It’s 2007, and at this point miRNA needs no introduction. Just in case you’re late to the party, though, miRNAs are small, single-stranded nucleotides, which were originally believed to have little impact on gene expression and protein formation.
In addition, miRNAs are accurate biomarkers for numerous diseases and are also involved in the expression of DNA. Scientists are still continuing to discover new roles for these nucleotides. How could we have possibly known that something so fascinating was to be found in the middle of absolutely nowhere?
Having established the importance of miRNA, the scientific community is now racing to identify regulatory miRNAs that control pathways involved in development and disease, and to fully characterize their regulatory behavior.
Because miRNAs are not mRNAs—they are smaller, not capped, and not polyadenylated—existing RNA-analysis tools have had to be adapted to work with miRNA. This includes nucleic acid isolation technology, miRNA detection and amplification technology, technologies for silencing and controlling miRNA signals, and computer software methods for profiling miRNA and analyzing the genome for miRNA sequences.
Qiagen’s (www.qiagen.com) contribution to the miRNA landscape has been in optimizing RNA-analysis tools for the smaller miRNAs—essentially creating lighter weight tackle for catching smaller fish. It has modified its industry-standard solid-phase mRNA purification system to create the miRNeasy system.
A related product, miRNeasy FFPE, enables purification of miRNAs from formalin-fixed, paraffin-embedded tissue samples. This is the form in which hospitals archive patient samples. These archives contain not only diseased tissue, but records pertaining to the treatment and outcome of the disease—a rich resource for mining miRNAs.
Following purification, Qiagen’s miScript system allows for the sensitive detection and precise quantification of hundreds of miRNAs from an RNA sample as small as 1 ng (which can be stored indefinitely for future access once converted to cDNA) via RT-PCR. Although microarrays can be used for analysis, RT-PCR has the advantage of being able to detect low-abundance miRNAs.
According to Eric Lader, director of R&D at Qiagen, no one knows whether extremely low abundance miRNAs are significant. “It might not be a great idea to ignore a whole class of miRNAs expressed at low levels.” RT-PCR picks up these low-abundance miRNAs. Microarray analysis, however, is still a valuable tool for large-scale miRNA profiling.
As Qiagen works to develop its toolbox for miRNA research, one area of development is the technology to identify true mRNA targets of miRNA. Current methods make use of miRNA mimics and inhibitors if there is already a predicted candidate for a miRNA target. However, it is difficult to link miRNA to its target without some prior association.
Silencing the Silencer
The biotech tool of RNA interference, or gene silencing, owes its existence to the miRNA pathway, revealed in 1998 by Andrew Fire and Craig Mello. They found that short pieces of double-stranded RNA could inhibit, or silence, genes. This discovery immediately instigated a push for therapeutics and tools to take advantage of the silencing effect. It is ironic, then, that gene silencing can actually be targeted at miRNA, basically turning off the cell’s natural turning-off device.
This was the plan of attack for Peng Jin, Ph.D., assistant professor, and his colleagues at Emory University. Dr. Jin’s team was drawn into the field of miRNA indirectly, via fragile X syndrome, a common cause of mental retardation.
One of the proteins lost in fragile X syndrome is FMRP, which is thought to regulate translation in neurons, and is known to associate with miRNAs as well as Dicer and Argonaute, key proteins in the miRNA pathway. Dr. Jin’s team wanted to know which genes were regulated by particular miRNAs, so they created an assay using siRNA to knock down miRNAs.
Using this method, they observed increases in target mRNA transcripts in response to knockdown of certain miRNAs. In addition to providing a valuable new method for analyzing miRNAs, this work has led to a potential breakthrough. “Basically, what we have taken is a chemical biology approach,” says Dr. Jin. “We have identified certain small molecules that modulate miRNA activity.” These results are not yet published.
The principle of miRNA knockdown can be used in the development of RNAi based therapeutics as well. Alnylam Pharmaceuticals (www.alnylam.com) entered into a joint venture with Isis Pharmaceuticals (www.isispharm.com) to form Regulus Therapeutics (www.regulusrx.com), dedicated to RNAi therapeutics targeted to miRNAs.
Regulus’ most advanced program targets mir122, which is expressed in hepatocytes and is required for replication of hepatitis C virus. “This whole approach opens a new frontier for pharmaceutical research. By antagonizing miRNAs, we can shut down an entire pathway of human disease. miRNAs control groups of genes that tend to be involved in disease. By targeting single miRNAs, we can have an impact on whole networks of genes involved in disease processes,” reports John Maraganore, Ph.D., president and CEO of Alnylam.
