Single molecule force spectroscopy on ligand-DNA complexes: from molecular binding mechanisms to biosensor applications

Recent developments in single molecule force spectroscopy (SMFS) allow direct observation and measurements of forces that hold protein-DNA complexes together. Furthermore, the mechanics of double-stranded (ds) DNA molecules in the presence of small binding ligands can be detected. The results elucidate molecular binding mechanisms and open the way for ultra sensitive and powerful biosensor applications.     

Malaria research: back to the future

One hundred years ago, Ross and Grassi provided us with the biological basis and fifty years ago, the human right declaration highlighted the moral responsibility of preventing death and reducing morbidity due to malaria. Yet one million children still die every year from the disease. The diversity in the parasite-host interaction surely represents a major obstacle, but modern scientific technology offers possibilities, at least partly or temporarily, to overcome the inherent ability of the parasite to evade chemotherapy and the immune system. True partnerships, North-South and others, represent the only way to respond to the scientific challenges. A strong commitment of different sectors in the global community to scientific achievements is necessary also with regards to malaria, a disease of the poor.     

Single molecule biology: coming of age

Cellular heterogeneity and stochastic fluctuation play key roles in biological processes. Single molecule approaches have the key advantage of avoiding ensemble averaging, allowing the observation of transient intermediates and heterogeneity (both static and dynamic). Thus they have revolutionised the way many biological questions are addressed. The challenge ahead is to develop integrated approaches such as the combination of single molecule imaging with single molecule manipulation to probe the dynamics of gene regulatory and cell signalling networks in living cells.     

Single molecule measurements and biological motors

Recent technological advances in lasers and optical detectors have enabled a variety of new, single molecule technologies to be developed. Using intense and highly collimated laser light sources in addition to super-sensitive cameras, the fluorescence of single fluorophores can now be imaged in aqueous solution. Also, laser optical tweezers have enabled the piconewton forces produced by pair of interacting biomolecules to be measured directly. However, for a researcher new to the field to begin to use such techniques in their own research might seem a daunting prospect. Most of the equipment that is in use is custom-built. However, most of the equipment is essence fairly simple and the aim of this article is to provide an entry point to the field for a newcomer. It focuses mainly on those practical aspects which are not particularly well covered in the literature, and aims to provide an overview of the field as a whole with references and web links to more detailed sources elsewhere. Indeed, the opportunity to publish an article such as this on the Internet affords many new opportunities (and more space!) for presenting scientific ideas and information. For example, we have illustrated the nature of optical trap data with an interactive Java simulation; provided links to relevant web sites and technical documents, and included a large number of colour figures and plots. Our group’s research focuses on molecular motors, and the bias of this article reflects this. It turns out that molecular motors have been a paradigm (or prototype) for single molecule research and the field has seen a rapid development in the techniques. It is hoped that the methods described here will be broadly applicable to other biological systems.This is an interactive contribution, which can be accessed at:     

Single molecule measurements and molecular motors

Single molecule imaging and manipulation are powerful tools in describing the operations of molecular machines like molecular motors. The single molecule measurements allow a dynamic behaviour of individual biomolecules to be measured. In this paper, we describe how we have developed single molecule measurements to understand the mechanism of molecular motors. The step movement of molecular motors associated with a single cycle of ATP hydrolysis has been identified. The single molecule measurements that have sensitivity to monitor thermal fluctuation have revealed that thermal Brownian motion is involved in the step movement of molecular motors. Several mechanisms have been suggested in different motors to bias random thermal motion to directional movement.     

Functional anatomy: from molecule to memory

The Drosophila memory gene amnesiac is expressed in neurons that project to mushroom body axons. Blockade of synaptic transmission in the amnesiac-expressing cells disrupts memory, but not learning, suggesting presynaptic and postsynaptic sites for memory formation.     

Motor control: from joints to objects and back

A new study shows that the nervous system has the flexibility to learn dynamics in object-centered coordinates - up to a limit.     

Single molecule techniques in DNA repair: A primer

A powerful new approach has become much more widespread and offers insights into aspects of DNA repair unattainable with billions of molecules. Single molecule techniques can be used to image, manipulate or characterize the action of a single repair protein on a single strand of DNA. This allows search mechanisms to be probed, and the effects of force to be understood. These physical aspects can dominate a biochemical reaction, where at the ensemble level their nuances are obscured. In this paper we discuss some of the many technical advances that permit study at the single molecule level. We focus on DNA repair to which these techniques are actively being applied. DNA repair is also a process that encompasses so much of what single molecule studies benefit – searching for targets, complex formation, sequential biochemical reactions and substrate hand-off to name just a few. We discuss how single molecule biophysics is poised to transform our understanding of biological systems, in particular DNA repair.     

Trypanosome alternative oxidase: from molecule to function

Trypanosome alternative oxidase (TAO) is the cytochrome-independent terminal oxidase of the mitochondrial electron transport chain. TAO is a diiron protein that transfers electrons from ubiquinol to oxygen, reducing the oxygen to water. The mammalian bloodstream forms of Trypanosoma brucei depend solely on TAO for respiration. The inhibition of TAO by salicylhydroxamic acid (SHAM) or ascofuranone is trypanocidal. TAO is present at a reduced level in the procyclic form of T. brucei, where it is engaged in respiration and is also needed for developmental processes. Alternative oxidases similar to TAO have been found in a wide variety of organisms but not in mammals, thus rendering TAO an important chemotherapeutic target for African trypanosomiasis.     

Interleukin 2: from immunostimulation to immunoregulation and back again

Interleukin 2 (IL-2) was one of the first cytokines to be discovered. However, the complex role of IL-2 and its receptor in the regulation of immune responses is only now emerging. This review explores the various signals triggered by IL-2 and discusses their translation into biological function. A model is outlined that accommodates the seemingly contradictory functions of IL-2, and explains how one cytokine can be an essential T-cell growth and differentiation factor and yet also be indispensable to maintain peripheral tolerance.     

One by one: Single molecule tools for genomics.

Much of the effort in any genomics programme arises from the need to generate and purify large numbers of identical molecules, since most analytical tools rely on the analysis of bulk DNA. Biological steps such as bacterial cloning--commonly used to prepare bulk samples of defined DNA fragments--are capricious and introduce their own restrictions and distortions. The analysis of single molecules, either directly or by in vitro enzymatic amplification, makes possible the examination of native genomic DNA without the complications and restrictions of biological propagation. Techniques already exist for the in vitro propagation of genomic fragments and for genome mapping, and offer the advantages of speed, flexibility and predictable behaviour. Single molecule sequencing, for which many approaches are being developed, is more challenging, but offers even greater rewards in terms of throughput and read length.