Scientists
at Harvard University have harnessed the prowess of fast-replicating
bacterial viruses, also known as phages, to accelerate the evolution of
biomolecules in the laboratory. The work, reported this week in the
journal Nature,
could ultimately allow the tailoring of custom pharmaceuticals and
research tools from lab-grown proteins, nucleic acids, and other such
compounds.
The
researchers, led by Professor David R. Liu, say their approach —
dubbed phage-assisted continuous evolution, or PACE — is roughly 100
times faster than conventional laboratory evolution, and far less
labor-intensive for scientists.
“Most
modern drugs are based on small organic molecules, but biological
macromolecules may be better suited as pharmaceuticals in some cases,”
says Liu, a professor of chemistry and chemical biology at Harvard and
an investigator with the Howard Hughes Medical Institute. “Our work
provides a new solution to one of the key challenges in the use of
macromolecules as research tools or human therapeutics: how to rapidly
generate proteins or nucleic acids with desired properties.”
Liu
and Harvard co-authors Kevin M. Esvelt and Jacob C. Carlson achieved up
to 60 rounds of protein evolution every 24 hours by linking laboratory
evolution to the life cycle of a virus that infects bacteria. This
phage’s life cycle of just 10 minutes is among the fastest known.
Because this generation time is so brief, the phage makes a perfect
vehicle for accelerated protein evolution. The PACE system uses E. coli
host cells to produce the resulting proteins, to serve as factories for
phage production, and to perform the key selection step that allows
phage-carrying genes encoding desired molecules to flourish.
In
three separate protein evolution experiments, PACE was able to generate
an enzyme with a new target activity within a week, achieving up to 200
rounds of protein evolution during that time. Conventional laboratory
evolution methods, Liu says, would require years to complete this many
rounds of evolution.
Evolution
of biomolecules is also a natural process, of course, but during
biological evolution generation times tend to be very long and
researchers have no control over the outcomes. Laboratory evolution
(also called directed evolution) has been practiced for decades to
generate biomolecules with tailor-made properties, but typically
proceeds at a rate of about one round of evolution every few days and
requires frequent sample manipulation by scientists or technicians
during that time.
In
addition to not requiring human intervention during the evolutionary
process, Liu’s new approach uses readily available components and is
designed to be resistant to “cheater” molecules that bypass the desired
selection process. Researchers can control PACE’s selection stringency
as well as its mutation rate.
“Laboratory
evolution has generated many biomolecules with desired properties, but a
single round of mutation, gene expression, screening or selection, and
replication typically requires days or longer with frequent human
intervention,” Liu, Esvelt, and Carlson write in Nature. “Since
evolutionary success is dependent on the total number of rounds
performed, a means of performing laboratory evolution continuously and
rapidly could dramatically enhance its effectiveness.”
Among
other achievements reported in Nature, Liu and colleagues used PACE to
recast an RNA polymerase normally activated by a T7 promoter to
recognize a T3 promoter instead. They also evolved polymerases that
initiate RNA transcripts with the genetic bases adenine (A) or cytosine
(C) instead of the usual guanine (G). In all cases, the PACE-generated
enzymes on their new targets matched or exceeded the activity of
wild-type enzymes.
This
work was supported by the National Institutes of Health, the Howard
Hughes Medical Institute, the Hertz Foundation, the National Science
Foundation, and the Harvard Chemical Biology Graduate Program.