From the Cancer Consortium Shechner lab, Department of Pharmacology, University of Washington

Mapping is crucial in understanding and contextualizing our environments. Mapping serves many purposes – it helps us establish efficient paths between destinations, locate new restaurants or find our friends’ apartments, or even predict which areas to avoid. Similarly, in biology, different part of cells can be mapped onto compartments that serve specific functions. Different cellular molecules can reside in each compartment. For example, DNA is only housed in the nucleus, while proteins and RNA are spread throughout all the compartments of the cell. Mapping where these molecules go and how they interact with one another helps researchers understand the function of any given molecule and how that function can be corrupted during disease. RNA interactions with proteins during splicing or DNA during transcription impact many diverse cellular processes.  Yet, to date, it has been difficult to literally map when and when these interactions occur or to identify RNA-interacting partners. To combat this problem and develop cellular maps of RNA interactions, the Shechner lab at the University of Washington Department of Pharmacology developed Oligonucleotide-mediated proximity-interactome MAPping (O-MAP).

To develop this novel method, Shechner and his team borrowed ideas from previous pioneers in the RNA field. Their workflow starts with DNA probes similar to those used in Fluorescence In Situ Hybridization (FISH) experiments. Like FISH probes, Shechner’s probes are designed to interact with a specific cellular RNA through complementary sequences. Here is the key difference: instead of the probe being fluorescently labelled as in FISH experiments, O-MAP probes contain another DNA sequence known as the “universal landing pad.” After the O-MAP probe hybridizes with the RNA, the group adds a secondary probe that latches onto the landing pad. The secondary probe also contains an enzyme that adds a small biotin tag to all the molecules in the RNA’s cellular neighborhood – a technique known as proximity biotinylation. From there, researchers can exploit the biotin tag to isolate all the tagged molecules. This approach allows researchers to map the cell neighborhoods of RNA molecules – something that has previously been impossible to do because of the transient nature of RNA.

“[O-MAP] is leveraging the precision and the modularity…and the low background that you can get from RNA FISH…and turning it into an interaction-discovery platform,” says Shechner.

O-MAP uses a primary and secondary HRP-conjugated probe to target a cellular RNA. After the RNA is targeted, HRP can add biotin tags to surrounding proteins or DNA. Streptavidin can then be used to isolate the biotin-tagged molecules.

After isolating the biotin-tagged molecules, many different techniques can be used to map the RNA neighborhoods. Mass spectrometry can be used to identify which proteins were tagged with biotin. Sequencing approaches can identify the specific RNA or DNA sequences the probed RNA molecule was interacting with. These techniques allow for an unbiased nanoscale mapping of the microenvironment surrounding a particular RNA. “O-MAP now lets you make interaction discoveries at this spatial scale where…there’s a lot of biology happening, but we haven’t been able to discovery it because of the lack of available technologies,” says Shechner. This new approach gives researchers a new level of resolution to understand how cellular molecules interact with each other.

Perhaps most exciting about this new technique is its accessibility. Because O-MAP borrows from well-established techniques like RNA FISH and proximity biotinylation, the tools are readily available for any group wanting to answer questions about RNA neighborhoods.

“What I like most about O-MAP is that anyone can do it…what’s special about it is it’s a new combination than many people wouldn’t think to do,” says Shechner.

Using O-MAP and tools like it, the group is laying the groundwork for a new bottom-up understanding of the molecular architecture of a cell at baseline and under stress.

Source – Fred Hutchinson Cancer Center