Stimulated Raman Spectroscopy: A new tool for profiling intracellular catalysis

Understanding how fast small molecules (such as drugs) or biomolecules (such as DNA) react inside cells is crucial to the development of new medicines and the design of successful industrial processes which rely on bio-transformations. And yet at the moment there are no tools which can give both the location of a biomolecule and its rate of reaction in live cells simply relying on the inherent properties of that molecule. Current strategies either require the cell or tissue under examination to be fixed (as for histological slides) so that the concentrations of compounds can be mapped by mass spectrometry (thus not applicable for examining reactions in living cells), or rely on the incorporation of isotopes to allow detection of the molecule under study by its nuclear magnetic resonance (a technique which does not allow the position of reacting molecule within the cell to be accurately determined).
A new technique, called stimulated Raman spectroscopy (SRS), allows the vibrational properties of a molecule to be measured in a way that is:
* quantitative - allowing accurate measurement of local concentrations;
* spatially resolved - allowing molecules to be mapped inside cells;
* chemically discriminating - allowing different types of molecules to be imaged at the same time;
* and uses non-destructive optical light for detection - meaning that the studies could eventually be transferred to fibre optic technologies to be used in patients.
We propose to use SRS to study the rate of one particular reaction inside cells so that we can demonstrate that it is possible to do this both in the presence of all of the other signals from the cell, and in a way that allows us to show exactly where the reacting molecules are inside a cell.

Rapid advances in the field of Raman imaging over the last decade, particularly in stimulated Raman spectroscopy (SRS), have the potential to revolutionise mechanistic understanding in biochemistry and medicinal chemistry.
* Raman is a non-destructive technique, employing relatively low energy laser irradiation, and water is only very weakly scattered in the Raman spectrum, thus live cell imaging is possible.
* Raman spectroscopy allows species-specific label-free visualisation; chemical contrast may be achieved when imaging a cell in its native environment without fixatives or stains.
* SRS can be used to give a quantitative readout (in contrast to more widely-known CARS and SERS techniques), allowing local intracellular concentrations to be determined.
In this project Raman spectroscopy will be used to measure directly the rate of reactions inside cells with spatial resolution. The thymidine analogue EdU will be incorporated into the DNA of cell nuclei and its reaction via the popular bioorthogonal copper(I) catalysed azide alkyne cycloaddition (CuAAC) click reaction will be used as the test platform. Rastering across the nucleus will allow EdU uptake to be monitored and quantified by SRS, and relative reaction rates to be mapped. The effects of enhanced local concentrations of catalyst and changes in chromatin structure on the rate of CuAAC reaction will be determined. Isotopic labelling experiments will be used to discriminate different subpopulations of the same species.

We anticipate that there will be 4 major areas which will benefit from this study into the use of stimulated Raman spectroscopy for profiling the rate of intracellular reactions:

1. Academics working in the fields of bioorthogonal chemistry, biotransformations, medicinal chemistry, chromatin biology and medical imaging will benefit from the knowledge which will be gained. To ensure maximum benefit publication of the project details will be in high impact journals and knowledge gained will be communicated at national and international scientific meetings.

2. The UK capacity of personnel trained to apply an in-depth chemical knowledge of the properties of molecules to complex biological problems will be enhanced and a highly skilled worker will be produced through this programme.

3. The translational impact of this research is likely to be in the medical and industrial biotechnology industries. Industrial medicinal chemists are likely to benefit from new methods to study the uptake and metabolism of drugs, whilst the industrial biotechnology industry might benefit from a new way to follow the rate of reactions catalysed in the presence of the biomass.

4. Through outreach activities such as the Edinburgh International Science Festival and Doors Open Days public engagement with the science behind the project will be advanced.