Loop dependence of the stability and dynamics of nucleic acid hairpins

Hairpin loops are critical to the formation of nucleic acid secondary structure, and to their function. Previous studies revealed a steep dependence of single-stranded DNA (ssDNA) hairpin stability with length of the loop (L) as ∼L8.5 ± 0.5, in 100 mM NaCl, which was attributed to intraloop stacking interactions. In this article, the loop-size dependence of RNA hairpin stabilities and their folding/unfolding kinetics were monitored with laser temperature-jump spectroscopy. Our results suggest that similar mechanisms stabilize small ssDNA and RNA loops, and show that salt contributes significantly to the dependence of hairpin stability on loop size. In 2.5 mM MgCl2, the stabilities of both ssDNA and RNA hairpins scale as ∼L4 ± 0.5, indicating that the intraloop interactions are weaker in the presence of Mg2+. Interestingly, the folding times for ssDNA hairpins (in 100 mM NaCl) and RNA hairpins (in 2.5 mM MgCl2) are similar despite differences in the salt conditions and the stem sequence, and increase similarly with loop size, ∼L2.2 ± 0.5 and ∼L2.6 ± 0.5, respectively. These results suggest that hairpins with small loops may be specifically stabilized by interactions of the Na+ ions with the loops. The results also reinforce the idea that folding times are dominated by an entropic search for the correct nucleating conformation

Stability and Kinetics of DNA Pseudoknots: Formation of T∗A•T Base-Triplets and Their Targeting Reactions

Pseudoknots have been found to play important roles in the biology of RNA. These stem-loop motifs are considered to be very compact and the targeting of their loops with complementary strands is accompanied with lower favorable free energy terms. We used a combination of spectroscopic (UV, CD and fluorescence), calorimetric (DSC, PPC and ITC) and kinetic (SPR) techniques to investigate: 1) Local base-triplet formation in pseudoknots; 2) energetic contributions for the association of pseudoknots with their complementary strands; and 3) the kinetic rates as a function of targeting strand length. We investigated a set of DNA pseudoknots with sequence: d(TCTCTTnAAAAAAAAGAGAT5TTTTTTT), where “Tn” is a thymine loop with n = 5, 7, 9, and 11. The favorable folding of each pseudoknot resulted in favorable enthalpy-entropy compensation, correlated to favorable base-pair stacking contributions and unfavorable uptakes of ions and water molecules. The increase in the length of the loop yielded higher TMs, 53°C to 59°C and folding enthalpies ranging from -60 to -110 kcal/mol, resulting in a significant stabilization, ΔG°(5) = -8.5 to -16.6 kcal/mol, which is consistent with the formation of 1-2 TAT/TAT base-triplet stacks. The PPC results yielded folding volume changes, ΔVs, ranging from 18 to 23 ml/mol, indicating the higher volume of the folded pseudoknots is due to the uptake of both water (ΔnW of -11 to -24 mol H2O/mol) and ions (Δnion of -2.5 to -4.1 mol Na+/mol). We use ITC and DSC to determine thermodynamic profiles for the reaction of pseudoknots with partially complementary strands. We obtained favorable reaction free energies terms. However, the targeting of compact pseudoknots containing local base-triplets is less favorable due to their larger folding free energy term. The SPR data indicated that the rate of association, kon, decreases while the rate of dissociation, koff, increases as the length of the targeting strand increases, which yielded increasing KD, app.. This indicates the affinity of the target strand to the pseudoknot decreases as the length of the target strand increases. A similar trend was obtained when dissociation constants, KD, DSC, were measured from DSC Hess cycles. However, the KD, DSC were much smaller. This apparent discrepancy between these techniques is that SPR is measuring both the initial association and initial dissociation rates of steady state equilibrium states, while DSC measures true equilibrium states of the entire molecules

Single-molecule experiments in biological physics: methods and applications

I review single-molecule experiments (SME) in biological physics. Recent technological developments have provided the tools to design and build scientific instruments of high enough sensitivity and precision to manipulate and visualize individual molecules and measure microscopic forces. Using SME it is possible to: manipulate molecules one at a time and measure distributions describing molecular properties; characterize the kinetics of biomolecular reactions and; detect molecular intermediates. SME provide the additional information about thermodynamics and kinetics of biomolecular processes. This complements information obtained in traditional bulk assays. In SME it is also possible to measure small energies and detect large Brownian deviations in biomolecular reactions, thereby offering new methods and systems to scrutinize the basic foundations of statistical mechanics. This review is written at a very introductory level emphasizing the importance of SME to scientists interested in knowing the common playground of ideas and the interdisciplinary topics accessible by these techniques. The review discusses SME from an experimental perspective, first exposing the most common experimental methodologies and later presenting various molecular systems where such techniques have been applied. I briefly discuss experimental techniques such as atomic-force microscopy (AFM), laser optical tweezers (LOT), magnetic tweezers (MT), biomembrane force probe (BFP) and single-molecule fluorescence (SMF). I then present several applications of SME to the study of nucleic acids (DNA, RNA and DNA condensation), proteins (protein-protein interactions, protein folding and molecular motors). Finally, I discuss applications of SME to the study of the nonequilibrium thermodynamics of small systems and the experimental verification of fluctuation theorems. I conclude with a discussion of open questions and future perspectives.Comment: Latex, 60 pages, 12 figures, Topical Review for J. Phys. C (Cond. Matt

