<p>Antisense RNAs regulate gene expression in many bacterial systems. The best characterized examples are from prokaryotic accessory elements such as phages, plasmids and transposons. Many of these antisense RNAs have been identified as plasmid copy number regulators where they regulate the replication frequency of the plasmid by negative feedback. Instability and fast binding kinetics is crucial for the regulatory efficiency of these antisense RNAs. </p><p>In this thesis, the interaction of the cis-encoded antisense RNA CopA with its target CopT was studied in detail using <i>in vivo</i> reporter gene fusion expression and different <i>in vitro </i>methods, such as surface plasmon resonance, fluorescence resonance energy transfer, and gel-shift assays.</p><p>Formation of inhibitory complexes differs from simple hybridization reactions between complementary strands. E.g., the binding pathway of CopA and CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts, resulting in a helix nucleus of two or three base pairs. This is followed by rapid unidirectional helix progression into the upper stems, resulting in a four-way helical junction structure. It had been suggested that the loop of CopT carries a putative U-turn, a structure first found in tRNA anticodon loops. We showed that this putative U-turn is one of the structural elements of CopA/CopT required to achieve fast binding kinetics. Furthermore, the hypothetical U-turn structure determines the direction of helix progression when the kissing complex progresses to a four-way helical junction structure. Another structural element in CopT is the helical stem adjacent to the recognition loop. This stem is important to present the recognition loop appropriately to provide a scaffold for the U-turn.</p><p>Furthermore, the role of protein Hfq in the interaction of antisense/target RNA was investigated, since several trans-encoded antisense RNAs had been shown to need this protein to exert their function. In contrast, studies of two cis-encoded antisense RNA systems showed that these antisense RNAs do not rely on Hfq for activity. In this study it was also shown that MicF, a trans-encoded antisense RNA which is dependent on Hfq, is greatly stabilized by this protein.</p>

Antisense RNAs regulate gene expression in many bacterial systems. The best characterized examples are from prokaryotic accessory elements such as phages, plasmids and transposons. Many of these antisense RNAs have been identified as plasmid copy number regulators where they regulate the replication frequency of the plasmid by negative feedback. Instability and fast binding kinetics is crucial for the regulatory efficiency of these antisense RNAs. In this thesis, the interaction of the cis-encoded antisense RNA CopA with its target CopT was studied in detail using in vivo reporter gene fusion expression and different in vitro methods, such as surface plasmon resonance, fluorescence resonance energy transfer, and gel-shift assays. Formation of inhibitory complexes differs from simple hybridization reactions between complementary strands. E.g., the binding pathway of CopA and CopT proceeds through a hierarchical order of steps. It initiates by reversible loop-loop contacts, resulting in a helix nucleus of two or three base pairs. This is followed by rapid unidirectional helix progression into the upper stems, resulting in a four-way helical junction structure. It had been suggested that the loop of CopT carries a putative U-turn, a structure first found in tRNA anticodon loops. We showed that this putative U-turn is one of the structural elements of CopA/CopT required to achieve fast binding kinetics. Furthermore, the hypothetical U-turn structure determines the direction of helix progression when the kissing complex progresses to a four-way helical junction structure. Another structural element in CopT is the helical stem adjacent to the recognition loop. This stem is important to present the recognition loop appropriately to provide a scaffold for the U-turn. Furthermore, the role of protein Hfq in the interaction of antisense/target RNA was investigated, since several trans-encoded antisense RNAs had been shown to need this protein to exert their function. In contrast, studies of two cis-encoded antisense RNA systems showed that these antisense RNAs do not rely on Hfq for activity. In this study it was also shown that MicF, a trans-encoded antisense RNA which is dependent on Hfq, is greatly stabilized by this protein.

Hybridization of two or more DNA or RNA strands is well documented for the process taking place with all strands free in solution or when one strand is immobilized on a substrate. This study contributes to the investigation of the hybridization process when two single DNA strands (ssDNA) are in close proximity. We took advantage of an X sensor in which hybridization of four DNA strands enables the formation of a DNA four-way junction (crossover or X) structure. We immobilized multiple layers of crossover structures to study its hybridization being triggered by short ssDNA coming from solution and further investigate how many layers of these structures can hybridize by the addition of only one ssDNA (called input). Using a molecular beacon as reporter, we combined crossover DNA strands that recognize the reporter sequence at one side and at the other, the sequence of its input or downward crossover layer. Fluorescent signal was detected by separation of the molecular beacon’s fluorophore and quencher, as it hybridizes with the system of layers. Immobilization of the X structures into the scaffold proved to increase their communication, in comparison to being free in solution. This evidence gives us significant information for the communication of hybridized layers in a localized system, showing a promising standard for development of multilayered logic gates. The potential of these crossover DNA strands using X structure include applications in the future of biological systems, nanotechnology, and target DNA recognition for its ability to quickly recognize a signal and propagate it through extended DNA nanostructure in a controlled manner.