Skoll is the name of the wolf, Who follows the shining priest, Into the desolate forest,
And the other is Hati, Hróðvitnir’s son, Who chases the bright bride of the sky
Snorri Sturluson’s Grímnismál
I chose Norse mythology as a description of the cause of solar eclipses rather than Greek because in this case the story is much less gruesome… Here we have a pair of wolves one obsessively chasing the moon, and the other the sun. Always on the verge of catching it. Every now and then they do catch their prey and it is necessary to distract them with noise so that sun and the moon can escape and shine again.
In this blog we will deal with the use of fluorophores on aptamers, and their eclipsing with quenchers. Aptamers can be directly synthesized with many common fluorophores (our favourite is FAM) available on the 5’ terminal phosphoramidite thus obviating the need for any post synthesis chemistry. We have found that for many fluorescently labeled aptamers simply binding to a target molecule modulates fluorescence expression either up or down. An increase in fluorophore expression can be caused when the binding event reduces water molecule access to the fluorophore, a decrease can be caused by physical blocking of excitation or emission by the bound target.
Fluorescence detection can also be achieved through fluorescence polarization. We commercialized an aptamer that bound to gluten proteins in wheat flour based on this principle. Fluorescence polarization (FP) works by measuring fluorescence excitation and emission in two orthogonal polarized wavelengths. The faster a molecule (and the fluorophore attached to such a molecule is tumbling) the lower the measurement of fluorescence. Given the relatively small size of aptamers their tumbling speed can be modified greatly by binding to a larger molecule such as a protein. Alternatively, we have also used fluorescently labeled antisense oligos and measured their displacement from aptamers that bind to small molecules. The most significant difficulty with FP analysis is that it is affected strongly by the viscosity of the solution analyzed. As such variability in the viscosity of the matrix that the target is present in will influence the analysis. In the past though, we did sell a single cuvette FP reader for less than $1,000.
We are often asked by prospective clients to develop aptamers that have a fluorophore on one end, and a quenching molecule on the other. The quenching molecules absorb the emission of the fluorophore (effectively turning it off, hence the eclipse analogy). There are examples in the literature where this approach has worked but we are a business, everything we do has to work, we cannot survive with rare examples. The difficulty with the combined approach is that this is tantamount to asking the aptamer to do two things, bind with high affinity to the target, and while binding to the target undergo an allosteric shift that alters the physical distance between the fluorophore and the quencher. I have suggested that there is a reason that eagles are good at catching rodents, but not pollinating flowers. At NeoVentures we suggest that it is more effective to focus on selecting an aptamer for binding affinity. Once the ideal aptamers are selected, we know the sequence, we can engineer the allosteric effect. In our opinion the best way to engineer the shift between quenched fluorescence in the absence of target, and expression of fluorescence in the presence of target is to label the aptamer with the fluorophore and an antisense oligonucleotide with the quencher. This approach also allows the end-user to optimize performance for commercialization by altering the molar ratio between antisense and aptamer. In general, higher sensitivity is achieved with fuller quenching, thus a molar excess of antisense is preferred.
At NeoVentures we have a lot of experience engineering fluorescence/quenching systems. The key is to develop a system that can be modulated to facilitate optimization. A wolf chasing the sun with its jaws open constantly grasping for the sun is an alluring image, but in our world we need to manage the art of the eclipse with more subtlety and consistent reproducibility.