Fabian Czerwinski, University of Copenhagen - Kopenhagen
Viele Stoffe, die auf sehr, sehr kleinen komplexen Nanopartikeln basieren, finden bereits kommerzielle Anwendung, so beispielsweise Zinkoxide in Sonnencreme. Dennoch ist sehr wenig bekannt über deren Einfluss auf die physikalischen Eigenschaften von Nukleidsäuren. Diese sind Träger des menschlichen Erbguts und unersetzlich für eine Reihe zellulärer Prozesse. Kleinste Veränderungen aufgrund von Wechselwirkungen zwischen Erbgut und Nanostrukturen können also große und potentiell giftige Konsequenzen für Organismen hervorrufen.
Da wenig bekannt ist über diese direkten Wechselwirkungen, sollen einzelne Nukleidsäuren in einer experimentellen Apparatur untersucht werden, wenn ihre Eigenschaften durch die Anwesenheit von bestimmten Nanopartikeln verändert wird. Diese hochpräzisen Experimente werden mit sogenannten optischen Pinzetten durchgeführt und durch komplementäre Techniken aus der Biochemie ergänzt. Die Ergebnisse werden im Anschluss mit Hilfe theoretischer Modelle analysiert, um möglichst generelle Beschreibungen für die untersuchten Wechselwirkungen herausarbeiten zu können.
Abstract of PhD Projekt
Nanoparticles with dimensions less than 100 nanometers are typically engineered, but also occur naturally. Physical and chemical properties of nanoparticles are very different from larger materials with the same composition. This results e. g. in exceptional conductivity, reactivity, and optical sensitivity of nanoparticles. While nanostructures hold enormous potential for commercial purposes, very little is known about their potential hazardous effects to human health and the environment. In this project we propose to use optical tweezers, confocal microscopy, advanced image analysis, in combination with genetically engineered nucleic-acid structures to probe the effect of selected nano-particles on individual nucleic acids.
The project can be divided into two parts: The first part focuses on structural changes of DNA induced by the fullerene C60 and the resulting toxic implications. Fullerenes bind into the minor groove of DNA thereby inhibiting the crucial processes of DNA replication and transcription.
The second part concentrates on naturally occurring mRNA structures, namely pseudoknots which de-termine the coding during translation, and the blocked dynamics of these structures by psoralens. Psoralens mimic base pairs. They also stabilize tertiary RNA structures whose mechanical strength is correlated to their translation into proteins. All these effects can result in toxic reactions to the host.
Detailed Description of PhD Project
Nanoparticles with dimensions less than 100 nanometers are typically engineered, but also occur naturally. Physical and chemical properties of nanoparticles are very different from larger materials with the same composition. This results e.g. in exceptional conductivity, reactivity, and optical sensitivity of nanoparticles. While nanostructures hold enormous potential for commercial purposes, very little is known about their potential hazardous effects to human health and the environment.
In this project we propose to use optical tweezers, confocal microscopy, advanced image analysis, in combination with genetically engineered nucleic-acid structures to probe the effect of selected nanoparticles on individual nucleic acids.
The project can be divided into two parts: The first part focuses on structural changes of DNA induced by the fullerene C60 and the resulting toxic implications. The second part concentrates on naturally occurring mRNA structures, namely pseudoknots which determine the coding during translation, and the blocked dynamics of these structures by psoralens.
Methods
The physical properties of nucleic-acid structures are measured as schematically shown in the figure: An optically trapped bead is mounted on a micropipette. Another bead, incubated with the nucleic-acid structure, is trapped and brought into vicinity of the first bead, thereby allowing specific attachment between the tether and the first bead.
Using the optical tweezers we obtain force-extension relations with sub-piconewton and nanometer resolution, thereby measuring the possible physical changes induced by the nanoparticles. Data analysis will include the methods presented in (PRL 95:158102, 2005).
Experiments are carried out inside a microfluidic device. This allows for precise and controlled setting of physiological conditions, defined concentrations of nanoparticles, etc.
To investigate how nano-particles influence torsional properties of nucleic acids probably magnetic tweezers will be used. Through his former research Fabian Czerwinski is very familiar with this technique and has contacts to research groups in this field. Therefore he will establish a fruitful collaboration pointing at this question.
Interaction between nanoparticles and DNA
The first type of nanoparticles which we will investigate is the fullerenes, which are fascinating symmetric carbon nanostructures, with interesting physical and chemical properties. Research until now has focused on possible applications of fullerenes with only a few investigations addressing the potential biological hazards. It has been theoretically predicted (BiophysJ 89:3856, 2005), that C60 can function as a minor-groove binder to double-stranded DNA and alter its elastic properties significantly. Thus potentially, it is able to cause severe damage inside living organisms by preventing enzymes from proper functioning or by leading to inaccessible regions for DNA repair mechanisms. Indeed, the fullerene C60 has been shown to cause severe brain damage in fish (Environ. Health Perspec. 112:1058, 2004).
