New system joins PC programming and cryo-EM to decide 3D constructions of RNA-only molecules

Overview

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• Source: DOE/SLAC National Accelerator Laboratory


• Date: 16 Aug,2021

The single-stranded gene material RNA, also known as RNA, is most well-known for its ability to guide the assembly of proteins and carry the genetic code for viruses such as HIV and SARS-CoV-2.

Scientists discovered a hidden talent 40 years ago: It can catalyze chemical reaction in cells, including joining and snipping RNA strands. This discovery gave rise to the notion that RNA was responsible for the evolution of large molecules, which ultimately led to human life.

Although scientists have made a lot of progress since then, it’s still difficult to obtain 3D images naked RNA molecules at a high enough resolution to view all the folds and pockets that are crucial to understanding their function. The molecules behave like fidgety children with floppy arms. They won’t stay still for photos unless they are part of a larger molecular structure that holds them in place.

This problem is solved by a new system that was developed at Stanford University. It also includes the Department of Energy’s SLAC National Accelerator Laboratory. This system combines computer software with cryogenic electron microscopy (or cryo-EM) to determine 3D structures of RNA molecules. It is extremely fast, precise, and accurate.

The research team of SLAC/Stanford Professor Wah Chu and Stanford Professor Rhiju das pushes the resolution to as high at 3.1 angstroms – just shy of where individual atoms are visible – and applies it to two RNA structures of deep interest to scientists.

Nature published the first study today. It revealed the first complete-length, near-atomic structure for a catalytic DNA or ribozyme from Tetrahymena, a single-celled creature that lives in pond waste. It was the first known ribozyme and has been used as a laboratory rat to study ribozymes since.

The second, which was published as a preprint reveals small pockets in an RNA fragment from SARS-CoV-2 known as the frameshift stimulation element (or FSE). The FSE subtly tricks infected cells to make alternative sets of virus proteins. It is so important in the virus’s ability of replication that it does not change even if other parts of the virus evolve to create new variants. This makes it a good potential target for drugs to treat COVID-19, its variants and maybe even other coronaviruses, and a number of research groups have been exploring that possibility.

FSE was conducted in 2020 at a time when Stanford and SLAC were closed down by the pandemic. Only essential work on the coronavirus response was permitted.

Based on insights from the 3D structure FSE, Das and his team at Stanford collaborated with Professor Jeff Glenn to create DNA molecules that can pair up and disrupt the structure of FSE.

Although researchers are still far from showing that this molecule can prevent viral infections in humans, scientists say the study points to a path towards developing a treatment.

We don’t know what the next pandemic virus will be, but we’re pretty confident it will be a single-strand RNA virus transmitted from animals to humans, and it will likely have a few bits of RNA that resist mutation. With this accelerated system we’ve developed, it now seems feasible to study viruses found in humans or animals, look for those conserved bits, quickly determine their 3D RNA structures and develop antivirals against them.”

Rhiju Das, Professor, Stanford University

Passionate pursuit of RNA
After Das saw Chiu’s talk about cryo-EM and solving the structure of RNA molecules, Das began to collaborate with Chiu in 2017.

“It blew me away,” Das recalled. “I had fallen in love with RNA in 2001. I thought it was the most important molecule of life. The first RNA molecule I looked at was this Tetrahymena ribozyme. Many, many people had worked on it – it was a bit of a cult molecule – and I spent five years of my PhD work trying to understand how it folds. So after hearing Wah’s talk I suggested that we work together to determine its structure.”

As far as scientists can tell, the ribozyme has no biological function in Tetrahymena, Das said: “It’s an inconsequential molecule in what some might consider an inconsequential organism.” However, 40 years ago Thomas Cech discovered that a tiny piece of RNA could be cut from a TetrahymenaRNA strand and then float away. “It was this miraculous thing that no one expected anRNA strand to do by itself,” Das stated. They realized immediately that this piece RNA was a multi-step machine, a catalyst. Cech was awarded the 1989 Nobel Prize in Chemistry in Chemistry.

Chiu began a similar relationship with cryo-EM while a graduate student at University of California, Berkeley in 1970. He is now the co-director of Stanford-SLAC CryoEM Facilities. This facility allows for imaging to be done. His career has been devoted to perfecting the technique.

Chiu stated that it was a long-held dream of his to use cryoEM to study RNA in all forms. “I consider obtaining these RNA structures to be one of my greatest accomplishments. We can theoretically do this with any number of molecules if we can do it with one.

Creating an RNA pipeline
Ribosolve is a pipeline that Chiu and Das developed in the years before the pandemic. It allows them to solve the structures of RNAs quickly, more accurately and with greater detail than ever before. It combines the computational tools of Kalli Kappel, a Stanford PhD student, with cryo-EM imaging advancements from Kaiming Zhang (postdoctoral researcher) and Zhaoming Su.

The team published a paper in Nature Methods last spring, reporting that they used the new approach to determine 3D structures for the Tetrahymena Ribozyme and 10 other molecules of RNA with greater than 10 angstrom resolution.

Jane S. Richardson (Duke University professor of biochemistry), wrote in a comment that was included with the report, “Each one of these eleven new structures turned out to offer biological or biochemical insight.” The approach is a “groundbreaking” new one that creates fast and reliable structures for RNA-only molecules. She also noted that a higher resolution of 2-4 angstroms would be a good demonstration of its utility for proteins and RNA.

The team reported in their Nature paper that they have now achieved a higher resolution for Tetrahymena’s ribozyme. They are hoping to push for FSE and will eventually produce atomic-resolution structures of these and possibly thousands of other RNAs.

Das stated, “I believe the Ribosolve pipeline can transform our understanding and possibly our ability to create medicines.” This could not have happened elsewhere. Access to top-quality cryo-EM instruments was crucial, as well as meeting Wah, who shared our belief that this could be important.