Nanotechnology enables visualization of RNA structures at near-atomic resolution

Date:
May 2, 2022
Source:
Wyss Institute for Biologically Inspired Engineering at Harvard
Summary:
Researchers have reported a fundamentally new approach to the
structural investigation of RNA molecules. ROCK, as it is called,
uses an RNA nanotechnological technique that allows it to assemble
multiple identical RNA molecules into a highly organized structure,
which significantly reduces the flexibility of individual RNA
molecules and multiplies their molecular weight. The team showed
that their method enables the structural analysis of the contained
RNA subunits with a technique known as cryo-electron microscopy
(cryo-EM).



FULL STORY ==========================================================================
We live in a world made and run by RNA, the equally important sibling of
the genetic molecule DNA. In fact, evolutionary biologists hypothesize
that RNA existed and self-replicated even before the appearance of
DNA and the proteins encoded by it. Fast forward to modern day humans:
science has revealed that less than 3% of the human genome is transcribed
into messenger RNA (mRNA) molecules that in turn are translated into
proteins. In contrast, 82% of it is transcribed into RNA molecules with
other functions many of which still remain enigmatic.


==========================================================================
To understand what an individual RNA molecule does, its 3D structure
needs to be deciphered at the level of its constituent atoms and
molecular bonds.

Researchers have routinely studied DNA and protein molecules by
turning them into regularly packed crystals that can be examined with
an X-ray beam (X-ray crystallography) or radio waves (nuclear magnetic resonance). However, these techniques cannot be applied to RNA molecules
with nearly the same effectiveness because their molecular composition
and structural flexibility prevent them from easily forming crystals.

Now, a research collaboration led by Wyss Core Faculty member Peng
Yin, Ph.D.

at the Wyss Institute for Biologically Inspired Engineering at Harvard University, and Maofu Liao, Ph.D. at Harvard Medical School (HMS), has
reported a fundamentally new approach to the structural investigation
of RNA molecules.

ROCK, as it is called, uses an RNA nanotechnological technique that
allows it to assemble multiple identical RNA molecules into a highly
organized structure, which significantly reduces the flexibility of
individual RNA molecules and multiplies their molecular weight. Applied to well-known model RNAs with different sizes and functions as benchmarks,
the team showed that their method enables the structural analysis of the contained RNA subunits with a technique known as cryo-electron microscopy (cryo-EM). Their advance is reported in Nature Methods.

"ROCK is breaking the current limits of RNA structural investigations and enables 3D structures of RNA molecules to be unlocked that are difficult
or impossible to access with existing methods, and at near-atomic
resolution," said Yin, who together with Liao led the study. "We expect
this advance to invigorate many areas of fundamental research and drug development, including the burgeoning field of RNA therapeutics." Yin
also is a leader of the Wyss Institute's Molecular Robotics Initiative
and Professor in the Department of Systems Biology at HMS.

Gaining control over RNA Yin's team at the Wyss Institute has pioneered
various approaches that enable DNA and RNA molecules to self-assemble into large structures based on different principles and requirements, including
DNA bricks and DNA origami. They hypothesized that such strategies could
also be used to assemble naturally occurring RNA molecules into highly
ordered circular complexes in which their freedom to flex and move is
highly restricted by specifically linking them together. Many RNAs fold
in complex yet predictable ways, with small segments base-pairing with
each other. The result often is a stabilized "core" and "stem-loops"
bulging out into the periphery.



==========================================================================
"In our approach we install 'kissing loops' that link different peripheral stem-loops belonging to two copies of an identical RNA in a way that
allows a overall stabilized ring to be formed, containing multiple copies
of the RNA of interest," said Di Liu, Ph.D., one of two first-authors
and a Postdoctoral Fellow in Yin's group. "We speculated that these higher-order rings could be analyzed with high resolution by cryo-EM,
which had been applied to RNA molecules with first success." Picturing stabilized RNA In cryo-EM, many single particles are flash-frozen
at cryogenic temperatures to prevent any further movements, and then
visualized with an electron microscope and the help of computational
algorithms that compare the various aspects of a particle's 2D surface projections and reconstruct its 3D architecture. Peng and Liu teamed
up with Liao and his former graduate student Franc,ois The'lot, Ph.D.,
the other co-first author of the study. Liao with his group has made
important contributions to the rapidly advancing cryo-EM field and the experimental and computational analysis of single particles formed by
specific proteins.

