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Using R2D2 to Understand RNA Folding

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If you love learning more about biology at a fundamental level, I have a great video for you! It simulates the 3D folding of RNA. RNA is a single stranded molecule, but it is still capable of forming internal loops that can be stabilized by base pairing, just like its famously double-stranded parent, DNA. Understanding more about RNA folding may be valuable in many different areas of biomedical research, including developing ways to help people with RNA-related diseases, such as certain cancers and neuromuscular disorders, and designing better mRNA vaccines against infectious disease threats (like COVID-19).

Because RNA folding starts even while an RNA is still being made in the cell, the process has proven hugely challenging to follow closely. An innovative solution, shown in this video, comes from the labs of NIH grantees Julius Lucks, Northwestern University, Evanston, IL, and Alan Chen, State University of New York at Albany. The team, led by graduate student Angela Yu and including several diehard Star Wars fans, realized that to visualize RNA folding they needed a technology platform that, like a Star Wars droid, is able to “see” things that others can’t. So, they created R2D2, which is short for Reconstructing RNA Dynamics from Data.

What’s so groundbreaking about the R2D2 approach, which was published recently in Molecular Cell, is that it combines experimental data on RNA folding at the nucleotide level with predictive algorithms at the atomic level to simulate RNA folding in ultra-slow motion [1]. While other computer simulations have been available for decades, they have lacked much-needed experimental data of this complex folding process to confirm their mathematical modeling.

As a gene is transcribed into RNA one building block, or nucleotide, at a time, the elongating RNA strand folds immediately before the whole molecule is fully assembled. But such folding can create a problem: the new strand can tie itself up into a knot-like structure that’s incompatible with the shape it needs to function in a cell.

To slip this knot, the cell has evolved immediate corrective pathways, or countermoves. In this R2D2 video, you can see one countermove called a toehold-mediated strand displacement. In this example, the maneuver is performed by an ancient molecule called a single recognition particle (SRP) RNA. Though SRP RNAs are found in all forms of life, this one comes from the bacterium Escherichia coli and is made up of 114 nucleotides.

The colors in this video highlight different domains of the RNA molecule, all at different stages in the folding process. Some (orange, turquoise) have already folded properly, while another domain (dark purple) is temporarily knotted. For this knotted domain to slip its knot, about 5 seconds into the video, another newly forming region (fuchsia) wiggles down to gain a “toehold.” About 9 seconds in, the temporarily knotted domain untangles and unwinds, and, finally, at about 23 seconds, the strand starts to get reconfigured into the shape it needs to do its job in the cell.

Why would evolution favor such a seemingly inefficient folding process? Well, it might not be inefficient as it first appears. In fact, as Chen noted, some nanotechnologists previously invented toehold displacement as a design principle for generating synthetic DNA and RNA circuits. Little did they know that nature may have scooped them many millennia ago!


[1] Computationally reconstructing cotranscriptional RNA folding from experimental data reveals rearrangement of non-naïve folding intermediates. Yu AM, Gasper PM Cheng L, Chen AA, Lucks JB, et. al. Molecular Cell 8, 1-14. 18 February 2021.


Ribonucleic Acid (RNA) (National Human Genome Research Institute/NIH)

Chen Lab (State University of New York at Albany)

Lucks Laboratory (Northwestern University, Evanston IL)

NIH Support: National Institute of General Medical Sciences; Common Fund

Caught on Camera: Neutralizing Antibodies Interacting with SARS-CoV-2

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Caption: Illustration showing the binding regions for the four classes of SARS-CoV-2 neutralizing antibodies. They bind to a part of the virus’s spike protein called the receptor binding domain (gray). Credit: Christopher Barnes, California Institute of Technology, Pasadena

As this long year enters its final month, there is good reason to look ahead to 2021 with optimism that the COVID-19 pandemic will finally be contained. The Food and Drug Administration is now reviewing the clinical trial data of the Pfizer and Moderna vaccines to ensure their safety and efficacy. If all goes well, emergency use authorization could come very soon, allowing immunizations to begin.

Work also continues on developing better therapeutics against SARS-CoV-2, the novel coronavirus that causes COVID-19. Though we’ve learned a great deal about this coronavirus in a short time, structural biologists continue to produce more detailed images that reveal more precisely where and how to target SARS-CoV-2. This research often involves neutralizing antibodies that circulate in the blood of most people who’ve recovered from COVID-19. The study of such antibodies and how they interact with SARS-CoV-2 offers critical biological clues into how to treat and prevent COVID-19.