Detection and Melting of Surface-Bound DNA using a Purely Electrochemical Approach

In this thesis, we demonstrate an electrochemical approach for monitoring the electric-field induced melting of surface-bound double-stranded DNA. The electrochemical routine involves repeated chronoamperometry pulses to melt the duplex and square wave voltammetry to monitor the extent of melting. We utilize a scanning potential and constant potential technique to generate melting curves and access the stability and kinetics of the DNA duplexes. Our method uses a mixed monolayer of thiol-modified DNA oligomers and mercaptohexanol on gold electrodes, are subsequently incubated with target DNA covalently modified with electroactive methylene blue. Under room temperature, 10 mM Tris, and optimized electrochemical parameters, DNA duplexes are discriminated based on factors such as base pairs, hairpins, and mismatches. As a proof of concept, this method was extended towards a label-free DNA melting method

Sensibilisierungsmechanismen, Reaktionsmechanimsmen und reaktive Zwischenzustände in photokatalytischen Reaktionen auf Zeitskalen von Femto- bis Mikrosekunden

The development of renewable energy sources depicts a constantly growing interdisciplinary research field. Beyond photovoltaics chemical photocatalysis plays a small role, but is gaining more and more importance. In photocatalysis, light serves as an energy source for the chemical conversion of certain molecules. However, not only the application of photocatalysis as energy source but also the utilization of photocatalysis in chemical synthesis has attracted a deep scien- tific interest. For the optimization of photocatalytic systems a fundamental understanding oft the underlying processes is more than essential. Thereby, transient absorption spectroscopy has proved to be a very useful tool. On the one hand, the operation of a setup for transient absorption spectroscopy and on the other hand the systematic data evaluation requires physical and mathe- matical skills whereas the results cannot be interpreted without deep chemical knowledge. With- in the framework of the present thesis the cooperation between the fields of organic chemistry and physics has turned out as a very productive cooperation. Sensitizing mechanisms, reaction mechanisms and reactive intermediate states in photocatalytic reactions on time scales from femto- to microseconds are the object of the present work. The present thesis will prove that the analysis of measurement data on the basis of established standard methods, such as the fitting of a sum of exponential functions to the temporal evolution of the measured signal, often is not sufficient for a complete interpretation of the data. Only a data analysis precisely adapted to the problem can lead to a fundamental understanding of the underlying processes. In the first part of the present thesis, the focus lies on light-induced intramolecular charge transfer processes. Marcus Theory, which depicts the theoretical background, will be briefly in- troduced. On the basis of a molecular donor-bridge-acceptor system it will be shown that the damping coefficient β is not sufficient to differ unambiguously between coherent tunneling and incoherent hopping mechanism. Flavin-capped DNA hairpins serve as a model for the investigation of intramolecular charge transfer processes. After photo-excitation, flavin induces a hole which migrates through the DNA strand. It will be shown that an adapted base sequence allows for quantum yields of ΦCS = 14% for long-lived charge separated states. In the next section it will be discussed if the building blocks of the DNA are adapted to serve as chiral backbone for enantioselective photocatalysis. The conformation-dependent charge- transfer dynamics in benzophenone-DNA dinucleotides will be put on solid ground with the help of Marcus Theory. It will be shown that these dinucleotides are generally not suited to serve as an inert backbone for every kind of photochemical reaction. In the following section a true bimolecular photocatalytic reaction will be discussed. Flavin serves as photocatalyst for the conversion of an alcohol to the corresponding aldehyde. A pre- cisely adapted data analysis allows and exact quantification of the diffusion controlled reaction dynamics on the ps time scale. The understanding of the process allows optimizing the reaction conditions. The targeted utilization of triplet chemistry within this reaction can help to increase the quantum yield for product formation. As photo-induced charge transfer processes have been intensively discussed, the focus in the second part of the thesis lies on the [2+2] photocycloaddition. As basis for the interpretation of subsequent measurements, the [2+2] photocycloaddition of substituted quinolones will be inves- tigated. The formation of the cyclobutane ring in which the quinolone triplet state plays the cen- tral role will be characterized and quantified on the time scale from ps to ns. Afterwards the [2+2] photocycloaddition of substituted quinolones will be initiated by a chiral xanthone-based photocatalyst. It will be shown that within this catalyst-substrate complex in which both constit- uents have a distance of only few