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‘Accelerated Evolution’ Converts RNA Enzyme To DNA Enzyme In Vitro

Posted by tumicrobiology on March 28, 2006

Scientists at The Scripps Research Institute have successfully converted an RNA enzyme (ribozyme) into a DNA enzyme (deoxyribozyme) through a process of accelerated in vitro evolution. The molecular conversion or transfer of both genetic information and catalytic function between these two different genetic systems, which are both based on nucleic acid-like molecules, is exactly what many scientists believe occurred during the very earliest period of earth's existence.

This "evolutionary conversion" provides a modern-day snapshot of how life as we understand it may have first evolved out of the earliest primordial mix of RNA-like molecules-sometimes referred to as the "pre-RNA world"-into a more complex form of RNA-based life (or the "RNA world") and eventually to cellular life based on DNA and proteins. Nucleic acids are large complex molecules that store and convey genetic information, but can also function as enzymes.

While the transfer of sequence information between two different classes of nucleic acid-like molecules-between RNA and DNA, for example-is straightforward because it relies on the one-to-one correspondence of the double helix pairing, transferring catalytic function is significantly more difficult because function cannot be conveyed sequentially. The present study demonstrates that the "evolutionary conversion" of an RNA enzyme to a DNA enzyme with the same function is possible, however, through the acquisition of a few critical mutations.

The study was released in an advance online version of the journal Chemistry & Biology.

Scripps Research Professor Gerald F. Joyce, a member of the Skaggs Institute for Chemical Biology whose laboratory conducted the study, said, "During early life on earth both genetic information and catalytic function were thought to reside only in RNA. In our study, the evolutionary transition from an RNA to a DNA enzyme represents a genuine change, rather than a simple expansion, of the chemical basis for catalytic function. This means that similar evolutionary pathways may exist between other classes of nucleic acid-like molecules. These findings could help answer some fundamental questions concerning the basic structure of life and how it evolved over time."

As Francis Crick, the Nobel laureate who, along with James Watson uncovered the double helix structure of DNA, articulated in 1970, all known organisms operate according to the central dogma of molecular biology-that the transfer of sequential genetic information proceeds from nucleic acid to nucleic acid, and from nucleic acid to protein. But a far different situation exists with regard to the transfer of catalytic function, which does not occur sequentially in contemporary biology. The new study shows that catalytic function can be transmitted sequentially between two different nucleic acid-like molecules, suggesting how it might have been conveyed from pre-RNA molecules to RNA during the simpler pre-RNA world period.

There are several candidates for the initial pre-RNA molecule, all of which have the ability to form base-paired structures with themselves and with RNA. Cross-pairing would allow genetic information to be transferred from these pre-RNA molecules to RNA. The catalytic function of these early enzymes might have been transferred to a corresponding RNA enzyme following the acquisition of a few critical mutations, the study said, just as the evolutionary change of a ribozyme to a deoxyribozyme with the same or similar catalytic functions might also have occurred through random mutation and selection.

For the study, an RNA ribozyme was converted to a corresponding deoxyribozyme through in vitro evolution. The ribozyme was first prepared as a DNA molecule of the same RNA sequence but with no detectable catalytic activity. A large number of randomized variations of this DNA were prepared, and repeated cycles of in vitro evolution were carried out. The result was a deoxyribozyme with about the same level of catalytic activity as the original ribozyme.

"The use of in vitro evolution provides the means to convert a ribozyme to a corresponding deoxyribozyme rapidly," Joyce said. "In the laboratory these procedures allow us to carry out many generations of test tube evolution. The resulting molecules have interesting catalytic properties, they teach us something new about evolution, and they have potential application as therapeutic and diagnostic agents."

Other authors of the study include Natasha Paul and Greg Springsteen. The study was supported by the National Aeronautics and Space Administration, the National Institutes of Health, and Johnson & Johnson Research.
Rice Bioengineers Pioneer Techniques For Knee Repair
A breakthrough self-assembly technique for growing replacement cartilage offers the first hope of replacing the entire articular surface of knees damaged by arthritis. The technique, developed at Rice University's Musculoskeletal Bioengineering Laboratory, is described in this month's issue of the journal Tissue Engineering

This has significant ramifications because we are now beginning to talk, for the first time, about the potential treatment of entire arthritic joints and not just small defects," said lead researcher and lab director Kyriacos Athanasiou, the Karl F. Hasselmann Professor of Bioengineering.

Athanasiou's new self-assembly method involves a break from conventional wisdom in bioengineering; almost all previous attempts to grow replacement transplant tissues involved the use of biodegradable implants that are seeded with donor cells and growth factors. These implants, which engineers refer to as scaffolds, foster the tissue growth process by acting as a template for new growth, but they always present a risk of toxicity due to the fact that they are made of materials that aren't naturally found in the body.

In the newly reported findings, Athanasiou and postdoctoral researcher Jerry Hu, using nothing but donor cells, grew dime-sized disks of cartilage with properties approaching those of native tissue. In a follow-up study due for publication soon, graduate student Christopher Revell refined the process to produce disks that are virtually identical to native tissue in terms of both mechanical and biochemical makeup. In a third, and perhaps most impressive breakthrough, Athanasiou and Hu used the self-assembly approach to grow the entire articular surface of the distal femur. Each of these unbroken samples were tailored three-dimensionally to fit a specific rabbit femur.

"If you told me 10 years ago that we would be making entire articular end caps via self assembly I would have said you were crazy," said Athanasiou. "The fact that we can do this is an indication of how far the discipline of tissue engineering has progressed."

Unlike cartilage, most tissues in our bodies — including skin, blood vessels and bone — regenerate themselves constantly. Tissue engineers try to capitalize on the body's own regenerative powers to grow replacement tissues that can be transplanted without risk of rejection. Donor cells from the patient are used as a starting place to eliminate rejection risks.

Most tissue engineering involves honeycombed plastic templates or hydrogels called scaffolds that are used to guide colonies of donor cells. Donor cells can be either adult stem cells or other immature cells. Athanasiou's latest work was done using chondrocytes, or cartilage cells.

Athanasiou, a former president of the international Biomedical Engineering Society, helped pioneer the development of coin-sized scaffolds in the early 1990s that are now the state-of-the-art clinical option for repairing small defects in articular knee cartilage.

His lab is also working on techniques to grow replacement knee menisci, the kidney shaped wedges of cartilage that sit between the femur and tibia and absorb the compressive shock that the bones undergo during walking and running. Over the past 18 months, he and his students Adam Aufderheide and Gwen Hoben have perfected methods of growing meniscus-shaped pieces of cartilage, but they are still trying to perfect the mechanical strength of the engineered meniscus tissue, which must be able to withstand up to an astounding 2,400 pounds per square inch of compressive pressure.

Athanasiou's research is funded by Rice University and the National Institutes of Health.

(TUMDF posted this news here for TU Microbiologists)

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