Dmitry Cherny: Research Interests

Single molecule tools for the analysis of complex biological reactions

Why single molecules?

The structural and functional dynamics of macromolecules are key elements in the majority of cellular processes, such as transcription, translation and splicing. These processes recruit DNA, RNA, proteins and other ligands into large macromolecular assemblies in a coordinated, time-specific fashion mainly by virtue of protein and nucleic acid interactions. Experimental approaches to follow the dynamics of these processes involve analysing a specific sub-set of macromolecular assemblies.  Ensemble (bulk) measurements provide valuable information about the averaged properties of these processes. However, random fluctuations of nucleic acids and proteins cause the reactions to become asynchronous, so the processes cannot be analyzed for long periods of time. Another problem is that assemblies may be heterogeneous, possibly because the selected sub-set lacks important regulatory elements, and this could exacerbate the loss of synchrony.

A simple solution to overcome some of the limitations of ensemble average measurements is to follow the dynamics, e.g. time trajectories, of individual molecules. Due to the stochastic nature of the molecules, the structural organization of individual complexes and/or their time trajectories will differ from each other. But, by analyzing many of them, an average dynamic over the population of the molecules can be built. Importantly, the differences between individual molecules reflects the differences occurring in nature, information not attainable from ensemble measurements.

Single molecule methods appeared a long time ago as imaging techniques, firstly as electron microscopy and later as scanning probe microscopy. Nowadays, there is an ever-expanding family of methods exploiting the mechanical, optical or chemical properties of the molecules. Often, modern devices combine more than one technique in a single unit. A real time measurement under ambient conditions is one of the main features of modern single molecule techniques.

Our mission

Our goal is two-fold. Firstly, we plan to analyze complex macromolecular assemblies using our main single molecule technique, i.e. total internal reflection fluorescence (TIRF) microscopy. Secondly, we plan to bring in more of these new methods via collaborations with departments of physics and chemistry. As a result, we hope to make single molecule methods available for many research groups working in the life sciences.

Currently, the main target for single molecule experiments is the splicing machinery. Splicing reactions can be studied only in nuclear extracts, and the pathways selected depend upon the contributions of numerous proteins that bind with ill-defined specificity to numerous sites along a pre-mRNA molecule. Attempts to follow the order of events and their kinetics as large macromolecular assembly have been thwarted by heterogeneity, dynamic or weak interactions, loss of synchrony and the difficulty of knowing the numbers of components involved. Above all, there are no general methods available for studying processes in real time.

At present, using TIRF we can detect various fluorophores (organic dyes or coloured proteins), localise them with 2-4 nm accuracy and detect Förster resonance energy transfer (FRET) occurring between two dyes in a close proximity (Fig. 1). We developed a general procedure for the analysis of FRET dynamics from single molecule experiments.

                   

Fig. 1a  Fig 1b

Figure 1. Detection of FRET at single molecule level. Two oligonucleotides labelled with Cy3 (donor, green) and Cy5 (acceptor, red) were annealed to specific RNA sequences separated by more than 130 nt. To detect FRET a third oligonucleotide was used to bring the two duplexes into close proximity (A). Note that the donor-acceptor distance is larger than one can deduce from the cartoon illustrating the structure of the molecule due to its 3D conformation. (B) Time traces for the donor (green) and acceptor (red) upon excitation of just the donor show alternating periods of high emission of the acceptor, corresponding to a FRET with efficiency of 0.89, and low emission of the acceptor, corresponding to its dark state. After ~530 sec of imaging the donor was bleached.

 

For splicing reactions we have developed specific procedures allowing the analysis of splicing complexes using pre-mRNA annealed with fluorescently-labelled oligonucleotides that is incubated in crude cellular extracts derived from cells expressing coloured splicing factors. We will monitor the topological state of pre-mRNA and its alterations during the splicing reaction. The kinetics of pre-mRNA interaction with regulatory proteins and the order of their assembly will be of primary interest. Since splicing reactions require the action of hundreds of proteins in a concerted fashion, multiplicity of association is a major uncertainty (Fig. 2). For the latter we have developed a method for the determination of RNA-protein stoichiometry.

 

Figure 2                                            

Fig. 2a

Fig 2bSingle molecule detection of splicing complexes formed in nuclear extract. Nuclear extract containing GFP‐PTB (polypyrimidine tract binding protein) was incubated with pre-mRNA previously annealed to Cy5‐labelled biotinylated oligonucleotide. The mixture was diluted and injected into an imaging chamber of TIRF microscope. Fluorescence images were acquired with 488 nm and 633 nm laser excitation and superimposed (A). White circles show colocalized Cy5 and GFP signals indicating for specific complex formation. Time-traces for GFP intensity from individual complexes showed multiple discrete steps (five, B) due to sequential bleaching of GFP molecules within a complex. 

 

Future developments will include optical manipulation with femtolitre aqueous droplets, TIRF microscopy with a new set-up designed for imaging of up to four fluorophores simultaneously, the use of new materials for protein/nucleic acid detection and manipulation (nanoparticles and quantum dots), and the enhancement of spatial and time resolution of signal detection by using fast, miniaturized CCD detectors. These new and current methods will be used to study the assembly of transcription complexes and intracellular dynamics. 

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Department of Molecular and Cell Biology

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E: MolCellBiol@le.ac.uk

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