A team of researchers from the University of Pennsylvania is developing a new kind of molecular probe that could provide real-time information about miRNA expression and localization.
According to Andrew Tsourkas, an assistant professor of bioengineering and radiology at UPenn who is heading the project, the probes have the potential for use in basic biology and with the new generation of miRNA-based diagnostics currently being developed by various companies and academic groups.
Already, one company has seen the promise of the miRNA probes. Tsourkas told RNAi News this week that Integrated DNA Technologies is collaborating with his lab on the work, although he noted the company has no formal contract with the researchers to commercialize the technology, which is still very early stage.
Tsourkas' work is being funded by a 3-year National Science Foundation grant. Thus far, the NSF has awarded Tsourkas roughly $355,000.
Tsourkas said his lab has focused on molecular imaging and dedicated most of its time to messenger RNA; the abundance of literature describing newly uncovered roles of miRNAs led him to begin developing probes that could be used with the small non-coding RNAs.
"Gene expression in general has been a long-standing interest" of the lab. "From an imaging standpoint, it makes a lot sense to go after [miRNAs since they] … are one of the mechanisms that control regulation of gene expression.
"Gene expression in general has been a long-standing interest" of the lab, he said. "From an imaging standpoint, it makes a lot of sense to go after [miRNAs since they] … are one of the mechanisms that control regulation of gene expression.
Undeniably, miRNAs are also "the hot topic right now," he noted. "It's a very new field where there are still a lot of unanswered questions. With microRNAs, things as simple as localization or even just expression levels [are important] … and although new tools are becoming available, there are very few imaging tools available."
To help fill in this gap in miRNA imaging, Tsourkas and his colleagues are developing a probe based on molecular beacons. Developed in the late 1990s, molecular beacons are single-stranded oligonucleotide hybridization probes that form a stem-and-loop structure.
According to the Public Health Research Institute, a non-profit research organization that out-licenses the molecular beacon technology, the probe's loop "contains a probe sequence that is complementary to a target sequence, and the stem is formed by the annealing of complementary arm sequences that are located on either side of the probe sequence.
"A fluorophore is covalently linked to the end of one arm and a quencher is covalently linked to the end of the other arm," PHRI said. "Molecular beacons do not fluoresce when they are free in solution. However, when they hybridize to a nucleic acid strand containing a target sequence they undergo a conformational change that enables them to fluoresce brightly."
This kind of activatable probe, Tsourkas said, allows for imaging within a live cell. "The problem with using unactivable probes is that you have to somehow remove or eliminate the probe that is not bound to your specific target," he said. "If something is activatable, it's typically quenched until your target becomes present.
"We're trying to adopt the molecular beacon format to be able to image a microRNA," he explained. "We're tweaking the molecular beacon design to get more quantitative information."
Tsourkas said his team has modified the original molecular beacon design by adding quantum dots, which permit ratiometric imaging.
"Typically, you'd get a bright signal or a low signal" depending on whether a target miRNA is present or not, he said. "But it's very hard to identify just how many microRNAs are in the cell."
The quantum dots, however, gives the researchers a point of reference for determining the number of miRNAs present in the cell, Tsourkas said.
"It really is a basic science research project to establish feasibility and make it work. And even when it reaches that point, I think it may be a little while before one can conceive of it being a commercialized kind of offering."
"We use the quantum dots to track our molecular beacons and also to quantify the number of molecular beacons in the cell," he explained. "We then use the ratio of molecular beacon fluorescence to the quantum dot fluorescence to tell how many molecular beacons are hybridized to miRNA. These two measurements can give us a fairly accurate count of the number of miRNA per cell."
Tsourkas noted that one of the biggest hurdles to developing the miRNA probes was "figuring out the best way to quantify the signals that we're getting. This required standardizing our microscopes so we know exactly what fluorescent signal corresponds to what number of microRNAs there are in a cell," he said.
"We completely characterized our probes on the microscope and in vitro, and we're starting to take measurements in cells right now," he added. "We're able to get the fluorescence ratios in cells, and we're in the process of correlating them with other methods like real-time PCR."
Ultimately, Tsourkas said he hopes the work will provide a tool for measuring real-time expression and localization patterns of miRNAs.
"Right now, there are a lot of assays out there that will provide fixed time-point expression levels," he said. "With [our] probes, you get a real-time readout on the actual microRNA expression. So instead of just looking at 5 minutes or 30 minutes, we'll be able to record the expression levels of different microRNAs in real time.
Tsourkas added that the new probes are expected to "allow us to look at localization patterns to complement in vitro hybridization. Again, in vitro hybridization seems to work okay, but it only provides a fixed time-point — it doesn't allow you to track where these microRNAs travel over time. [Our probes] give you a fuller picture of what's going on inside the cell," he said.
Another area where Tsourkas sees great potential for the miRNA probes is in diagnostics.
"The wealth of literature [now associated with] microRNA expression with different diseases really opens up the door for ushering in these types of probes to see if we can correlate [miRNAs] to different diagnoses," he said.
He said that Prabodh Gupta and Zubair Baloch from the department of pathology and laboratory medicine at the Hospital of the University of Pennsylvania will be providing his lab with samples of normal and cancerous lung tissue for use in developing assays that could be used to rapidly detect the over-expression of miRNAs associated with particular malignancies.
"We're hoping we can [develop a test that could be performed] during the surgeries themselves," Tsourkas said.
This work, he added, is expected to begin before the end of the year.
Although Tsourkas' research is largely being conducting in-house, he noted that some assistance is coming from Integrated DNA Technologies.
"They've been a huge support," he said.
Mark Behlke, vice president of molecular genetics at IDT, told RNAi News this week that "there are a lot of aspects of this technology that are exciting … and any technologies that can improve [the visualization] of … microRNAs, or any gene-expression events, in living cells is … going to be a boon for researchers. But there are a lot of challenges too."
According to Behlke, additional work needs to be done to establish the methodologies required to use the probes quantitatively, as well as explore what modifications are required to make the probes nuclease stable.
Further, it remains unclear what delivery techniques will work best with the probes, he said.
"Getting simple DNAs or RNAs into cells — there are approaches for that today," Behlke said. "But these quantum dot complexes … [are] probably going to be a different kind of compound to transfect."
"At this juncture, we're more interested in [Tsourkas' work] more at the basic science level," he added. "It really is a basic science research project to establish feasibility and make it work. And even when it reaches that point, I think it may be a little while before one can conceive of it being a commercialized kind of offering."
— Doug Macron ([email protected])