A recent study in the journal Nature brings more progress, providing the most in-depth analysis yet of how human neutralizing antibodies physically grip SARS-CoV-2 to block it from binding to our cells [1]. To conduct this analysis, a team of NIH-supported structural biologists, led by postdoc Christopher Barnes and Pamela Björkman, California Institute of Technology, Pasadena, used the power of cryo-electron microscopy (cryo-EM) to capture complex molecular interactions at near-atomic scale.

People infected with SARS-CoV-2 (or any foreign substance, for that matter) generate thousands of different versions of attack antibodies. Some of these antibodies are very good at sticking to the coronavirus, while others attach only loosely. Barnes used cryo-EM to capture highly intricate pictures of eight different human neutralizing antibodies bound tightly to SARS-CoV-2. Each of these antibodies, which had been isolated from patients a few weeks after they developed symptoms of COVID-19, had been shown in lab tests to be highly effective at blocking infection.

The researchers mapped all physical interactions between several human neutralizing antibodies and SARS-CoV-2’s spike protein that stud its surface. The virus uses these spiky extensions to infect a human cell by grabbing on to the angiotensin-converting enzyme 2 (ACE2) receptor. The molecular encounter between the coronavirus and ACE2 takes place via one or more of a trio of three protein domains, called receptor-binding domains (RBDs), that jut out from its spikes. RBDs flap up and down in the fluid surrounding cells, “reaching up” to touch and enter, or “laying down” to hide from an infected person’s antibodies and immune cells. Only an “up” RBD can attach to ACE2 and get into a cell.

Taken together with other structural information known about SARS-CoV-2, Barnes’ cryo-EM snapshots revealed four different types of shapes, or classes, of antibody-spike combinations. These high-resolution molecular views show that human neutralizing antibodies interact in many different ways with SARS-CoV-2: blocking access to either one or more RBDs in their “up” or “down” positions.

These results tell us a number of things, including underscoring why strategies that combine multiple types of antibodies in an “antibody cocktail” might likely offer broader protection against infection than using just a single type of antibody. Indeed, that approach is currently being tested in patients with COVID-19.

The findings also provide a molecular guide for custom-designing synthetic antibodies in the lab to foil SARS-CoV-2. As one example, Barnes and his team observed that one antibody completely locked all three RBDs into closed (“down”) positions. As you might imagine, scientists might want to copy that antibody type when designing an antibody-based drug or vaccine.

It is tragic that hundreds of thousands of people have died from this terrible new disease. Yet the immune system helps most to recover. Learning as much as we possibly can from those individuals who’ve been infected and returned to health should help us understand how to heal others who develop COVID-19, as well as inform precision design of additional vaccines that are molecularly targeted to this new foe.

While we look forward to the arrival of COVID-19 vaccines and their broad distribution in 2021, each of us needs to remember to practice the three W’s: Wear a mask. Watch your distance (stay 6 feet apart). Wash your hands often. In parallel with everyone adopting these critical public health measures, the scientific community is working harder than ever to meet this moment, doing everything possible to develop safe and effective ways of treating and preventing COVID-19.


[1] SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies. Barnes CO, Jette CA, Abernathy ME, et al. Nature. 2020 Oct 12. [Epub ahead of print].


Coronavirus (COVID-19) (NIH)

Combat COVID (U.S. Department of Health and Human Services, Washington, D.C.)

Freezing a Moment in Time: Snapshots of Cryo-EM Research (National Institute of General Medical Sciences/NIH)

Björkman Lab (California Institute of Technology, Pasadena)

NIH Support: National Institute of General Medical Sciences; National Institute of Allergy and Infectious Diseases

Speeding COVID-19 Drug Discovery with Quantum Dots

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Viruses with nanoparticles attached
Credit: Ethan Tyler and Alan Hoofring/NIH Medical Arts

These round, multi-colored orbs in the illustration above may resemble SARS-CoV-2, the coronavirus responsible for COVID-19. But they’re actually lab-made nanocrystals called quantum dots. They have been specially engineered to look and, in some ways, act like the coronavirus while helping to solve a real challenge for many labs that would like to study SARS-CoV-2.

