An experiment in open notebook science – Sortase mediated protein-DNA ligation
In a recent post I extolled the possible virtues of Open Notebook Science in avoiding or ameliorating the risk of being scooped. I also made a virtue of the fact that being open encourages you to take a more open approach; that there is a virtuous circle or positive feedback. However much of this is very theoretical. We don’t have good case studies to point at that show that Open Notebook Science generates positive outcomes in practice. To take a more cynical perspective where is the evidence that I am willing to take risks with valuable data? My aim with this post is to do exactly that, put something out there that is (as far as I know) new and exciting, and kick off a process that may help us to generate a positive example.
I mentioned in the previous post that we have been scooped not once, but twice, on this project. I will come back to the second scooping later but my object here is to try and avoid getting scooped a third time. As I mentioned in the previous post we are using the S. aureus Sortase enzyme to attach a range of molecules to proteins. We have found that this provides a clean, easy, and most importantly general method for attaching things to proteins. Labelling of proteins, attaching proteins to solid supports, and generating various hybrid-protein molecules has a very wide range of applications and new and easy to use methods are desperately needed. We have recently published the use of this to attach proteins to solid supports and others have described the attachment of small molecules, peptides, PNA, PEG and a range of other things.
One type of protein-conjugate that is challenging to generate is one in which a protein is linked to a DNA molecule. Such conjugates have a wide range of potential applications particularly as analytical tools where the very strong and selective binding that can often be found in a protein is linked to the wide range of extremely sensitive techniques available for DNA detection and identification. Such techniques have been limited because it is difficult to find a general and straightforward technique for making such conjugates.
We have used our Sortase mediated ligation to successfully attach oligonucleotides to proteins and I have put up the data we have that supports this in my lab book (see here for an overview of what we have and here for some more specific examples with conditions). I should note that some of this is not strictly open notebook science because this is data from a student which I have put up after the event.
We are confident that it is possible to get reasonable yields of these conjugates and that the method is robust and easy to apply. This is an exciting result with some potentially exciting applications. However to publish we need to generate some data on applications of these conjugates. One obvious target here is to use a DNA array and differently coloured fluorescent proteins attached to different oligonucleotides to form an image on the array. The problem is that we are not well set up to do this in my lab and don’t have the expertise or resources to do this experiment efficiently. We could do it but it seems to me that it would be quicker and more efficient for someone else with the expertise and experience to do this. In return they obviously get an authorship on the paper.
Other experiments we are interested in doing:
- Analytical experiment using the binding of a protein-DNA conjugate that utilises the DNA part for detection.
- Pull down of peptide-DNA conjugates onto an array after exposure of the peptides to a protease
- Attachment of proteins to a full length PCR product containing the gene for the protein. Select one of the protein and then re-amplifity the desired gene. (I had a quick go at this but it didn’t work)
So what I am asking is this:
- If any reader of this blog is interested in doing these (or any other) experiments to aid us in getting the published paper then get in touch
- If you feel so inclined then publicise this call wider on your own blog and let’s see whether using the blogosphere to make contacts can really aid the science
We will send the reagents to anyone who would like to do the experiments along with any further information required. In principle people ought to be able to figure out everything they need from the lab book but this will probably not be the case in practise. The idea here is to see whether this notion of a loose collaboration of groups with different resources and expertise that is driven by the science can work and whether it is a competitive way of doing science.
My criteria in accepting collaborators will be as follows:
- Willingness to adopt an Open Notebook Science approach for this experiment (ideally using our lab book system but not necessarily)
- Interest in and willingness to engage in the development of the published paper (including proposing and/or carrying out any new experiments that would be cool to include)
- Ability to actually carry out the experiment in reasonable time (ideally looking for a couple of months here)
So this is notionally a win-win situation for me. We will be getting on and doing our own thing as well but by working with other groups we may be able to get this paper out more efficiently and effectively. Maybe others will come up with clever experiments that would add to the value of the paper. The worst case scenario is that someone comes along and sees this, copies the results, and publishes ahead of us. The best case scenario is that someone else already working in a similar direction may come across this and propose working together on this.
In any case, the results promise to be interesting…
 Chan et al, 2007, Covalent attachment of proteins to solid supports via Sortase-mediated ligation, PLoS ONE, e1164
 Popp et al, 2007, Sortagging: a versatile method for protein labelling, Nat Chem Biol, 3:707
 Mao et al, 2004, Sortase-mediated protein ligation: a new method for protein engineering, J Am Chem Soc, 126:2670
 Pritz et al, 2007, Synthesis of biologically active peptide nucleic acid-peptide conjugates by sortase-mediated ligation, J Org Chem, 72:3909
 Parasarathy et al, 2007, Sortase A as a novel molecular “stapler” for sequence specific protein conjugation, Bioconj Chem, 18:469
 Barbulis et al, 2005, Using protein-DNA chimeras to detect and count small numbers of molecules, Nature Methods, 2:31