidung von Match- und Mismatch-Zielen in visko-sem Zelllysat besser bei. Zudem konnte gezeigt werden, dass das FIT2-Konzept durch Hinzufügen eines hybridisie-rungsunempfindlichen Cyanin 7 Farbstoffs zu den Sonden dahingehend erweitert werden kann, dass eine ratiometrische Detektion der hybridisierten Sonde möglich ist und Hellig-keitsunterschiede aufgrund von lokalen Schwankungen der Sondenkonzentration bei der Bildgebung lebender Zellen korrigiert werden können. Mit diesen qFIT2-Sonden konnten Jurkat und CCRF-CEM T-Zellen in einem Mikroskopie-basierten Experiment unterschieden werden. / The ability to detect and visualize nucleic acids in living organisms is crucial for under-standing cellular processes. For this purpose, the research group of Prof. Dr. Oliver Seitz has introduced fluorogenic forced intercalation (FIT) hybridization probes, which exploit the unique property of the cyanine dyes thiazole orange and quinoline blue to exhibit increased fluorescence when placed in the constrained environment of a nucleic acid du-plex formed between probe and specific target sequence. Although FIT probes demon-strate solid fluorescence enhancement and specificity, further improvement of their abso-lute brightness and signal-to-background ratio would be desirable. To achieve this, the present thesis investigated an approach that equips FIT probes with two identical fluorophores (FIT2 strategy). This should on the one hand increase probe brightness, while simultaneously reducing fluorescence in the single strand and when hy-bridized to mismatched RNA, through a combination of contact-mediated quenching and non-radiant energy transfer. Various probe lengths, dye-dye distances and positions were screened, and it could be confirmed that FIT2 probes have higher extinction coefficients, greater fluorescence enhancement and better selectivity than their mono-dye counter-parts. Moreover, they better retain their ability to discriminate match and mismatch tar-gets in viscous cell lysate. Finally, it was demonstrated that the FIT2 concept can be extended by adding a hybridiza-tion-insensitive Cyanine 7 dye to the probes, allowing ratiometric detection of hybridized probe and correction of brightness differences due to local fluctuations in probe concen-tration during live-cell imaging. Using these qFIT2 probes, Jurkat and CCRF-CEM T-cells could be distinguished in a microscopy-based experiment.