Quantum dots, which have been around since the mid-1980s, are designed with special optical properties that allow them to fluoresce when exposed to ultraviolet light. The two pictured here are about 10 nanometers in diameter, about 3,000 times smaller than the width of a human hair. The quantum dot consists of a semi-conductive cadmium selenide inner core (orange) surrounded by a zinc sulfide outer shell (teal). Molecules on its surface (yellow) allow researchers to attach the viral spike protein (purple), which SARS-CoV-2 depends on to infect human cells.

To the left is a human cell (gray) studded with the ACE2 receptors (blue) that those viral spike proteins bind to before SARS-CoV-2 enters and infects our cells. In the background, you see another spike protein-studded quantum dot. But human neutralizing antibodies (pink) are preventing that one from reaching the human cell.

Because SARS-CoV-2 is so highly infectious, basic researchers without access to specially designed biosafety facilities may be limited in their ability to study the virus. But these harmless quantum dots offer a safe workaround. While the quantum dots may bind and enter human cells just like the virus, they can’t cause an infection. They offer a quick, informative way to assess the potential of antibodies or other compounds to prevent the coronavirus from binding to our cells.

In work published in the journal ACS Nano, a team that included Kirill Gorshkov, NIH’s National Center for Advancing Translational Sciences (NCATS), Rockville, MD, along with Eunkeu Oh and Mason Wolak, Naval Research Laboratory, Washington, D.C., demonstrated how these quantum dots may serve as a useful new tool to speed the search for new COVID-19 treatments. The dots’ fluorescent glow enabled the researchers to use a microscope to observe how these viral mimics bind to ACE2 in real time, showing how SARS-CoV-2 might attach to and enter our cells, and suggesting ways to intervene.

Indeed, imagine thousands of tiny wells in which human cells are growing. Imagine adding a different candidate drug to each well; then imagine adding the loaded quantum dots to each well and using machine vision to identify the wells where the dots could not enter the cell. That’s not science fiction. That’s now.

With slightly different versions of their quantum dots, the NCATS researchers and their colleagues at the Naval Research Laboratory will now explore how other viral proteins are important for the coronavirus to infect our cells. They also can test how slight variations in the spike protein may influence SARS-CoV-2’s behavior. This work provides yet another stunning example of how scientists with widely varying expertise have banded together—using all the tools at their disposal—to forge ahead to find solutions to COVID-19.


[1] Quantum dot-conjugated SARS-CoV-2 spike pseudo-virions enable tracking of angiotensin converting enzyme 2 binding and endocytosis. Gorshkov K, Susumu K, Chen J, Xu M, Pradhan M, Zhu W, Hu X, Breger JC, Wolak M, Oh E. ACS Nano. 2020 Sep 22;14(9):12234-12247.


What are Quantum Dots? (National Institute of Biomedical Imaging and Bioengineering/NIH)

Coronavirus (COVID-19) (NIH)

I Am Translational Science: Kirill Gorshkov (National Center for Advancing Translational Sciences/NIH)

U. S. Naval Research Laboratory (Washington, D.C.)

NIH Support: National Center for Advancing Translational Sciences

The Prime Cellular Targets for the Novel Coronavirus

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Credit: NIH

There’s still a lot to learn about SARS-CoV-2, the novel coronavirus that causes COVID-19. But it has been remarkable and gratifying to watch researchers from around the world pull together and share their time, expertise, and hard-earned data in the urgent quest to control this devastating virus.

That collaborative spirit was on full display in a recent study that characterized the specific human cells that SARS-CoV-2 likely singles out for infection [1]. This information can now be used to study precisely how each cell type interacts with the virus. It might ultimately help to explain why some people are more susceptible to SARS-CoV-2 than others, and how exactly to target the virus with drugs, immunotherapies, and vaccines to prevent or treat infections.

This work was driven by the mostly shuttered labs of Alex K. Shalek, Massachusetts Institute of Technology, Ragon Institute of MGH, MIT, and Harvard, and Broad Institute of MIT and Harvard, Cambridge; and Jose Ordovas-Montanes at Boston Children’s Hospital. In the end, it brought together (if only remotely) dozens of their colleagues in the Human Cell Atlas Lung Biological Network and others across the U.S., Europe, and South Africa.

