Hyperpolarized Xenon-Based Molecular Sensors for Label-Free Detection of analytes.
Garimella PD, Meldrum T, Witus LS, Smith M, Bajaj VS, Wemmer DE, Francis MB, Pines A.The full citation is
A few months ago I reviewed a very nice paper by Perrone et al. regarding NMR-based sensors (see http://sitspinnmr.blogspot.com/2013/10/nmr-chemosensing.html). This weeks paper describes an alternative approach to designing an NMR-based sensor. Garimella et al. design a sensor with two binding sites. One site will bind a specific analyte, in this case the dye Rhodamine 6G. The other site will bind xenon gas molecules dissolved in solution. It turns out that xenon NMR is so sensitive that subtle changes on the other end of the sensor, such as the binding of a ligand, alter the chemical shift. Below it the TOC graphic to explain the concept visually.
What do the author's actually do?
They begin by creating a peptide receptor library with 1.6e5 (=20^4) molecules based on the sequence KXXPGXXGWKKG. Their hope is that some of these molecules (~2% or so - which is 3,200) will bind with a dye. These peptides are bound to polystyrene beads at the C-terminus and pyrene at the N-terminus. Since the peptides are attached to beads, the authors incubate the bead library with 10 uM dye for 1 hour, wash away the dye and manually sort the colored beads. (I am glad I don't have that job.) They screen the bead library multiple times and sequence the hits by mass spectrometry. I am sure that this part is pretty clever, but the details are a bit lost on me. In the end, their consensus sequence to bind Rhodamine 6G is H2N-KDDPGDEDWKKG-CO2H, which they call the "D-peptide." The make another peptide as a control with the sequence H2N-KNNPGNQGWKKG-CO2H. They call this peptide the "N-peptide." The figure below shows visual confirmation that the D-peptides binds with Rhodamine 6G using the bead incubation assay, whereas the N-peptides does not show any color change. So are we the audience to assume that the N-peptide does not bind the dye? At this stage, I think the answer is yes.
Garimella et al. can now move away from the beads and focus on the peptides. They do some smart controls like replacing the pyrene with the Xenon binding cryptophane cage at the N-terminus to confirm that the dye binds to the peptide, not the pyrene. They also confirm specificity by trying many other dyes, none of which bind with the D-peptide (no word about the N-peptide, though).
Now we can get into some spectroscopy. The authors use NOESY to confirm the interaction between the dye and D-peptide. Their sample is 250 uM dye and 200 uM D-peptide-cage. They also record a NOESY of 250 uM dye and 200 uM N-peptide-cage. Here is what the authors report:
"The spectra of both the D- and N-peptides in the presence of dye revealed cross peaks in the region corresponding the dye-peptide interactions. However, there were many more cross peaks in the D-peptide-cage sample, and they were more intense than those in the N-peptide-cage sample."
and then a bit later
"The NOE data .. verifie(s) that there was a difference in interaction of the dye with the D- and N-peptides, confirming our visual evidence that the library produced a receptor for the Rhodamine 6G analyte."
So much for a negative control! Still you have to give the authors a lot of credit for turning a bug into a feature. (I am stealing that phrase from my friend Todd, who used it to describe a research projects around here. It seems to me that the ability to turn a bug into a feature is a key trait in good science.) So the N-peptide binds Rhodamine 6G, albeit with less affinity than the D-peptide. As a final note on this topic, it seems to me that there is something funny going on with the interactions between the dye and peptide. I spent some time staring at the NOESY spectra in the SI and I can't make heads or tails of this interaction.
On to the Xenon-based detection - The Pines lab has developed a really cool system to deliver optically hyperpolarized xenon gas into their NMR samples. The net result is a HUGE signal. In the introduction to this paper the authors say that the NMR signal has the "strength compatible to those of water in conventional experiments." I am assuming that they only need one scan!
At any rate, the authors make ten NMR samples, five for the D-peptide-cage, five for the N-peptide-cage each with increasing dye concentration from 0 to 1000 uM in steps of 250 uM. The peptide concentration equals 200 uM. Let's first consider the Xe NMR spectrum for the peptide-cage samples with no dye. There are three peaks. One for hyperpolarized Xe bound to the cage at ~60 ppm. There is a sample for aqueous Xe (some the gas pumped into the solution dissolves in the solvent like CO2 in soda pop) at ~200 ppm. There is also so undissolved Xe gas signal, which is set to 0 ppm. (Since these signals are in slow exchange, I guess the dissolution process is slow on the NMR time scale.) The gaseous signal is used as a reference and set to zero ppm in all spectra. What I find strange is that the exact chemical shift of the aqueous Xe is at ~190.75 for the D-peptide and 191.25 for the N-peptide. This is not a good quality for a sensor to possess. I think that you would want any extra unbound sensor to be impervious to other conditions in the solution. Is Xe sensing the pH of the solution (presumably the pKa of the D- and N- peptide is different)? The authors did warn us that Xe NMR was sensitive. The exact chemical shift of the Xe bound to cage equals ~60.5 ppm for the D-peptide and 62.25 ppm for the N-peptide. I'll say it again - The authors did warn us that Xe NMR was sensitive.
Let's first consider the D-peptide. When dye is added to the solution both peaks shift. The aqueous Xe moves upfield and the Xe-cage moves downfield. I think it is strange that the aqueous Xe shifts, but it is what it is. The Xe-cage moves quite a bit more than the aqueous, Xe by the way. Now let's consider the N-peptide. As the dye concentration increases, the Xe-cage peak move upon binding with the N-peptide, albeit less than with the D-peptide. The authors advocate using the difference between the peaks as a readout of the assay. Using this metric, the Xe peaks move 0.78 and 0.33 ppm for the D-peptide-cage and N-peptide-cage upon binding 1 mM dye. See figure below ...
I am going to criticize this paper a bit, so I'll issue my usual caveat: I am a nobody doing LN2 fills and teaching 1st year graduate students how to acquire 1D 1H spectra, whereas the authors of this paper are real scientists doing the hard, creative work of producing scientific knowledge. My purpose in starting this blog is not to trash people's work so that I feel better about my life. I wanted to force myself (on a very public forum) to critique great science in hope of improving my science. So here is my critique in one sentence: the negative control is not so negative. Traditionally, in a paper like this one, you would have one positive and one negative control, proving that you get a signal when the analyte is present and no signal when the wrong analyte present. In this paper the authors see a delta delta of 0.78 ppm for a carefully chosen strong binder and a delta delta of 0.33 ppm for a weaker binder. If they could make a non-binder would the delta delta equal 0 ppm? What would they see if they tried another dye molecule?
Overall, I give the authors a lot credit for using a SELEX type procedure to hunt out a binder. I kind of glossed over this point earlier to get to the spectroscopy, but it was a substantial effort to find a needle in a stack with 20^4 pieces of hay. Clearly, this paper is a starting point. The authors set out to show that their sensor-cage-Xe trick would is feasible. I think they succeeded. I wonder if their problem isn't going to be that Xe NMR is just too sensitive, though.