"Cryo-EM has great advantages over traditional methods in seeing
high- resolution details of biological molecules including proteins,
DNAs and RNAs, but the small size and moving tendency of most RNAs
prevent successful determination of RNA structures. Our novel method of assembling RNA multimers solves these two problems at the same time,
by increasing the size of RNA and reducing its movement," said Liao,
who also is Associate Professor of Cell Biology at HMS. "Our approach
has opened the door to rapid structure determination of many RNAs by
cryo-EM." The integration of RNA nanotechnology and cryo-EM approaches
led the team to name their method "RNA oligomerization- enabled cryo-EM
via installing kissing loops" (ROCK).

To provide proof-of-principle for ROCK, the team focused on a large intron
RNA from Tetrahymena, a single-celled organism, and a small intron RNA
from Azoarcus, a nitrogen-fixing bacterium, as well as the so-called
FMN riboswitch.

Intron RNAs are non-coding RNA sequences scattered throughout the
sequences of freshly-transcribed RNAs and have to be "spliced" out in
order for the mature RNA to be generated. The FMN riboswitch is found
in bacterial RNAs involved in the biosynthesis of flavin metabolites
derived from vitamin B2. Upon binding one of them, flavin mononucleotide
(FMN), it switches its 3D conformation and suppresses the synthesis of
its mother RNA.

"The assembly of the Tetrahymena group I intron into a ring-like structure
made the samples more homogenous, and enabled the use of computational
tools leveraging the symmetry of the assembled structure. While our
dataset is relatively modest in size, ROCK's innate advantages allowed
us to resolve the structure at an unprecedented resolution," said
The'lot. "The RNA's core is resolved at 2.85 AA [one AAngstro"m is one ten-billions (US) of a meter and the preferred metric used by structural biologists], revealing detailed features of the nucleotide bases and
sugar backbone. I don't think we could have gotten there without ROCK
-- or at least not without considerably more resources." Cryo-EM also
is able to capture molecules in different states if they, for example,
change their 3D conformation as part of their function. Applying ROCK
to the Azoarcus intron RNA and the FMN riboswitch, the team managed to
identify the different conformations that the Azoarcus intron transitions through during its self-splicing process, and to reveal the relative conformational rigidity of the ligand-binding site of the FMN riboswitch.

"This study by Peng Yin and his collaborators elegantly shows
how RNA nanotechnology can work as an accelerator to advance other
disciplines. Being able to visualize and understand the structures of
many naturally occurring RNA molecules could have tremendous impact on
our understanding of many biological and pathological processes across different cell types, tissues, and organisms, and even enable new drug development approaches," said Wyss Founding Director Donald Ingber, M.D., Ph.D., who is also the Judah Folkman Professor of Vascular Biology at
Harvard Medical School and Boston Children's Hospital, and Professor of Bioengineering at the Harvard John A. Paulson School of Engineering and
Applied Sciences.

The study was also authored by Joseph Piccirilli, Ph.D., an expert
in RNA chemistry and biochemistry and Professor at The University of
Chicago. It was supported by the National Science Foundation (NSF;
grant# CMMI-1333215, CCMI- 1344915, and CBET-1729397), Air Force Office
of Scientific Research (AFOSR; grant MURI FATE, #FA9550-15-1-0514),
National Institutes of Health (NIH; grant# 5DP1GM133052, R01GM122797,
and R01GM102489), and the Wyss Institute's Molecular Robotics Initiative.


========================================================================== Story Source: Materials provided
by Wyss_Institute_for_Biologically_Inspired_Engineering_at
Harvard. Original written by Benjamin Boettner. Note: Content may be
edited for style and length.


========================================================================== Journal Reference:
1. Di Liu, Franc,ois A. The'lot, Joseph A. Piccirilli, Maofu Liao,
Peng Yin.

Sub-3-AA cryo-EM structure of RNA enabled by engineered
homomeric self- assembly. Nature Methods, 2022; DOI:
10.1038/s41592-022-01455-w ==========================================================================

Link to news story: https://www.sciencedaily.com/releases/2022/05/220502120510.htm

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