The project began when Shalek, Ordovas-Montanes, and others read that before infecting human cells, SARS-CoV-2 docks on a protein receptor called angiotensin-converting enzyme 2 (ACE2). This enzyme plays a role in helping the body maintain blood pressure and fluid balance.

The group was intrigued, especially when they also learned about a second enzyme that the virus uses to enter cells. This enzyme goes by the long acronym TMPRSS2, and it gets “tricked” into priming the spike proteins that cover SARS-CoV-2 to attack the cell. It’s the combination of these two proteins that provide a welcome mat for the virus.

Shalek, Ordovas-Montanes, and an international team including graduate students, post-docs, staff scientists, and principal investigators decided to dig a little deeper to find out precisely where in the body one finds cells that express this gene combination. Their curiosity took them to the wealth of data they and others had generated from model organisms and humans, the latter as part of the Human Cell Atlas. This collaborative international project is producing a comprehensive reference map of all human cells. For its first draft, the Human Cell Atlas aims to gather information on at least 10 billion cells.

To gather this information, the project relies, in part, on relatively new capabilities in sequencing the RNA of individual cells. Keep in mind that every cell in the body has essentially the same DNA genome. But different cells use different programs to decide which genes to turn on—expressing those as RNA molecules that can be translated into protein. The single-cell analysis of RNA allows them to characterize the gene expression and activities within each and every unique cell type. Based on what was known about the virus and the symptoms of COVID-19, the team focused their attention on the hundreds of cell types they identified in the lungs, nasal passages, and intestines.

As reported in Cell, by filtering through the data to identify cells that express ACE2 and TMPRSS2, the researchers narrowed the list of cell types in the nasal passages down to the mucus-producing goblet secretory cells. In the lung, evidence for activity of these two genes turned up in cells called type II pneumocytes, which line small air sacs known as alveoli and help to keep them open. In the intestine, it was the absorptive enterocytes, which play an important role in the body’s ability to take in nutrients.

The data also turned up another unexpected and potentially important connection. In these cells of interest, all of which are found in epithelial tissues that cover or line body surfaces, the ACE2 gene appeared to ramp up its activity in concert with other genes known to respond to interferon, a protein that the body makes in response to viral infections.

To dig further in the lab, the researchers treated cultured cells that line airways in the lungs with interferon. And indeed, the treatment increased ACE2 expression.

Earlier studies have suggested that ACE2 helps the lungs to tolerate damage. Completely missed was its connection to the interferon response. The researchers now suspect that’s because it hadn’t been studied in these specific human epithelial cells before.

The discovery suggests that SARS-CoV-2 and potentially other coronaviruses that rely on ACE2 may take advantage of the immune system’s natural defenses. When the body responds to the infection by producing more interferon, that in turn results in production of more ACE2, enhancing the ability of the virus to attach more readily to lung cells. While much more work is needed, the finding indicates that any potential use of interferon as a treatment to fight COVID-19 will require careful monitoring to determine if and when it might help patients.

It’s clear that these new findings, from data that weren’t originally generated with COVID-19 in mind, contained several potentially important new leads. This is another demonstration of the value of basic science. We can also rest assured that, with the outpouring of effort from members of the scientific community around the globe to meet this new challenge, progress along these and many other fronts will continue at a remarkable pace.


[1] SARS-CoV-2 receptor ACE2 is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues. Ziegler, CGK et al. Cell. April 20, 2020.


Coronaviruses (National Institute of Allergy and Infectious Diseases/NIH)

Human Cell Atlas (Broad Institute, Cambridge, MA)

Shalek Lab (Harvard Medical School and Massachusetts Institute of Technology, Cambridge)

Ordovas-Montanes Lab (Boston Children’s Hospital, MA)

NIH Support: National Institute of Allergy and Infectious Diseases; National Institute of General Medical Sciences; National Heart, Lung, and Blood Institute

Bringing Needed Structure to COVID-19 Drug Development

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SARS-Cov-2 Molecular Map
Caption: Molecular map showing interaction between the spike protein (gold) of the novel coronavirus and the peptidase domain (blue) of human angiotensin-converting enzyme 2 (ACE2). Credit: Adapted from Yan R., Science, 2020.

