Scientists at The Scripps Research Institute (TSRI) have taken a big step toward the laboratory re-creation of the "RNA world," which is generally believed to have preceded modern life forms based on DNA and proteins.
"This is probably the first time some of these complex RNA molecules have been synthesized with a ribozyme [a special RNA enzyme] since the end of the RNA world four billion years ago," said TSRI Professor Gerald F. Joyce, the senior author of the study.
The results from the study, reported this week in the online Early Edition of the Proceedings of the National Academy of Sciences, show the scientists have succeeded in creating a ribozyme that can basically serve both to amplify genetic information and to generate functional molecules.
The new ribozyme can replicate short lengths of RNA efficiently and perform transcription on even longer RNAs to make functional RNA molecules with complex structures--coming close to what scientists imagine in terms of an RNA replicator that could have supported life before modern biology, where protein enzymes now handle gene replication and transcription.
Researchers including Feng Zhang have confirmed that a bacterial protein hypothesized as a tool for targeted editing of RNA, similar to how CRISPR-Cas9 targets DNA, can indeed be used as an alternate editing approach. The finding holds important implications for a range of biological applications, such as marking, modifying and modulating RNA. Roughly half of all bacteria species utilize an immune system called the Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes (CRISPR-Cas), which protects the microbes from viruses and other invading DNA. While much focus in recent years has been directed at the ability to harness CRISPR-Cas systems to edit DNA, systems that specifically target RNA have been less studied. Here, Zhang and colleagues build on previous work to characterize the behavior of a new type of CRISPR-CAS system, C2c2, suspected to target RNA. Through a series of experiments, the researchers demonstrate that C2c2 can be used to cleave single-stranded RNA, but not double-stranded RNA; as well, it can be used to knockout messenger RNA of bacteria in vivo. In testing the ability of C2c2 to target specific RNA in Escherichia coli, the team found that it initially focuses on its target RNA, followed by a second phase in which it degrades RNA in a less specific way. Other RNA-targeting immune systems likely exist, the authors say, and further research will lead to the development of programmable molecular tools for in vivo RNA manipulation. One example includes modulating RNA function and translation, which could be used for large-scale screening of biological molecules, construction of synthetic regulatory circuits and other purposes, the authors say.
The crucibles that bore out early building blocks of life may have been, in many cases, modest puddles.
Now, researchers working with that hypothesis have achieved a significant advancement toward understanding an evolutionary mystery -- how components of RNA and DNA formed from chemicals present on early Earth before life existed.
In surprisingly simple laboratory reactions in water, under everyday conditions, they have produced what could be good candidates for missing links on the pathway to the code of life.
And when those components joined up, the result even looked like RNA.
As the researchers' work progresses, it could reveal that much of the original chemistry that led to life arose not in fiery cataclysms and in scarce quantities, but abundantly and gradually on quiet, rain-swept dirt flats or lakeshore rocks lapped by waves.
In turn, their work could increase our understanding of the probability of life's existence elsewhere in the universe.
The research from the NSF/NASA Center for Chemical Evolution, headquartered at the Georgia Institute of Technology, is generously funded through a grant from the National Science Foundation and NASA. The recent results were published on April 25, 2016 in Nature Communications.
Pursuing the origins specifically of RNA, the close chemical relative of DNA, a research team led by Nicholas Hud, a professor in the School of Chemistry and Biochemistry at the Georgia Institute of Technology and director of the CCE, worked with a pair of potential chemical ancestors of the nucleobases of RNA.
For roughly half a century, scientists have hypothesized that life, which uses DNA to store genetic information, was preceded by life forms that used RNA very broadly. And RNA may have had a precursor, proto-RNA, with different but similar nucleotides (the "N" in RNA).
"Early Earth was a messy laboratory where probably many molecules like those needed for life were produced. Some survived and prospered, while others eventually vanished," Hud said. "That goes for the ancestors of RNA, too."
Using two molecules known as barbituric acid and melamine, the researchers formed proto-nucleotides so strongly resembling two of RNA's nucleotides that it is tempting to speculate that they are indeed their ancestors.
