Bendy DNA Speeds Research
Sarah Loftus
26th March 2012
A new method for studying the way genes are controlled has been developed by a team at the University of Oxford, potentially dramatically decreasing the amount of time required to carry out experiments.
The research team, from the University’s Biological Physics Research Group, exploited the bendy properties of DNA to develop a device which can simply detect when a gene is being switched on and off, potentially cutting experiment times down from several days to just 15 minutes and even allowing the study of gene regulation at a single-cell level (most current techniques require a large number of cells). The study was published recently in the journal ChemPhysChem and demonstrates how a length of DNA was combined with a detector to show the presence of molecules involved in switching on a gene. This combination of biological component with detector is called a biosensor.
DNA contains all the information required by the cells that make up our bodies to build all of their components. These components are proteins and the instructions required to build each protein are found along a short section of DNA called a gene. Since cells don’t require all proteins all of the time, the timing and quantity of their production is very carefully regulated. This regulation comes in the form of proteins called transcription factors which bind to genes, controlling whether the protein they hold information about is made or not by switching the gene on or off. One of the ways they do this is by bending the DNA to which they bind, making the gene more accessible to the parts of the cell that read the gene and build the protein. The new biosensor uses this bending to detect the presence or absence of a transcription factor by combining it with a well established tool, known as FRET.
FRET, or Förster resonance energy transfer, behaves like a molecular ruler, by producing a signal when two molecules called chromophores are very close together. Chromophores are molecules that absorb and emit light energy. The signal is generated by the exchange of energy between the chromophores. A chromophore is attached to each end of a length of DNA with the gene of interest situated in between. The biosensor is built such that when the transcription factor is absent, the chromophores are in close proximity and a signal is produced. When a DNA-bending transcription factor binds to the DNA sensor, the chromophores are pulled apart so that there is no longer energy exchange thereby providing a means of detection for the transcription factor.
The research team, from the University’s Biological Physics Research Group, exploited the bendy properties of DNA to develop a device which can simply detect when a gene is being switched on and off, potentially cutting experiment times down from several days to just 15 minutes and even allowing the study of gene regulation at a single-cell level (most current techniques require a large number of cells). The study was published recently in the journal ChemPhysChem and demonstrates how a length of DNA was combined with a detector to show the presence of molecules involved in switching on a gene. This combination of biological component with detector is called a biosensor.
DNA contains all the information required by the cells that make up our bodies to build all of their components. These components are proteins and the instructions required to build each protein are found along a short section of DNA called a gene. Since cells don’t require all proteins all of the time, the timing and quantity of their production is very carefully regulated. This regulation comes in the form of proteins called transcription factors which bind to genes, controlling whether the protein they hold information about is made or not by switching the gene on or off. One of the ways they do this is by bending the DNA to which they bind, making the gene more accessible to the parts of the cell that read the gene and build the protein. The new biosensor uses this bending to detect the presence or absence of a transcription factor by combining it with a well established tool, known as FRET.
FRET, or Förster resonance energy transfer, behaves like a molecular ruler, by producing a signal when two molecules called chromophores are very close together. Chromophores are molecules that absorb and emit light energy. The signal is generated by the exchange of energy between the chromophores. A chromophore is attached to each end of a length of DNA with the gene of interest situated in between. The biosensor is built such that when the transcription factor is absent, the chromophores are in close proximity and a signal is produced. When a DNA-bending transcription factor binds to the DNA sensor, the chromophores are pulled apart so that there is no longer energy exchange thereby providing a means of detection for the transcription factor.
Diagram showing how transcription factor binding bends DNA and causes inactivation of the biosensor
In order for the biosensor to be effective, “kinks” had to be introduced into the length of DNA comprising the biosensor to bring the chromophores into close proximity. This had to be done because DNA is usually straight meaning that if the chromophores had been simply added to either end, the sensor would have been restricted for use in the study of transcription factors that introduce extremely large bends, for example greater than 110°. Kinks were generated by taking advantage of the double stranded composition of DNA. The two strands making up a DNA molecule each consist of a row of molecules called amino acids. These amino acids are like the hooks and eyes of a Velcro strip in that the amino acids making up one strand stick to the amino acids on the other, holding the strands together. A kink is introduced by including five amino acids in a row that do not pair with the amino acids on the other strand (like missing out five rows of hooks on the Velcro strip) which makes the DNA bend at an angle of 73°. Three of these kinks in the biosensor mean that the overall structure is almost C-shaped, with chromophores at either end. By having the gene of interest away from the kinks, the abnormal structure of the DNA making up the biosensor does not interfere with transcription factor binding.
The study demonstrated how the biosensor can be used to study gene regulation by the catabolite activator protein (CAP) which causes a bend angle of about 80-90° upon DNA binding, but the authors suggest that the biosensor may be used to study binding of other transcription factors that bend DNA. They also suggest that the biosensor could be used to determine concentrations of a particular transcription factor by modifying the DNA sequence of the gene of interest making it more or less favourable for transcription factor binding. For example if the gene is made less favourable for transcription factor binding, a higher concentration of the transcription factor would be required for binding and detection by the sensor. The research group are currently developing degradation-resistant DNAs that may be introduced into bacteria to directly detect changes in transcription factor concentration in vivo, further broadening the use of their biosensor.
The study demonstrated how the biosensor can be used to study gene regulation by the catabolite activator protein (CAP) which causes a bend angle of about 80-90° upon DNA binding, but the authors suggest that the biosensor may be used to study binding of other transcription factors that bend DNA. They also suggest that the biosensor could be used to determine concentrations of a particular transcription factor by modifying the DNA sequence of the gene of interest making it more or less favourable for transcription factor binding. For example if the gene is made less favourable for transcription factor binding, a higher concentration of the transcription factor would be required for binding and detection by the sensor. The research group are currently developing degradation-resistant DNAs that may be introduced into bacteria to directly detect changes in transcription factor concentration in vivo, further broadening the use of their biosensor.
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