With so much information swirling around these days about the coronavirus disease 2019 (COVID-19) pandemic, it would be easy to miss one of the most interesting and significant basic science reports of the past few weeks. It’s a paper published in the journal Science [1] that presents an atomic-scale snapshot showing the 3D structure of the spike protein on the novel coronavirus attached to a human cell surface protein called ACE2, or angiotensin converting enzyme 2. ACE2 is the receptor that the virus uses to gain entry.

What makes this image such a big deal is that it shows—in exquisite detail—how the coronavirus attaches to human cells before infecting them and making people sick. The structural map of this interaction will help guide drug developers, atom by atom, in devising safe and effective ways to treat COVID-19.

This new work, conducted by a team led by Qiang Zhou, Westlake Institute for Advanced Study, Hangzhou, China, took advantage of a high-resolution imaging tool called cryo-electron microscopy (cryo-EM). This approach involves flash-freezing molecules in liquid nitrogen and bombarding them with electrons to capture their images with a special camera. When all goes well, cryo-EM can solve the structure of intricate macromolecular complexes in a matter of days, including this one showing the interaction between a viral protein and human protein.

Zhou’s team began by mapping the structure of human ACE2 in a complex with B0AT1, which is a membrane protein that it helps to fold. In the context of this complex, ACE2 is a dimer—a scientific term for a compound composed of two very similar units. Additional mapping revealed how the surface protein of the novel coronavirus interacts with ACE2, indicating how the virus’s two trimeric (3-unit) spike proteins might bind to an ACE2 dimer. After confirmation by further research, these maps may well provide a basis for the design and development of therapeutics that specifically target this critical interaction.

The ACE2 protein resides on the surface of cells in many parts of the human body, including the heart and lungs. The protein is known to play a prominent role in the body’s complex system of regulating blood pressure. In fact, a class of drugs that inhibit ACE and related proteins are frequently prescribed to help control high blood pressure, or hypertension. These ACE inhibitors lower blood pressure by causing blood vessels to relax.

Since the COVID-19 outbreak, many people have wondered whether taking ACE inhibitors would be helpful or detrimental against coronavirus infection. This is of particular concern to doctors whose patients are already taking the medications to control hypertension. Indeed, data from China and elsewhere indicate hypertension is one of several coexisting conditions that have consistently been reported to be more common among people with COVID-19 who develop life-threatening severe acute respiratory syndrome.

In a new report in this week’s New England Journal of Medicine, a team of U.K. and U.S. researchers, partly supported by NIH, examined the use of ACE inhibitors and other angiotensin-receptor blockers (ARBs) in people with COVID-19. The team, led by Scott D. Solomon of Brigham and Women’s Hospital and Harvard Medical School, Boston, found that current evidence in humans is insufficient to support or refute claims that ACE inhibitors or ARBs may be helpful or harmful to individuals with COVID-19.

The researchers concluded that these anti-hypertensive drugs should be continued in people who have or at-risk for COVID-19, stating: “Although additional data may further inform the treatment of high-risk patients … clinicians need to be cognizant of the unintended consequences of prematurely discontinuing proven therapies in response to hypothetical concerns.” [2]

Research is underway to generate needed data on the use of ACE inhibitors and similar drugs in the context of the COVID-19 pandemic, as well as to understand more about the basic mechanisms underlying this rapidly spreading viral disease. This kind of fundamental research isn’t necessarily the stuff that will make headlines, but it likely will prove vital to guiding the design of effective drugs that can help bring this serious global health crisis under control.


[1] Structural basis for the recognition of the SARS-CoV-2 by full-length human ACE2. Yan R, Zhang Y, Li Y, Xia L, Guo Y, Zhou Q. Science. 27 March 2020. [Epub ahead of publication]

[2] Renin–Angiotensin–Aldosterone System Inhibitors in Patients with Covid-19. Vaduganathan M, Vardeny O, Michel T, McMurray J, Pfeffer MA, Solomon SD. 30 NEJM. March 2020 [Epub ahead of Publication]


Coronavirus (COVID-19) (NIH)

COVID-19, MERS & SARS (National Institute of Allergy and Infectious Diseases/NIH)

Transformative High Resolution Cryo-Electron Microscopy (Common Fund/NIH)

Qiang Zhou (Westlake Institute for Advanced Study, Zhejiang Province)

Scott D. Solomon (Brigham and Women’s Hospital, Boston)

NIH Support: National Center for Advancing Translational Sciences; National Heart, Lung, and Blood Institute

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