The two ingredients would have been readily abundant for reactions on a prebiotic Earth, Hud said. "And they would have been well suited for primitive information coding," he added.
Because of the resemblances and properties, some scientists already have speculated on an ancestral role for melamine and barbituric acid.
But the CCE scientists are careful not to jump to that conclusion just yet.
"To claim ancestry, we would have to show a mechanism by which these nucleotides we made in the lab could turn into the existing nucleotides in RNA," said Ram Krishnamurthy, Hud's collaborator from the Scripps Research Institute in La Jolla, California. "It's a complex path that we'd have to at least design on paper, and we're not there."
NASA-funded researchers at the Georgia Institute of Technology are tapping information found in the cells of all life on Earth, and using it to trace life's evolution. They have learned that life is a master stenographer - writing, rewriting and recording its history in elaborate biological structures.
Some of the keys to unlocking the origin of life lie encrypted in the ribosome, life's oldest and most universal assembly of molecules. Today's ribosome converts genetic information (RNA) into proteins that carry out various functions in an organism. But the ribosome itself has changed over time. Its history shows how simple molecules joined forces to invent biology, and its current structure records ancient biological processes that occurred at the root of the Tree of Life, some 3.8 billion years ago.
By examining variations in the ribosomal RNA contained in modern cells, scientists can visualize the timeline of life far back in history, elucidating molecular structures, reactions and events near the biochemical origins of life.
A cross-chiral RNA polymerase ribozyme
Authors:
Sczepanski et al
Abstract:
Thirty years ago it was shown that the non-enzymatic, template-directed polymerization of activated mononucleotides proceeds readily in a homochiral system, but is severely inhibited by the presence of the opposing enantiomer. This finding poses a severe challenge for the spontaneous emergence of RNA-based life, and has led to the suggestion that either RNA was preceded by some other genetic polymer that is not subject to chiral inhibition or chiral symmetry was broken through chemical processes before the origin of RNA-based life. Once an RNA enzyme arose that could catalyse the polymerization of RNA, it would have been possible to distinguish among the two enantiomers, enabling RNA replication and RNA-based evolution to occur. It is commonly thought that the earliest RNA polymerase and its substrates would have been of the same handedness, but this is not necessarily the case. Replicating d- and l-RNA molecules may have emerged together, based on the ability of structured RNAs of one handedness to catalyse the templated polymerization of activated mononucleotides of the opposite handedness. Here we develop such a cross-chiral RNA polymerase, using in vitro evolution starting from a population of random-sequence RNAs. The d-RNA enzyme, consisting of 83 nucleotides, catalyses the joining of l-mono- or oligonucleotide substrates on a complementary l-RNA template, and similar behaviour occurs for the l-enzyme with d-substrates and a d-template. Chiral inhibition is avoided because the 106-fold rate acceleration of the enzyme only pertains to cross-chiral substrates. The enzyme’s activity is sufficient to generate full-length copies of its enantiomer through the templated joining of 11 component oligonucleotides.
pop sci write up
here.
When Francis Crick and James Watson discovered the double helical structure of deoxyribonucleic acid (DNA) in 1953, it began a genetic revolution to map, study, and sequence the building blocks of living organisms.
DNA encodes the genetic material passed on from generation to generation. For the information encoded in the DNA to be made into the proteins and enzymes necessary for life, ribonucleic acid (RNA), single-stranded genetic material found in the ribosomes of cells, serve as intermediary. Although usually single-stranded, some RNA sequences were predicted to have the ability to form a double helix, much like DNA.
In 1961, Alexander Rich along with David Davies, Watson, and Crick, hypothesized that the RNA known as poly (rA) could form a parallel-stranded double helix based on the results of fibre diffraction experiments.
Fifty years later, scientists from McGill University successfully crystallized a short RNA sequence, poly (rA)11, and used data collected at the Canadian Light Source (CLS) and the Cornell High Energy Synchrotron to confirm the hypothesis of a poly (rA) double-helix.
