Forum for Science, Industry and Business

Sponsored by:     3M 
Search our Site:

 

Researchers develop new way to see single RNA molecules inside living cells

08.04.2009
Biomedical engineers have developed a new type of probe that allows them to visualize single ribonucleic acid (RNA) molecules within live cells more easily than existing methods. The tool will help scientists learn more about how RNA operates within living cells.

Techniques scientists currently use to image these transporters of genetic information within cells have several drawbacks, including the need for synthetic RNA or a large number of fluorescent molecules. The fluorescent probes developed at the Georgia Institute of Technology circumvent these issues.

"The probes we designed shine bright, are small and easy to assemble, bind rapidly to their targets, and can be imaged for hours. These characteristics make them a great choice for studying the movement and location of RNA inside a single cell and the interaction between RNA and binding proteins," said Philip Santangelo, an assistant professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University.

Details of the probe production process and RNA imaging strategy were published online in the journal Nature Methods on April 6. In addition to Santangelo, Georgia Tech graduate student Aaron Lifland, Emory University associate professor Gary Bassell and Vanderbilt University professor James Crowe Jr. also contributed to this research. This research was funded by new faculty support from Georgia Tech.

In the study, the probes – produced by attaching a few small fluorescent molecules called fluorophores to a modified nucleic acid sequence and combining the sequences with a protein – exhibited single-molecule sensitivity and allowed the researchers to target and follow native RNA and non-engineered viral RNA in living cells.

"The great thing about these probes is that they recognize RNA sequences and bind to them using the same base pairing most people are familiar with in regards to DNA," explained Santangelo. "By adding only a few probes that would bind to a region of RNA, we gained the ability to distinguish a targeted RNA molecule from a single unbound probe because the former lit up two or three times brighter."

For their experiments, the team used a bacterial toxin to transport the probes into living cells – a delivery technique that when combined with the high affinity of the probes for their targets, required significantly fewer probes than existing techniques. The toxin created several tiny holes in the cell membrane that allowed the probes to enter the cell's cytoplasm.

The researchers tested the sensitivity of conventional fluorescence microscopy to image individual probes inside a cell. Previous studies showed that these techniques were able to image an accumulation of probes inside a cell, but the current study demonstrated that individual probes without cellular targets could be observed homogenously distributed in the cytoplasm with no localization or aggregation.

With single-molecule sensitivity accomplished, the researchers investigated whether they could visualize individual RNA molecules using the probes. To do this, they simultaneously delivered probes designed to target a human messenger RNA (mRNA) sequence region and a probe designed with no target in the human genome. They were able to image unbound probes of both types as well as individual RNA molecules that had attached to the former probes.

The imaging technique also allowed the researchers to observe a process called dynamic RNA-protein co-localization, which is the joining of RNA molecules and RNA binding proteins in a single cell.

"We observed substantial transient interactions between proteins and viral RNA molecules that I don't think had ever been seen before with non-engineered RNA," noted Santangelo. "We saw one of the proteins move into a viral RNA granule and reside within it for over a minute before it was released, and we also saw another protein that appeared to dock with a viral RNA granule."

Santangelo is currently trying to improve the probes by making them smaller and brighter, while also using them to investigate viral pathogenesis and other biological phenomena.

"We are excited to use this imaging strategy to study how single viral RNAs travel from the nucleus of a cell to a virus assembly site, how mRNAs are regulated by location and time, and RNA trafficking in neurons," added Santangelo.

Abby Vogel | EurekAlert!
Further information:
http://www.gatech.edu

More articles from Life Sciences:

nachricht Rainbow colors reveal cell history: Uncovering β-cell heterogeneity
22.09.2017 | DFG-Forschungszentrum für Regenerative Therapien TU Dresden

nachricht The pyrenoid is a carbon-fixing liquid droplet
22.09.2017 | Max-Planck-Institut für Biochemie

All articles from Life Sciences >>>

The most recent press releases about innovation >>>

Die letzten 5 Focus-News des innovations-reports im Überblick:

Im Focus: The pyrenoid is a carbon-fixing liquid droplet

Plants and algae use the enzyme Rubisco to fix carbon dioxide, removing it from the atmosphere and converting it into biomass. Algae have figured out a way to increase the efficiency of carbon fixation. They gather most of their Rubisco into a ball-shaped microcompartment called the pyrenoid, which they flood with a high local concentration of carbon dioxide. A team of scientists at Princeton University, the Carnegie Institution for Science, Stanford University and the Max Plank Institute of Biochemistry have unravelled the mysteries of how the pyrenoid is assembled. These insights can help to engineer crops that remove more carbon dioxide from the atmosphere while producing more food.

A warming planet

Im Focus: Highly precise wiring in the Cerebral Cortex

Our brains house extremely complex neuronal circuits, whose detailed structures are still largely unknown. This is especially true for the so-called cerebral cortex of mammals, where among other things vision, thoughts or spatial orientation are being computed. Here the rules by which nerve cells are connected to each other are only partly understood. A team of scientists around Moritz Helmstaedter at the Frankfiurt Max Planck Institute for Brain Research and Helene Schmidt (Humboldt University in Berlin) have now discovered a surprisingly precise nerve cell connectivity pattern in the part of the cerebral cortex that is responsible for orienting the individual animal or human in space.

The researchers report online in Nature (Schmidt et al., 2017. Axonal synapse sorting in medial entorhinal cortex, DOI: 10.1038/nature24005) that synapses in...

Im Focus: Tiny lasers from a gallery of whispers

New technique promises tunable laser devices

Whispering gallery mode (WGM) resonators are used to make tiny micro-lasers, sensors, switches, routers and other devices. These tiny structures rely on a...

Im Focus: Ultrafast snapshots of relaxing electrons in solids

Using ultrafast flashes of laser and x-ray radiation, scientists at the Max Planck Institute of Quantum Optics (Garching, Germany) took snapshots of the briefest electron motion inside a solid material to date. The electron motion lasted only 750 billionths of the billionth of a second before it fainted, setting a new record of human capability to capture ultrafast processes inside solids!

When x-rays shine onto solid materials or large molecules, an electron is pushed away from its original place near the nucleus of the atom, leaving a hole...

Im Focus: Quantum Sensors Decipher Magnetic Ordering in a New Semiconducting Material

For the first time, physicists have successfully imaged spiral magnetic ordering in a multiferroic material. These materials are considered highly promising candidates for future data storage media. The researchers were able to prove their findings using unique quantum sensors that were developed at Basel University and that can analyze electromagnetic fields on the nanometer scale. The results – obtained by scientists from the University of Basel’s Department of Physics, the Swiss Nanoscience Institute, the University of Montpellier and several laboratories from University Paris-Saclay – were recently published in the journal Nature.

Multiferroics are materials that simultaneously react to electric and magnetic fields. These two properties are rarely found together, and their combined...

All Focus news of the innovation-report >>>

Anzeige

Anzeige

Event News

“Lasers in Composites Symposium” in Aachen – from Science to Application

19.09.2017 | Event News

I-ESA 2018 – Call for Papers

12.09.2017 | Event News

EMBO at Basel Life, a new conference on current and emerging life science research

06.09.2017 | Event News

 
Latest News

Rainbow colors reveal cell history: Uncovering β-cell heterogeneity

22.09.2017 | Life Sciences

Penn first in world to treat patient with new radiation technology

22.09.2017 | Medical Engineering

Calculating quietness

22.09.2017 | Physics and Astronomy

VideoLinks
B2B-VideoLinks
More VideoLinks >>>