Hertz Fellow David Horning's Breakthrough Research Helps Re-Create What May Have Happened at the Beginning of Life

September 29, 2016

Researchers at The Scripps Research Institute (TSRI) in La Jolla, California, including Hertz Fellow David Horning, have made important progress in re-creating some of the biochemical processes and ingredients that might have enabled early RNA molecules to replicate and evolve. The work has generated considerable attention, and in recent weeks, Horning and his laboratory director, Gerald Joyce, have been explaining what they did to a number of publications that follow this research such as Science, Quanta and The Scientist.

Their findings, reported last month in the Proceedings of the National Academy of Sciences (PNAS), titled "Amplification of RNA by an RNA Polymerase Ribozyme", support key aspects of the widely accepted “RNA World” hypothesis, in which single-stranded RNA molecules are thought to have constituted one of the earliest forms of life – if not the earliest form of life -- some 4 billion years ago.

In this view, RNA replicated itself in a fashion able to experience mutations that could lead to evolution. It could both store genetic information and catalyze chemical reactions in primitive cells. This is a process that was taken over much later by RNA’s more stable, double-stranded cousin DNA, which is essential to the replication of modern cells but must be aided by proteins, which act as catalysts, and RNA, which is required for information transfer.

Both DNA and RNA encode information in molecules, called nucleotides: the familiar ATCG in DNA and AUCG in RNA. The nucleotides are represented by the first letters of their molecular names. They bind in exclusive pairs: In DNA, A always to T and C always to G. In RNA the mandatory pairs are AU and CG.

Many biologists see the presence of RNA in modern cells as living fossils of the ancient RNA World. Since the 1990s, scientists have been creating RNA enzymes, called ribozymes, that can generate complementary RNA from short template sequences of RNA nucleotides, a process called polymerization. Like a mirror image of a mirror image, the complement to the complement would then be a fresh copy of the original sequence – in other words, RNA replication. Although there have been important advances in ribozyme polymerization studies, all were limited to using a small set of template sequences composed mostly of C, for cytosine, in the list of RNA nucleotides. Since most ribozymes have more complex sequences, ribozyme polymerization could not make other ribozymes, nor generate complements of complements to achieve RNA replication.

Horning thought the emphasis should be on whether the ribozymes could make other functional RNAs – including RNA that could serve, for example, as a catalyst (i.e. another ribozyme) – rather than whether they could copy a particular sequence more quickly. For the RNA World hypothesis to have been a functioning place billions of years ago, ribozymes must have existed that could generate a wide range of longer, functional RNA sequences.

David had become interested in the field as an undergraduate at Harvard. Although his initial interest was in physics, his focus changed when he found a summer position in the laboratory of Jack Szostak, a leader in the field of molecular biology. In a few years, Szostak would win a Nobel Prize for his role in discovering how telomeres protect human chromosomes. In the meantime, however, working in Szostak’s lab “basically got me hooked” on the challenge of trying to understand and re-create aspects of evolution and the origins of life, Horning says. By the time he was ready for graduate school, he was making plans to generate the kinds of functional ribozymes that might more realistically mirror the ancient world.

It was a highly ambitious scientific effort, made possible by the support of the Hertz Fellowship – and, of course, by acceptance at Scripps, into the laboratory of Gerald Joyce, another eminent molecular biologist. “Even three-quarters of the way through my PhD, it wasn’t clear that things were going to work or that we’d end up with something better than before,” he said. The Hertz Fellowship “gave me multiple years to work on this very difficult project rather than going for something easier but not as groundbreaking in terms of moving toward synthetic life.”

His PhD work took him partway, laying the groundwork for what was to come. The support he had received from Joyce and the sense that things were just reaching fruition when his degree was awarded tipped the scales in favor of staying on at Scripps for postdoctoral work rather than moving elsewhere.

The achievement reported in PNAS was achieved by generating trillions of variations of an existing ribozyme, introducing slight, random changes to its molecular sequence. The result was a very large, diverse pool of ribozymes which could be tested for their ability to create two different RNA molecules. In a process known generically as "directed evolution", those ribozymes that were successful were then separated out to undergo further mutation, testing and selection to undergo the process again. After 24 such cycles, each one generating more complex ribozymes, they had a ribozyme labeled 24-3 polymerase that could copy nearly any other RNA and increase the presence of specific RNA strands 10,000-fold. As reported in Science, citing the PNAS article, the work “provided the first RNA version of the polymerase chain reaction, a widely used technique to make copies of DNA.”

Joyce and Horning are now working on improvements to 24-3 polymerase, which is tightly folded and cannot copy itself, a necessary step in creation of a self-replicating system that has the potential to evolve on its own. Horning is optimistic that they are on the right track to do so, but he notes how often high hopes have been disappointed in the past.

Like other experts in the field, he recognizes that there might have been very early cells that had characteristics of life before RNA. The Scripps effort is not aimed at creating those but at something with more staying power, “the simplest imaginable Darwinian system that can exist on Earth.

“What we like about where we are right now is that all the pieces that you would want in a self-replicating system are there. They’re just not good enough. Evolution is really, really good at improving things which exist -- that is, at optimizing existing functions.”

 

 

science image
(A) Diagram of the 24-3 polymerase ribozyme copying RNA. Template RNA is shown as tan, RNA synthesized by the polymerase as cyan. (B) Complex functional RNAs made by the 24-3 polymerase: a ligase ribozyme (top) that can join pieces of RNA together and a transfer RNA (bottom) from yeast, used to translate RNA into protein. Coloring as in (A). (C) Amplification of RNA by an RNA enzyme. Small amounts of input RNA template are amplified thousands of fold in a polymerase chain reaction (PCR) using only RNA and no proteins.