Ångströms, new electronic properties appear. The photo- excitation of a new electronic state not only initiates the [2+2] photocycloaddition of the quino- lone but also depicts a new sensitizing mechanism, which has to the author’s best knowledge not been observed in photocatalysis of organic molecules. The quinolone triplet state does not appear in this mechanism. The question, if this mechanism can be transferred to other photocatalytic systems has to be answered within the framework of further studies.Die Erforschung nachhaltiger und ressourcenschonender Energiequellen bildet ein stetig wachsendes, interdisziplinäres Forschungsfeld. Neben der Photovoltaik, die inzwischen eine etablierte Energiequelle darstellt, ist die chemische Photokatalyse noch ein kleines, aber stets wachsendes Teilgebiet. In der Photokatalyse dient das einfallende Licht dazu, chemische Ver- bindungen umzuformen. Nicht nur die Anwendung der Photokatalyse für die Energiegewinnung, sondern auch der Einsatz in der chemischen Synthese stößt dabei auf wachsendes Interesse. Um photokatalytische Systeme zu optimieren ist ein fundamentales Verständnis der Prozesse erfor- derlich. Die transiente Absorptionsspektroskopie hat sich dabei als geeignetes Werkzeug erwie- sen. Der Betrieb eines Aufbaus zur Messung transienter Spektren auf verschiedenen Zeitskalen und die gezielte Datenauswertung erfordert fundiertes physikalisches und mathematisches Ver- ständnis, wohingegen die Interpretation der Ergebnisse nicht ohne chemisches Wissen erfolgen kann. Im Rahmen dieser Arbeit hat sich die Kooperation zwischen der organischen Chemie und der Physik als erfolgreiche Zusammenarbeit erwiesen. Gegenstand der vorliegenden Arbeit sind die Sensibilisierungsmechanismen, die Reaktionsmechanismen und die reaktiven Intermediate in photokatalytischer Reaktionen auf Zeitskalen von Femto- bis Mikrosekunden. Es wird gezeigt, dass die Auswertung von Datensätzen mit Standardmethoden, wie der Anpas- sung einer Summe von Exponentialfunktionen an die zeitliche Entwicklung des Signals oft nicht ausreichend ist, um die Messdaten hinreichend zu interpretieren. Erst eine der Problemstellung exakt angepasste Datenanalyse führt zum Verständnis der zugrundeliegenden Prozesse. Zunächst werden lichtinduzierte intramolekulare Ladungstransferprozesse behandelt. Die the- oretische Basis für die Beschreibung solcher Prozesse bildet die Marcus-Theorie, die kurz einge- führt wird. Anhand eines molekularen Donor-Bridge-Acceptor-Systems wird gezeigt, dass der exponentielle Dämpfungskoeffizient β oft ungeeignet ist um der Reaktion einen kohärenten Tun- nelprozess oder einen inkohärenten Transfermechanismus zuzuweisen. DNS-Haarnadelstrukturen, welche kovalent mit einem Flavin-Chromophor verbunden sind, dienen als Modellsystem für die Untersuchung intramolekularer Ladungstransferprozesse. Nach Anregung induziert Flavin eine Elektronenfehlstelle in dem benachbarten DNS-Strang, die den Strang entlangwandern kann. Es wird gezeigt, dass durch die geeignete Wahl der Basensequenz eine Quantenausbeute von ΦCS = 14 % für langlebige ladungsgetrennte Zustände erreicht wird. In einem weiteren Abschnitt wird untersucht, ob die Bausteine der DNS als chirale Umgebung für die enantioselektive Photokatalyse geeignet sind. Mit Hilfe einer auf der Marcus-Theorie basierenden Interpretation der Messergebnisse wird die konformationsabhängige Ladungstrans- ferdynamik in Benzophenon-DNS-Dinukleotiden beschrieben. Es wird gezeigt, dass die Dinuk- leotide nicht uneingeschränkt für die enantioselektive Photokatalyse geeignet sind. Schließlich wird eine echte bimolekuare photokatalytische Reaktion untersucht. Dabei wird mit Hilfe von Flavin ein Alkohol in ein Aldehyd umgesetzt. Mit einer angepassten Datenauswer- tung werden diffusive Prozesse auf der ps-Zeitskala genau quantifiziert. Die gewonnen Informa- tionen dienen dazu, die Reaktionsbedingungen zu optimieren um über einen Triplett- Reaktionsmechanismus höhere Quantenausbeuten zu erzielen. Nachdem photoinduzierte Ladungstransferprozesse ausgiebig diskutiert wurden, liegt der Fo- kus im zweiten Teil auf der [2+2] Photocycloaddition: Als Grundlage für die Interpretation spä- terer Messungen wird zunächst die [2+2] Photocykloaddition an substituierten Chinolonen unter- sucht. Die einzelnen Reaktionsschritte der Ringbildung werden auf der Zeitskala von ps bis ns quantifiziert und charakterisiert, wobei der Triplettzustand den zentralen Zustand bildet. An- schließend wird die [2+2] Photocycloaddition an substituierten Chinolonen durch einen chiralen, auf dem organischen Chromophor Xanthon basierenden Photokatalysator initiiert. Es wird ge- zeigt, dass innerhalb des Katalysator-Substrat-Komplexes, in dem beide Moleküle einen Abstand von wenigen Ångström haben, neue elektronische Eigenschaften auftreten. Die Anregung eines neuen Zustands initiiert nicht nur die [2+2] Photocycloaddition sondern stellt auch einen neuen Sensibilisierungsmechanismus dar, der bisher in photokatalytischen Reaktion organischer Mole- küle nicht beobachtet wurde. Der Triplettzustand des Chinolons tritt hierbei nicht mehr auf. Ob sich dieser Sensibilisierungsmechanismus auch auf andere Systeme übertragen lässt, muss durch weitere Arbeiten auf diesem Gebiet geklärt werden

