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