We will perform optical studies of the physical properties of DNA in the presence and absence of fullerenes as described above. Evaluating the findings, we will presumably extend the studies using other nanoparticles such as ZnO –used e.g. in commercial sunscreens– which are believed to function in a comparable manner.
Vital life processes as e.g. DNA replication and transcription, involve the traffic of molecular machines along nucleic acids. These processes exert torque and consequently bend DNA. It is therefore likely that bound nanoparticles or induced DNA stiffening will hinder the normal traffic of molecular motors and decrease their efficiency and reliability. Additionally, the presence of nanoparticles leads to DNA regions which are not accessible for repair mechanisms at all. Obtaining quantitative information of how nanoparticles interact with DNA is a crucial step towards deriving the possible toxic impact. If the investigations show significantly altered physical properties of DNA, the studies will be extended to investigations of the traffic of RNA polymerase along a DNA in the presence of nanoparticles. This will be done both in vitro at the single molecule level and in vivo on the ensemble level, with the goal of shedding light onto the question of whether the altered elastic properties of DNA or the physical presence of nanoparticles affect the enzymatic activity.
Stabilizing viral mRNA by psoralens
In addition to studying how unfolded nucleic structures interact with invading nanoparticles we will study the interactions between nanoparticles and folded nucleic structures.
Viral mRNA contains a wealth of three-dimensional structures called pseudoknots. These pseudoknots are used for regulatory purposes while the virus is exploiting the translation system of the hosting cell. Upon encounter with a pseudoknot with some probability the ribosome will shift reading frame, and hence produce a different protein. How the degree of frameshifting is related to the mechanical strength of the mRNA pseudoknot has previously been shown at NBI (PNAS 104:5830, 2007). In this project we wish to take the system one step further, to see how nanoparticles influence the stability of tertiary RNA structures using available pseudoknots derived from the Infectious Bronchitis Virus. Psoralens are nanocompounds that intercalate into DNA and RNA. They serve as a cross-linking reagent. Upon radiation with UV light it psoralens can forms a covalent link between pyrimindine residues on complementary strands aligned in a particular helical geometry. Covalent linking can also be achieved between bases on closely juxtaposed single strands in macromolecular assemblies. Thus, we expect psoralens to have a stabilizing effect on the pseudoknots. Using the optical tweezers as described above enables us to investigate the mechanical stability of the pseudoknots upon induced stabilization.
Psoralens terminate translation, but we will also investigate psoralen-like compounds, aiming for discovering those that only partially terminate translation. In addition, we will study the correlation between mechanical stability and frameshifting with the long-term purpose of intervening into the regulatory pathway of viruses. If time permits, we would also like to genetically construct pseudoknots with the goal of investigating how specific base pairings within the pseudoknot affect both frameshifting and mechanical stability.
Non-equilibrium thermodynamics
If a nucleic structure is mechanically unfolded by applying a directed force, the unfolding is a non-equilibrium process, rendering the corresponding data analysis non-trivial. To this end, we will employ recently developed non-equilibrium thermodynamics such as the theorems of Jarzynski (PRL 78:2690, 1997) and Crooks (PRE 60:2721, 1999). However, these theories assume structures which occupy only two states (typically folded or unfolded). We wish to extend these theories to nucleic-acids structures containing intermediate steps in the folding pathway.
References
(PNAS 104:5830, 2007) Hansen, T. M., Reihani, S. N. S., Oddershede, L. B. and Sørensen, M. A.: Correlation between mechanical strength of messenger RNA pseudoknots and ribosomal frameshifting. PNAS, 104, 5830-5835, April 2007.
(PRL 95:158102, 2005) Vladescu, I. D., McCauley, M. J., Rouzina, I. and Williams, M. C.: Mapping the Phase Diagram of Single DNA Molecule Force-Induced Melting in the Presence of Ethidium. Physical Review Letters, 95, 158102, October 2005.
(BiophysJ 89:3856, 2005) Zhao, X., Striolo, A. and Cummings, P. T.: C60 Binds to and Deforms Nucleotides. Biophysical Journal, 89, 3856–3862, December 2005.
(Environ. Health Perspec. 112:1058, 2004) Oberdörster, E.: Manufactured Nanomaterials (Fullerenes, C60) Induce Oxidative Stress in the Brain of Juvenile Largemouth Bass. Environmental Health Perspectives, 112(10), 1058-1062, July 2004.
(PRE 60:2721, 1999) Crooks, G. E.: Entropy production fluctuation theorem and the nonequilibrium work relation for free energy differences. Physical Review E, 60(3), 2721-2726, September 1999.
(PRL 78:2690, 1997) Jarzynski, C.: Nonequilibrium Equality for Free Energy Differences. Physical Review Letters, 78(14), 2690-2693, April 1997.