Move over, Frankenstein! Your 21st-century
counterpart has just been announced.
In true sci-fi fashion, a team of researchers from The Scripps
Research Institute (TSRI)
in La Jolla, Calif., has created a brand-new bacteria based on a
genetic structure found nowhere on Earth.
According to lead researcher Floyd Romesberg, the feat
involved artificially engineering a unique combination of DNA
material - a combination not found in any living creature - and then
successfully inserting it into a living cell that usually contains
only natural combinations of DNA.
"Life on Earth in all its diversity
is encoded by only two pairs of DNA bases, A-T and C-G,"
Romesberg explained in an institute news release. "And what
we've made is an organism that stably contains those two plus a
third, unnatural pair of bases."
"This shows that other solutions to storing [genetic]
information are possible," he added, "and, of course, takes us
closer to an expanded-DNA biology that will have many exciting
applications - from new medicines to new kinds of
nano-technology."
The product of more than 15 years of work, the current effort builds
on a proof-of-concept study conducted in 2008. At that time,
investigators had shown that hooking up natural and unnatural
pairings of DNA was possible in a test-tube setting.
The next challenge was to replicate the process inside a living
cell.
The cell chosen by the TSRI team was the
common
E. coli bacterium, and into it they
inserted what they considered to be the best unnatural DNA pairing
they could construct: a combination of two molecules called "d5SICS"
and "dNaM".
After leaping through a series of highly complex technical problems,
the study authors finally managed to pull off their goal:
the fashioning on a half-synthetic
organism that could actually replicate its unnatural self as
long as scientists continuously supplied it with the necessary
molecular material.
Romesberg said that, in principle, his
team's high-concept work has a very practical purpose: to gain a
"greater power than ever" to fashion new treatments by harnessing
the power of genetics.
Illustration
highlighting the expansion of the genetic alphabet
by comparison with
natural DNA and Watson's and Crick's original paper.
Credit: Synthorx
Scientists at The Scripps Research Institute (TSRI)
have engineered a bacterium whose genetic material includes an added
pair of DNA "letters," or bases, not found in nature.
The cells of this unique bacterium can
replicate the unnatural DNA bases more or less normally, for as long
as the molecular building blocks are supplied.
"Life on Earth in all its diversity
is encoded by only two pairs of DNA bases, A-T and C-G, and what
we've made is an organism that stably contains those two plus a
third, unnatural pair of bases," said TSRI Associate Professor
Floyd E. Romesberg, who led the research team.
"This shows that other solutions to
storing information are possible and, of course, takes us closer
to an expanded-DNA biology that will have many exciting
applications - from new medicines to new kinds of
nanotechnology."
The report on the achievement appears
May 7, 2014, in an advance online publication of the journal Nature.
Many
Challenges
Floyd E. Romesberg and his laboratory have been working since
the late 1990s to find pairs of molecules that could serve as new,
functional DNA bases - and, in principle, could code for proteins
and organisms that have never existed before.
The task hasn't been a simple one. Any functional new pair of DNA
bases would have to bind with an affinity comparable to that of the
natural nucleoside base-pairs adenine-thymine and cytosine-guanine.
Such new bases also would have to line
up stably alongside the natural bases in a zipper-like stretch of
DNA. They would be required to unzip and re-zip smoothly when worked
on by natural polymerase enzymes during DNA replication and
transcription into RNA.
And somehow these nucleoside interlopers
would have to avoid being attacked and removed by natural DNA-repair
mechanisms.
Scientists create
first living organism that transmits added letters in DNA 'alphabet'
Expanding the genetic alphabet. Credit: Synthorx
Despite these challenges, by 2008 Romesberg and his colleagues had
taken a big step towards this goal.
In a study published that year, they
identified sets of nucleoside molecules that can hook up across a
double-strand of DNA almost as snugly as natural base pairs and
showed that DNA containing these unnatural base pairs can replicate
in the presence of the right enzymes.
In a study that came out the following
year, the researchers were able to find enzymes that transcribe this
semi-synthetic DNA into RNA.
But this work was conducted in the simplified milieu of a test tube.
"These unnatural base pairs have
worked beautifully in vitro, but the big challenge has been to
get them working in the much more complex environment of a
living cell," said Denis A. Malyshev, a member of the Romesberg
laboratory who was lead author of the new report.
The
plasmid DNA contained natural T-A and C-G base pairs along with
the best-performing unnatural base pair Romesberg's laboratory
had discovered, two molecules known as d5SICS and dNaM. The goal
was to get the E. coli cells to replicate this semi-synthetic
DNA as normally as possible.
The greatest hurdle may be reassuring to those who fear the
uncontrolled release of a new life form:
the molecular building
blocks for d5SICS and dNaM are not naturally in cells.
Thus, to
get the E. coli to replicate the DNA containing these unnatural
bases, the researchers had to supply the
molecular building blocks artificially, by adding them to
the fluid solution outside the cell.
Then, to get the building
blocks, known as nucleoside triphosphates, into the cells, they
had to find special triphosphate transporter molecules that
would do the job.
How to expand the
genetic alphabet.
Credit: Synthorx
The researchers eventually were able to find a triphosphate
transporter, made by a species of microalgae, that was good
enough at importing the unnatural triphosphates.
"That was a big
breakthrough for us - an enabling breakthrough," said Malyshev.
Though the completion of the project took another year, no
hurdles that large arose again.
The team found, somewhat to
their surprise, that the semi-synthetic plasmid replicated with
reasonable speed and accuracy, did not greatly hamper the growth
of the E. coli cells, and showed no sign of losing its unnatural
base pairs to DNA repair mechanisms.
"When we stopped the flow of the unnatural triphosphate
building blocks into the cells, the replacement of d5SICS-dNaM
with natural base pairs was very nicely correlated with the cell
replication itself - there didn't seem to be other factors
excising the unnatural base pairs from the DNA," Malyshev said.
"An important thing to note is that these two breakthroughs also
provide control over the system. Our new bases can only get into
the cell if we turn on the 'base transporter' protein.
Without
this transporter or when new bases are not provided, the cell
will revert back to A, T, G, C, and the d5SICS and dNaM will
disappear from the genome."
The next step will be to demonstrate the in-cell
transcription of the new, expanded-alphabet DNA into the RNA
that feeds the protein-making machinery of cells.
"In principle,
we could encode new proteins made from new, unnatural amino
acids - which would give us greater power than ever to tailor
protein therapeutics and diagnostics and laboratory reagents to
have desired functions," Romesberg said.
"Other applications,
such as nano-materials, are also possible."
