Single biomolecule studies using optical tweezers

Single biological molecule studies enable to probe and visualize exciting details of the events in physiological in vivo processes. The basic underlying question of this dissertation is to understand biological processes at a single molecule level. In contrast to ensemble techniques, advances in single molecule manipulation (e.g. optical and magnetic tweezers, atomic force microscopy) and / or fluorescence techniques allow to investigate the properties of individual molecules in real time with a possibility to change external conditions (buffers) in situ and modulate inter- and intra-molecular interactions. This thesis reports the application of a single molecule technique, dual beam optical tweezers, for the study of single biomolecules. A range of single molecule systems was investigated such as i)VirE2 protein DNA machinery, ii) DNA-surfactant, EtBr (ethidium bromide), SYBR® Green-DNA interactions and iii) dsDNA denaturation studies. In addition the development of the present experimental setup is described to enable combined force measurement as well as single molecule fluorescence studies. The presented biomolecular results provide new and complementary information on the different biological systems demonstrating the diversity of experiments that can be performed on single DNA molecules using optical tweezers. Chapter one gives a brief introduction to optical tweezers, describes how optical tweezers work, the physics behind it, details of the experimental setup and the method of force calibration required in micromanipulation. Optical tweezers have opened exciting avenues of research, especially in biology. Biologists will be able to investigate the nature of molecular machines one by one, and infer from their behavior those properties common to the population. In chapter 2, we show how optical tweezers were employed to study the change in the mechanical properties of single DNA molecules upon binding of small agents. The first part of this chapter reports on the changes in mechanics of single dsDNA in the presence of cationic and anionic surfactants (used as non-viral vectors in gene therapy). The second part describes the interaction of DNA binding ligands (SYBR® Green, EtBr) with individual DNA strands. Agrobacterium tumefaciens (AT), a Gram-negative bacterium, evolved a complex and unique mechanism to transfer a long single stranded DNA (ssDNA) molecule from its cytoplasm to the eukaryotic host plant cell nucleus. Central to this mechanism, chapter 3 discusses the results of the measurements on VirE2 protein interacting with single stranded DNA (ssDNA). VirE2 protein is a multifunctional protein from AT that coat the transferred-ssDNA (T-DNA), interacts with host factors assisting nuclear import of the complex, forms channels in lipid bilayers and displays a highly cooperative binding to ssDNA. The biological findings are presented in a new generic model which can be used to explain how generation of forces helps bacterial DNA to enter the plant cell based on our single molecule data. Single molecule dsDNA denaturation, relevant in many molecular biological experiments, induced by NaOH and mechanical pulling are studied in chapter 4. Here optical tweezers experiments give access to the ‘melting’ of hydrogen bonds by mechanical forces or alkali denaturation (NaOH) of dsDNA in real time. The mechanical stability and the transition of dsDNA to ssDNA is investigated at different ionic strength as well as in buffers. Fluorescent images of single λ DNA labeled with SYBR® Green were observed up to forces ≥ 65 pN and indicate a B-DNA to S −DNA transition. Chapter 5 describes the implementation of single-molecule fluorescence detection (SMF) in optical tweezers. The design and instrumental capabilities of optical tweezers combined with SMF are discussed in detail. The development of this instrument provides a worldwide unique experimental setup and opens up new possibilities in the studies of complex biological systems. Finally chapter 6 summarizes the results of this thesis and discusses future experimental applications. The appendices provide further details for DNA sample preparation, molecular biology and chemical surface activation recipes, an instruction manual for the setup and the list of currently published papers

Evidence of robust 2D transport and Efros-Shklovskii variable range hopping in disordered topological insulator (Bi2Se3) nanowires

We report the experimental observation of variable range hopping conduction in focused-ion-beam (FIB) fabricated ultra-narrow nanowires of topological insulator (Bi2Se3). The value of the exponent in the hopping equation was extracted as ~ 1/2 for different widths of nanowires, which is the proof of the presence of Efros-Shklovskii hopping transport mechanism in a strongly disordered system. High localization lengths (0.5nm, 20nm) were calculated for the devices. A careful analysis of the temperature dependent fluctuations present in the magnetoresistance curves, using the standard Universal Conductance Fluctuation theory, indicates the presence of 2D topological surface states. Also, the surface state contribution to the conductance was found very close to one conductance quantum. We believe that our experimental findings shed light on the understanding of quantum transport in disordered topological insulator based nanostructures.Comment: 14pages, 4 figure

Robust broad spectral photodetection (UV-NIR) and ultra high responsivity investigated in nanosheets and nanowires of Bi2Te3 under harsh nano-milling conditions

