SmartFlares™ – single-molecule fluorescence of RNA in-vivo

At last week’s American Association for Cancer Research (AACR) meeting in Washington D.C., it is the serendipitous discovery from walking around the exhibit hall that gets the creative thinking going. And at such a large venue – there were about 17,000 scientists in attendance – you never know what you’ll find among the 22 rows of exhibitors.

Such was my visit to the EMD / Millipore booth. Asking whether a former colleague from my days years ago from QIAGEN happened to be there (a fellow product manager that I shared an office with), I came across a new product called SmartFlares. And of all the people I spoke with at that show, and all the technologies I noticed (many new to me across the protein / RNA / DNA / whole cell analysis space) this one captured my attention. So much so that I headed over to EMD’s separate presentation area where one of the developers gave a short talk about the technology.

SmartFlares works on the principle of colloidal gold nanoparticles (on the order of 13 nm or so), acting as a quenching agent when a fluor is in close proximity. The gold particle is derivatized in such a way that a gene-specific oligonucleotide can be covalently bound to the particle at high density, and a short fluor-labeled oligonucleotide (the ‘reporter’ agent) can hybridize to that oligo, with the fluor in close proximity to the particle where the signal can be effectively quenched.

These SmartFlare reagents can then simply enter the cell via endocytosis (an incubation on the order of 18 hours), where this reagent can interact with the cytoplasm (they do not enter the nucleus) and release the labeled fluor. This article (Seferos and Mirkin et al., J. Am. Chem. Soc. Nov 2007, “Nano-flares: Probes for Transfection and mRNA Detection in Living Cells”) shows the signal characteristics of SmartFlares™, and the live cells can be sorted on the basis of endogenous fluorescence. EMD provides ‘nonsense’ controls (SmartFlares with sequence that does not exist naturally) as a background control, and other controls are available from them.

The beauty of the SmartFlare is its ability to look at relative RNA levels in-situ, and then separate these cells with a flow sorter for any downstream analysis. And rather than being a destructive technology, the cells endocytose via a native process, and excrete the reagent after a period of time, so a sorted population can be analyzed and recovered.

Another nice aspect of this technology is that it examines RNA level without any transfection, in-situ amplification or RNA purification. (I’ve written before about Advanced Cell Diagnostics, which looks at RNA levels in-situ from FFPE samples, that uses signal amplification technology.)

EMD / Millipore has produced some 250 genes, and there is an ability to do a 2-plex gene analysis (with two fluors, FAM and Cy3).

There has been a great interest in single-cell analysis, however it is clear that many experiments are required for a population of cells (i.e. many single cells need to be assayed as the level of gene expression can vary widely across individual cells). The beauty of this technology is its ability to easily separate a subpopulation of cells that have a particular gene expression profile or even a pair of genes that are differentiated in their co-expression. I didn’t ask explicitly, but it would appear that selected cells could go through another round of selection if desired. (The scientist I spoke with said that Millipore is working on developing a third color to work with.)

Speaking with a colleague at AACR about Fluidigm’s C1 system, it’s clear to me that that platform is unique and currently they have that selection process for single cell handling to themselves. A major differentiator for the SmartFlare is its non-destructive nature. For the C1, you have a single-cell population-level view for your gene(s) of interest, and many are using that platform as a front-end for single-cell genomic analysis (in particular very well suited for RNA-Seq due to the use of Life Technologies Cells to Ct additive single-cell reagents).

This has a lot to do with next-generation sequencing. Working with Innocentive, Life Techologies way back in 2010 announced a ‘Grand Challenge‘ – with a $1M purse in prize money – for the successful sequencing of an entire genome of a single cell, simultaneous to an entire transcriptome from a single cell.

And if market research surveys are to be believed, way back in 2009 I remember reading some research that RainDance conducted to assess the single-cell genomics market, and identified a genuine market opportunity there. (If my memory serves correctly, it was on the order of a majority of genomics researchers would conduct single-cell research ‘if the tools were available to simplify handling and identification of single cells of interest’.) Given the amount of interest at the Fluidigm booth at AACR in the C1 instrument, I would concur with this sentiment. And to quote an applications specialist that I met there on the exhibit hall, ‘everyone in the Northeast wants one’ (she covers the Boston and New York areas, with a high concentration of genomics researchers).

About RainDance – they have another opportunity there for single cell handling in the context of emulsion droplets, but a fair amount of development work remains to be done from my understanding. (As a side-note, RainDance is currently putting their resources behind a digital PCR offering; development effort on a single-cell application will depend on the relative success of their efforts in a marketplace that includes BioRad’s QX100, and the upcoming Life Technologies QuantStudio 3D.)

I’ll be sure to write about another single-cell technology I discovered at AACR – soon. (Stay tuned!)

The ability of the SmartFlare to recover live cells as a subset of a population, and then do further characterization and/or selection, makes it a winner. I’ll be sure to follow this technology (in terms of publications etc.) in the months and years to come.