The detailed 3D structure of poly (rA)11 was published by the laboratory of Dr. Kalle Gehring, McGill University, in collaboration with George Sheldrick, University of Göttingen, and Christopher WIlds, Concordia University. The paper appeared in the journal Angewandte Chemie International Edition under the title of "Structure of the Parallel Duplex of Poly (A) RNA: Evaluation of a 50 year-Old Prediction."
"After 50 years of study, the identification of a novel nucleic acid structure is very rare. So when we came across the unusual crystals of poly (rA), we jumped on it," said Dr. Gehring.
Gehring said identifying the double-helical RNA will have interesting applications for research in biological nanomaterials and supramolecular chemistry. Nucleic acids have astounding properties of self-recognition and their use as a building material opens new possibilities for the fabrication of bionanomachines – nanoscale devices created using synthetic biology.
link.
The St. Laurent Institute, a non-profit medical research institute focused on the systems biology of disease, today announced in a study published in the July edition of Genome Biology, that genetic matter, previously ignored by the scientific community, may play an important role in cancer. The study, "VlincRNAs controlled by retroviral elements are a hallmark of pluripotency and cancer" found that novel non-coding parts of the human genome known as vlincRNAs (very long intergenic, non-coding RNAs) triggered by ancient viruses, participate in the biology of stem cells, and in the development of cancer. Importantly, the group of researchers from U.S., Europe and Russia found that the elimination of these vlincRNAs caused the death of cancer cells.
"Understanding this previously ignored part of the human genome, its role in human development, and how it may be taken over by disease, opens a new frontier in science with important implications for medical advances," said Philipp Kapranov, Ph.D., lead researcher at the St. Laurent Institute. "Future research into the role and function of vlincRNAs holds promise for both highly targeted diagnostic tests and more precise cancer treatments.
Up to 98 percent of human genomic matter is known as "junk" or "dark matter" non-coding DNA, and had for years attracted little interest among scientists who doubted its role in human health and disease. Recent research has begun to identify that part of that non-coding DNA is used by the cell to make RNA such as vlincRNA, highly tissue-specific RNA chains of unusually large lengths, many of which are only found in embryonic or cancerous cells. VlincRNAs found in these two types of cells tend to be expressed based upon genetic signals from ancient viruses that invaded our ancestors' genome millions of years ago and were gradually "domesticated" over evolutionary time. The number of vlincRNAs expressed by these domesticated viral sequences correlates with both embryonic development and malignant cancers.
A new study shows how complex biochemical transformations may have been possible under conditions that existed when life began on the early Earth.
The study shows that RNA is capable of catalyzing electron transfer under conditions similar to those of the early Earth. Because electron transfer, the moving of an electron from one chemical species to another, is involved in many biological processes – including photosynthesis, respiration and the reduction of RNA to DNA – the study's findings suggest that complex biochemical transformations may have been possible when life began.
There is considerable evidence that the evolution of life passed through an early stage when RNA played a more central role, before DNA and coded proteins appeared. During that time, more than 3 billion years ago, the environment lacked oxygen but had an abundance of soluble iron.
"Our study shows that when RNA teams up with iron in an oxygen-free environment, RNA displays the powerful ability to catalyze single electron transfer, a process involved in the most sophisticated biochemistry, yet previously uncharacterized for RNA," said Loren Williams, a professor in the School of Chemistry and Biochemistry at the Georgia Institute of Technology.
The results of the study were scheduled to be published online on May 19, 2013, in the journal Nature Chemistry. The study was sponsored by the NASA Astrobiology Institute, which established the Center for Ribosomal Origins and Evolution (Ribo Evo) at Georgia Tech.
Free oxygen gas was almost nonexistent in the Earth's atmosphere more than 3 billion years ago. When free oxygen began entering the environment as a product of photosynthesis, it turned the earth's iron to rust, forming massive banded iron formations that are still mined today. The free oxygen produced by advanced organisms caused iron to be toxic, even though it was – and still is – a requirement for life. Williams believes the environmental transition caused a slow shift from the use of iron to magnesium for RNA binding, folding and catalysis.