The Invader Concept
Third Wave Technologies (www.twt.com) is focused on nucleic acid testing and diagnostics. It has applied its technology to miRNA analysis with intriguing results. Third Wave’s Invader chemistry is based on a two-step reaction.
In the first step, an oligonucleotide binds to the target sequence, and then a probe also binds to it. In the case of a point mutation or other variation from the reference sequence, a single-base overlap is created. The cleavase enzyme then cleaves off the 5´ flap of the overlapping sequence. The released 5´ flap then binds to another probe, this time labeled with FRET molecules.
Subsequent cleavage of this complex results in a fluorescence signal that can be detected. Since both reactions happen over and over again, the signal from a single miRNA can be amplified repeatedly, leading to ten-million-fold amplification of a single signal sequence. Detection is carried out in real time, or as an endpoint.
“We actually detect the miRNA in the sample without altering the miRNA,” says Hatim Allawi, Ph.D., director of discovery and advanced technologies for Third Wave. Other advantages that Dr. Allawi cites for Invader assays are short assay times—as little as two hours, large (six log) quantitative dynamic range, and the ability to multiplex. Multiple targets can be assayed in the same tube using Invader chemistry.
“Our miRNA Invader assay comes in handy,” notes Dr. Allawi, “when screening through patient samples or clinical samples. Invader is highly quantitative and highly specific. The answer is not a false positive or a false negative.”
Bioinformatics for miRNA Analysis
One of the most powerful tools of all for analyzing mRNA is the computer. Using specialized algorithms, researchers can analyze gene sequences for miRNAs and predict miRNA targets and pathways. Since unbiased miRNA analysis is difficult to impossible in vitro, a powerful software package to narrow down the hypotheticals is indispensible.
Actigenics (www.actigenics.com), now a division of Cepheid (www.cepheid.com), offers a comprehensive miRNA analysis platform (MiRgate).
Actigenics’ miRNA analysis begins with bioinformatic predicitions of thousands of novel miRNA candidates, followed by a custom microarray analysis to create a microarray expression profile for clinical samples. Then the MiRgate software suite correlates microarray results with significant pathways (using the company’s extensive database), producing a custom report for each sample. MiRgate will also analyze noncoding DNA for miRNAs, search for SNPs, and predict miRNA target sequences.
There is considerable affinity between Cepheid and Actigenics, explains David Persing, M.D., Ph.D., chief medical and technology officer and executive vice president for Cepheid. “We managed to acquire the expertise of a group that was extremely talented at being able to predict the presence of miRNA candidates within the human genome.
“Dr. Bernard Michot, formerly of INSERM, joined us as head of the group in the Cepheid France facility. He and his group of bioinformatics specialists developed a software package that is capable of producing accurate predictions of miRNA candidates and potential diagnostic markers not yet represented within the public domain data set.”
For Cepheid, this was an opportunity to access proprietary diagnostic targets for a broad array of diseases, and for Actigenics, it was an opportunity to commercialize a unique technology.
“We have reasons to believe miRNA markers might be among the most accurate for the diagnosis of cancer, inflammation, and autoimmune conditions such as systemic lupus, and there are a lot of areas yet to be explored by our system,” notes Dr. Persing. “What we expect to get out of it in practical terms is a short list of diagnostic markers that we can incorporate into tests on our GeneXpert diagnostic platform.”
In some cases, Cepheid has identified miRNA targets within regions of amplified and deleted chromosomes in cancers. For example, the region 17q12-22 is commonly amplified in breast cancer. It contains the gene for ErbB2, which is the target of Herceptin.
Cepheid predicted that two miRNAs from the same region would be overexpressed in breast cancer with potential regulatory roles apart from ErbB2 amplification. Overexpression of these two molecules was later demonstrated on microarrays. Interestingly, no miRNAs from the public domain data set were found to map to this region of the chromosome. These miRNAs may themselves be good diagnostic markers and they may in turn point to downstream pathways that may also be good candidates for new companion diagnostics in the mold of Herceptin/ErbB2.
Happily, the greatest obstacle to full exploitation of miRNA biology has been removed—that of simply not knowing it existed. miRNAs have begun to explain mysteries such as the mechanisms of embryonic development and the haywire genetics of cancer. And yet, the mind-boggling complexity of tiny regulatory elements that control hundreds of genes at a time may make us nostalgic for the days when noncoding DNA was junk and gene regulation consisted of a comfortably large protein stationed like a parking lot attendant on top of a very obvious promoter region.
But as with most nostalgia, we’d have to admit that the world is a more interesting place with miRNA in it, and as better tools become available for analyzing miRNA, those dizzying networks of interactions should slowly begin to make sense.