RNA EXPLORED IN EFFORT TO DEVELOP ARTIFICIAL ENZYMES AND TO UNDERSTAND THE ORIGINS OF LIFE

June 12, 2012 | Denise Henderson Vaughn

Can molecules of ribonucleic acid - RNA - be modified so that they can instruct cells to do things not currently being done by living organisms, such as teaching human cells to ward off viruses, or causing bacteria to "eat" toxic waste and process it into harmless by-products?

What roles might RNA have played in assisting proliferation among the one-cell organisms that constituted the earliest forms of life on Earth?

These two questions about RNA - one looking to the future and one looking to the past - are contained within current research in the lab of Bond Life Sciences Center investigator Donald Burke. He's looking to answer these questions and others, funded by two new grants that together total close to $1.5 million. One is from the National Science Foundation and the other from NASA.

Burke is working in a new field, synthetic biology, and in it "we're building artificial component parts of cells that can be used by cells in new and exciting ways," he said.

Donald Burke

While Burke's two questions are at opposite ends of the spectrum in terms of past and future, they have in common an investigation into evolution. He smiles when he calls his research "evolution in a bottle." This evocative description reflects the sophisticated techniques Burke uses to mimic nature's evolutionary process to select RNA components that are best suited for certain tasks.

Burke begins with a "polylibrary" of a quadrillion random molecules; they are strings of nucleotides manufactured by a research lab, and each string has a different genetic sequence. These nucleotides represent potential RNA components that might perform a desired function. Burke uses a biochemical process to screen these random molecules for characteristics he is seeking. Promising molecules are physically separated out, reproduced in large numbers and the "offspring" molecules are tested again. This screening and narrowing procedure continues - sometimes up through 20 generations - with processing for each generation taking as little as a day or as much as two weeks. Thus, Burke's process resembles evolution over eons, but it is compressed into a comparatively short time frame.

Any desired function that RNA might be likely to accomplish could be searched for using this technique, Burke said.

With this process, scientists hope to design controllable molecules that would carry out some of the same reactions that cells do naturally. For example, the research might result in being able to artificially regulate genes. One possibility Burke can foresee is RNA interacting with a cell's own control mechanisms, "so that stem cells develop the way you want them to, in a way a doctor can control from outside the patient," he said.

Alternately, some newly designed molecules might be able to carry out functions previously not available anywhere. For example, if biologists could engineer new ways that cells deal with nutrients and other chemicals they encounter, then that might lead to "teaching bacteria to turn environmental toxins into food, and to break them down into by-products," Burke said. This could serve as a means of bioremediation for toxic waste sites.

Possibilities abound. One is harnessing RNA to improve energy extraction from biofuels, Burke said. Or, "you could use it (RNA) to engineer crops, or bacteria to make useful pharmaceutical agents for drugs, or anti-malaria components."

Admittedly, some of those potential directions are several years off, Burke said. But first, the question is: "Can RNA do any of these things? That's the nature of the experiments we're doing," he said.

Recognition of RNA's many roles has expanded in recent years

Much has been learned about RNA since the first catalytic RNA molecules were discovered 30 years ago. Originally, RNA was thought to only encode proteins, meaning an RNA molecule serves as a copy of a segment of DNA, which is used to determine how to build a protein that does the work in a cell. Providing cells with protein-making machinery is indeed RNA's predominant role, but in the years since its discovery, research has demonstrated that RNA can carry out many other tasks. For example, a type of RNA known as Xist is responsible for inactivating one of a female's two X-chromosomes.

Burke, an MU associate professor in Molecular Microbiology and Immunology/Biochemistry, has contributed to RNA advancements. Along with labs at Harvard and Yale, his lab has successfully coaxed RNA into attaching a phosphate molecule onto itself. Burke's recent work has fundamentally advanced understanding of how RNA can achieve those reactions.

The wave of the future, Burke said, is to fine-tune RNA in ways that biologists care about, to see what RNA can do artificially, to create RNA that never existed in nature, and to discover new ways that RNA can participate in the workings of cells.

The movement of phosphates is fundamental to life processes

Within any living organism, complex biochemical interchanges take place at the cellular level that can direct the cell's behavior and sometimes will influence the whole organism. High in importance among these chemical interchanges is the movement of phosphates. Attaching a phosphate to a protein is often the trigger that "turns on" or "turns off" that protein's ability to perform a certain function. Enzymes called kinases are the vehicles that attach the phosphates.

This method of controlling cell function, known as phosphorylation, "is ubiquitous," Burke said. Nature uses it in hundreds of ways to trigger specific actions. For example, phosphorylation helps the body maintain balanced water content; also, phosphates transfer the signals that enable cells in the retina to process incoming light.

At this time, scientists can observe but cannot control the manner in which enzymes, proteins, phosphates and cells work together to accomplish various missions. "If we can control the process that attaches or removes phosphates from a protein, then we can control that protein's activities - get it to do or not do whatever it was designed to accomplish," Burke said.

That's where Burke's research with RNA comes in. In that polylibrary of RNA parts, Burke is looking for those needle-in-a-haystack RNA sequences that behave in a similar manner as the kinases that move phosphates. "We want to build artificial enzymes made of RNA molecules" that can carry out those reactions, he said. Burke hopes to "create artificial metabolic pathways that do biochemical reactions."

Drug companies are watching this type of medical research. "Phosphorylation of specific proteins controls cell division, which is very important for cancer research," Burke said. "It (phosphorylation) also activates immune responses, which is important for protection from pathogens and for controlling diseases of autoimmunity and allergy. As such, there is a major push in pharmaceutical research to find compounds that control the enzymes that add phosphates to, and remove phosphates from, specific proteins."

NSF and NASA: different uses for similar research

The two agencies that have recently funded Burke's work have different goals for closely related research. Both grants ask: "What is possible for RNA to do?" Both explore the biochemistry of using RNA to move phosphates around. The NFS grant emphasizes proteins, in particular developing artificial RNA molecules that can turn proteins on and off by manipulating their phosphorylation state. This work is expected to yield results that could aid development of artificial components for cells, as described above.

In contrast, the NASA grant emphasizes small molecules: putting phosphates on the building blocks of RNA and DNA.

RNA somewhat resembles DNA and is thought to have been its precursor billions of years ago. Many theories of how life began on Earth emphasize RNA's role, saying it was the "molecule of life" in an "RNA world." Thus, RNA is believed to have been long ago the predominant genetic molecule (instead of DNA) and the predominant enzymatic molecule (instead of protein).

With the NASA grant, Burke is looking to simulate ancient conditions that might have allowed early RNA to foster cell reproduction. In addition, Burke is exploring whether RNA can assist cells in utilizing environmental sources of phosphates (rather than internal biological sources), because early life forms would have needed at some point to harvest raw phosphorus.

NASA is interested because the knowledge might inform searches for extraterrestrial life, Burke said. "If you look for life around other stars, you need to know what to look for. Understanding origins of life on Earth helps; it informs the exploration mission," he said.

With these two new grants, Burke is simultaneously delving into RNA's involvement in reproducing the first life on Earth - evolution - and he is also looking for ways to artificially evolve RNA to carry out helpful new tasks that could alter the Earth's future.