Due to miniaturization of device dimensions, the next generations photodetector based devices are expected to be fabricated from robust nanostructured materials. Hence there is an utmost requirement of investigating exotic optoelectronic properties of nanodevices fabricated from new novel materials and testing their performances at harsh conditions. The recent advances on 2D layered materials indicate exciting progress on broad spectral photodetection (BSP) but still there is a great demand for fabricating ultra-high performance photodetectors made from single material sensing broad electromagnetic spectrum since the detection range 325 nm to 1550 nm is not covered by the conventional Si or InGaAs photodetectors. Alternatively, Bi2Te3 is a layered material, possesses exciting optoelectronic, thermoelectric, plasmonics properties. Here we report robust photoconductivity measurements on Bi2Te3 nanosheets and nanowires demonstrating BSP from UV to NIR. The nanosheets of Bi2Te3 show the best ultra-high photoresponsivity (~74 A/W at 1550 nm ). Further these nanosheets when transform into nanowires using harsh FIB milling conditions exhibit about one order enhancement in the photoresponsivity without affecting the performance of the device even after 4 months of storage at ambient conditions. An ultra-high photoresponsivity and BSP indicate exciting robust nature of topological insulator based nanodevices for optoelectronic applications.Comment: 14 pages, 6 figure

ssDNA Binding Reveals the Atomic Structure of Graphene

We used AFM to investigate the interaction of polyelectrolytes such as ssDNA and dsDNA molecules with graphene as a substrate. Graphene is an appropriate substrate due to its planarity, relatively large surfaces that are detectable via an optical microscope, and straightforward identification of the number of layers. We observe that in the absence of the screening ions deposited ssDNA will bind only to the graphene and not to the SiO2 substrate, confirming that the binding energy is mainly due to the pi-pi stacking interaction. Furthermore, deposited ssDNA will map the graphene underlying structure. We also quantify the pi-pi stacking interaction by correlating the amount of deposited DNA with the graphene layer thickness. Our findings agree with reported electrostatic force microscopy (EFM) measurements. Finally, we inspected the suitability of using a graphene as a substrate for DNA origami-based nanostructures

Interaction of cationic surfactants with DNA: a single-molecule study

The interaction of cationic surfactants with single dsDNA molecules has been studied using force-measuring optical tweezers. For hydrophobic chains of length 12 and greater, pulling experiments show characteristic features (e.g. hysteresis between the pulling and relaxation curves, force-plateau along the force curves), typical of a condensed phase (compaction of a long DNA into a micron-sized particle). Depending on the length of the hydrophobic chain of the surfactant, we observe different mechanical behaviours of the complex (DNA-surfactants), which provide evidence for different binding modes. Taken together, our measurements suggest that short-chain surfactants, which do not induce any condensation, could lie down on the DNA surface and directly interact with the DNA grooves through hydrophobic–hydrophobic interactions. In contrast, long-chain surfactants could have their aliphatic tails pointing away from the DNA surface, which could promote inter-molecular interactions between hydrophobic chains and subsequently favour DNA condensation

VirE2: A Unique ssDNA-Compacting Molecular Machine

The translocation of single-stranded DNA (ssDNA) across membranes of two cells is a fundamental biological process occurring in both bacterial conjugation and Agrobacterium pathogenesis. Whereas bacterial conjugation spreads antibiotic resistance, Agrobacterium facilitates efficient interkingdom transfer of ssDNA from its cytoplasm to the host plant cell nucleus. These processes rely on the Type IV secretion system (T4SS), an active multiprotein channel spanning the bacterial inner and outer membranes. T4SSs export specific proteins, among them relaxases, which covalently bind to the 5' end of the translocated ssDNA and mediate ssDNA export. In Agrobacterium tumefaciens, another exported protein—VirE2—enhances ssDNA transfer efficiency 2000-fold. VirE2 binds cooperatively to the transferred ssDNA (T-DNA) and forms a compact helical structure, mediating T-DNA import into the host cell nucleus. We demonstrated—using single-molecule techniques—that by cooperatively binding to ssDNA, VirE2 proteins act as a powerful molecular machine. VirE2 actively pulls ssDNA and is capable of working against 50-pN loads without the need for external energy sources. Combining biochemical and cell biology data, we suggest that, in vivo, VirE2 binding to ssDNA allows an efficient import and pulling of ssDNA into the host. These findings provide a new insight into the ssDNA translocation mechanism from the recipient cell perspective. Efficient translocation only relies on the presence of ssDNA binding proteins in the recipient cell that compacts ssDNA upon binding. This facilitated transfer could hence be a more general ssDNA import mechanism also occurring in bacterial conjugation and DNA uptake processes

DNA nanomechanics allows direct digital detection of complementary DNA and microRNA targets

Over the past decade, several techniques have been developed to improve the detection of small amounts of DNA and RNA molecules, but detection of DNA molecules at concentrations below the femtomolar level requires amplified detection schemes. A unique nanomechanical response of hybridized DNA and RNA molecules that serves as an intrinsic molecular label is now reported; nanomechanical measurements allow direct detection and counting of hybridized molecules