Williams and Georgia Tech School of Chemistry and Biochemistry postdoctoral fellow Chiaolong Hsiao used a standard peroxidase assay to detect electron transfer in solutions of RNA and either the iron ion, Fe2+, or magnesium ion, Mg2+. For 10 different types of RNA, the researchers observed catalysis of single electron transfer in the presence of iron and absence of oxygen. They found that two of the most abundant and ancient types of RNA, the 23S ribosomal RNA and transfer RNA, catalyzed electron transfer more efficiently than other types of RNA. However, none of the RNA and magnesium solutions catalyzed single electron transfer in the oxygen-free environment.
"Our findings suggest that the catalytic competence of RNA may have been greater in early Earth conditions than in present conditions, and our experiments may have revived a latent function of RNA," added Williams, who is also director of the RiboEvo Center.
In modern biology, all life, with the exception of some viruses, uses DNA as its genetic storage mechanism. According to the "RNA-world" hypothesis, RNA appeared on Earth first, serving as both the genetic-storage material and the functional molecules for catalyzing chemical reactions, then DNA and proteins evolved much later. Unlike DNA, RNA can adopt many different molecular conformations and so it is functionally interactive on the molecular level. In the soon-to-be-published research paper, two professors of chemistry, Christine Keating and Philip Bevilacqua, and two graduate students, Christopher Strulson and Rosalynn Molden, probe one of the nagging mysteries of the RNA-world hypothesis.
"A missing piece of the RNA-world puzzle is compartmentalization," Bevilacqua said. "It's not enough to have the necessary molecules that make up RNA floating around; they need to be compartmentalized and they need to stay together without diffusing away. This packaging needs to happen in a small-enough space -- something analogous to a modern cell -- because a simple fact of chemistry is that molecules need to find each other for a chemical reaction to occur."
To test how early cell-like structures could have formed and acted to compartmentalize RNA molecules even in the absence of lipid-like molecules that make up modern cellular membranes, Strulson and Molden generated simple, non-living model "cells" in the laboratory. "Our team prepared compartments using solutions of two polymers called polyethylene glycol (PEG) and dextran," Keating explained. "These solutions form distinct polymer-rich aqueous compartments, into which molecules like RNA can become locally concentrated."
The team members found that, once the RNA was packed into the dextran-rich compartments, the molecules were able to associate physically, resulting in chemical reactions. "Interestingly, the more densely the RNA was packed, the more quickly the reactions occurred," Bevilacqua explained. "We noted an increase in the rate of chemical reactions of up to about 70-fold. Most importantly, we showed that for RNA to 'do something' -- to react chemically -- it has to be compartmentalized tightly into something like a cell. Our experiments with aqueous two-phase systems (ATPS) have shown that some compartmentalization mechanism may have provided catalysis in an early-Earth environment."
Keating added that, although the team members do not suggest that PEG and dextran were the specific polymers present on the early Earth, they provide a clue to a plausible route to compartmentalization -- phase separation. "Phase separation occurs when different types of polymers are present in solution at relatively high concentrations. Instead of mixing, the sample separates to form two distinct liquids, similar to how oil and water separate." Keating explained. "The aqueous-phase compartments we manufactured using dextran and PEG can drive biochemical reactions by increasing local reactant concentrations. So, it's possible that some other sorts of polymers might have been the molecules that drove compartmentalization on the early Earth." Strulson added that, "In addition to the RNA-world hypothesis, these results may be relevant to RNA localization and function in non-membrane compartments in modern biology."
The team members also found that the longer the string of RNA, the more densely it would be packed into the dextran compartment of the ATPS, while the shorter strings tended to be left out. "We hypothesize that this research result might indicate some kind of primitive sorting method," Bevilacqua said. "As RNA gets shorter, it tends to have less enzyme activity. So, in an early-Earth system similar to our dextran-PEG model system, the full-length, functional RNA would have been sorted and concentrated into one phase, while the shorter RNA that is not only less functional, but also threatens to inhibit important chemical reactions, would not have been included."