Structure, dynamics and hydration in drug-DNA recognition

The role of deoxyribonucleic acids in the cell has made DNA an attractive target for drug molecules. The anthracycline antitumour antibiotics are potent cytotoxic agents that have found widespread use in cancer chemotherapy. Nogalamycin binds DNA through intercalation, preferentially to 5'-TpG and 5'-CpG sites, by threading through the DNA helix and interacting with both the major and minor grooves simultaneously. In this thesis, the interaction of nogalamycin with the 5'-TpG site has been investigated using synthetic oligonucleotide duplexes and a combination of high-resolution NMR techniques and NOE-restrained molecular dynamics simulations. The solution structure of the 1: 1 complex with d(ATGCAT)2 is described with NOE data unambiguously identifying the position and orientation of the bound drug molecule, allowing conclusions to be drawn regarding the specificity for the TpG site. Binding at one TpG site sterically blocks the interaction at the symmetrically equivalent CpA site. The structural studies are extended to investigate by NMR the role of solvation in drug- DNA recognition and binding. Based on the sign and magnitude of solute-solvent NOEs, it is shown that only a small subset of water molecules visible in the crystal and MD structures are found to be bound in the solution complex, and that a number of these are involved in mediating drug-DNA interactions. The role of the dynamic network of water molecules in stabilising the complex in solution is discussed. Finally, the binding of nogalamycin at a TpG site carrying a DNA strand break has been investigated using a novel designed single-stranded intermolecular duplex consisting of two hairpins stabilised by GAA loops [d(ACGAAGTGCGAAGC)]. Although stacking of the two hairpins is weak, nogalamycin is shown to bind and stabilise a 1: 1 complex by binding at the intercalation site. The complex is discussed in terms of the mechanism by which nogalamycin is able to bind to premelted duplex DNA

Overstretching short DNA – Single-molecule force spectroscopy studies

Structural deformation of DNA has a central role in many biological processes. It occurs, for example, during replication, transcription, and regulation of the activity of the genome. To understand these fundamental processes it is necessary to have a detailed knowledge of the mechanical properties of DNA. How DNA responds to longitudinal stress has been studied on the single-molecule level for over two decades. Early it was discovered that torsionally relaxed double-stranded (ds) DNA undergoes a structural transition when subjected to forces of about 60-70 pN. During this overstretching transition the contour length of the DNA increases by up to 70% without complete strand dissociation. Since its discovery, a debate has arisen as to whether the DNA molecule adopts a new form or denatures under the applied tension. In the work of this thesis the overstretching transition is studied using optical tweezers to extend individual dsDNA molecules of 60 – 122 base pairs. By stretching short designed molecules of variable base-composition and with structural modification, factors determining the outcome of the process could be isolated and investigated. The structural changes induced during the transition vary depending on the stability of the dsDNA. Sequences that have a high GC-content are demonstrated to undergo a reversible overstretching transition into a longer form that remains base-paired. At high salt concentrations, this form of DNA, referred to as S-form, is found to be stable for extended periods of time, while at low salt it quickly denatures. AT-rich sequences are found to denature under tension in two different ways: if the AT-rich domain has one free end, melting will occur by progressive peeling of one strand from the other. When peeling is inhibited, here using synthetic inter-strand crosslinks, melting instead occurs internally within the sequence. The results presented here refine our knowledge of DNA mechanics, essential for understanding how proteins in our cells interact with DNA