Ivan Dmochowski and XinJing Tang, researchers at the University of Pennsylvania, US, shed light on gene regulation.

 

Researchers have made significant strides recently in developing molecules and methods to turn genes on and off with light. Yesterday's science fiction is quickly becoming reality, and none too soon, as the completion of genome sequencing projects is ushering in a new era of biological exploration. 

One important goal is to determine the functions of all 30 000 human genes and to look at the complex interplay between the proteins they encode. This interplay is affected not only by the proteins' locations and concentrations but also by the timing of their expression from genes. So, quantitative molecular methods for controlling gene expression will assist greatly in these efforts, and open new realms for investigation. 

Light-activated groups inserted into DNA can be used to study genes in zebrafish embryos

Researchers have already used light-responsive neurotransmitters, hormones, and other signalling molecules in experiments in model systems, such as neurons, fruit flies, and zebrafish. Extending this idea, light-activated molecules that control protein expression will make it possible to tackle a wide range of problems of even greater complexity. 

Imagine, for example, introducing light-activated compounds into a living animal and then being able to regulate which proteins are made in the brain, just by shining a laser! This technique poses no injury to the animal, while probing the role of the proteins involved in brain development and regulation. By varying the position, timing, and power of the laser beam, it will be possible to affect cell signalling pathways to elucidate, and possibly even enhance, brain function. 

One of the most general, and promising, methods for photoregulating genes uses light-activated DNA and RNA oligonucleotides. For almost a decade, researchers have used light-activated (nitrobenzyl or coumarin) groups to modify protein-coding DNA and RNA strands and so block them from functioning. However, strands labelled with several of these blocking groups have been introduced into cells and larger organisms, and shining light on them has only partially restored their biological activity; labelled strands can remain inactive if they diffuse away from the light path. 

There are other problems with the oligonucleotide method. Until now the range of available oligonucleotides has been limited to those activated by near-ultraviolet (UV) light (wavelengths around 365nm). This light must be applied carefully to minimise its toxic effects, and rival chromophores within the cell can compete for the near-UV photons. Despite these hurdles, labelled messenger RNA (mRNA) has been used to study the role of the gene Lhx2 in developing zebrafish embryo brains. Researchers are building on this promising example. 

Recently, light-activated blocking groups have been incorporated into specific sites within DNA and RNA oligonucleotides. In one example, an efficient way to control the biological activity of a DNA molecule was to attach it to a complementary strand using a light-activated linker. In this way, the original DNA molecule remained bound to the complementary strand, which blocked its binding to an mRNA target, until its release was triggered with light. Using this approach, genes have been turned on and off in zebrafish embryos. 

Related studies have demonstrated the potential of using light-activated oligonucleotides to control important cellular processes such as RNA degradation and protein recognition. A different strategy, which uses small catalytic DNA molecules, called DNAzymes, to control RNA degradation, has also gained momentum. DNAzymes can be cycled repeatedly between on and off states using light and could provide the ultimate control over mRNA levels in cells.   

Finally, protecting groups that can be removed selectively by two-photon excitation using infrared light (wavelengths over 700nm), offer tremendous opportunities for biological investigations. Infrared light can penetrate deeply into biological tissue and the two-photon method allows light-activation to be limited to a specific depth within the sample. This will aid efforts towards controlling gene expression in individual cells in large animals and could one day offer treatment options for human patients. 

A convergence of new technologies has led researchers to develop light-activated DNA and RNA molecules that hold considerable promise for investigations into cell, neuro , developmental, and cancer biology. The next few years promise to be fruitful, as photochemical, gene-targeted strategies are applied to solve myriad problems of biological significance.

Read Dmochowski and Tang's highlight 'Regulating gene expression with light-activated oligonucleotides' in February's issue of Molecular BioSystems.