U

C G

U C

U UA

G A

C

U GG

C A

C C UU

C U

AC G

G AC

A
^C U G U ^G U CU^GAUGA G _AG GCAGG
CG

G

UCCGGU GUCC U C AGGCCA CAGG

U_GU _{C UAA}AG A

G

GC UA

||_GC AAAAA CG

L2bulge1, aptamer unbound, ribozyme active conformation

G
UC
C
U
A AG
U
U
CU
GG

C A

C GU

C UC

AC U

G A

C

^C U ^G _CU_U CU^GAUGA _{G A}G ^GA _G

C

G UCCGGU GUCC U C
AGGCCA CAGG G
U_GU _{CUA G A AG}
GC G_{C G}
A
A
UA
||CG
_GC AAAAA

×

L2bulge1, aptamer bound, ribozyme inactive conformation

an example of a synthetic riboswitch engineered by maung nyan Win and Christina smolke in which the ribozyme is turned off when the aptamer binds ligand.

that might enable completely new approaches to medical technology.

In 2004, for example, Shapiro and his colleagues created a test-tube “ molecular computer” consisting of three interconnected modules. The first module sensed the concentrations of four types of messenger RNA, the working copies of the genetic instructions in DNA, which are used to produce proteins. The second module performed a “diagnosis,” computing whether two of the messenger RNA levels decreased while two others increased, a signature that might indicate a disease. Depending on the results of the computation, the third module dispensed a drug molecule. “We demonstrated the whole process, beginning to end,” Shapiro notes, “but in a test tube.”

To both sense specific strands of messenger RNA and to perform the computation, the Weizmann researchers exploited the sequence-specific matching of DNA strands. So far, though, they have not operated their molecular computer in the complex environment of a living cell. Other teams have had success with different schemes. For instance, a group including Shapiro’s former collaborator Yaakov Benenson, now a researcher at the FAS Center for Systems Biology at Harvard University, demonstrated computation—but not sensing—in cultured human kidney cells. They exploited the newly discovered phenomenon of RNA interference, in which the presence of short RNA templates activates cellular mechanisms that suppress protein synthesis for matching messenger RNA.

A Caltech team led by Christina Smolke, now a professor of bioengineering at Stanford University, designed complex RNA molecules that included three separate sections, performing sensing, computation, and actuation. Although all three modules are part of the same molecule, they act independently, so the function of each part can be separately modified, she says. “You have this plug-and-play type capability to build many types of functions from a smaller set of modular components.” The RNA molecules they design are manufactured by yeast or even mammal cells after the researchers insert the corresponding DNA.

In addition to computing Boolean logic operations, Smolke’s team has demonstrated other signal-processing functions, including bandpass filtering and adjustable signal gain, with their RNA platform. But she acknowledges

in addition to
computing Boolean
logic operations,
a Caltech team has
demonstrated other
signal-processing
functions with its
Rna platform.

that the field has yet to settle on the best approach. “Ultimately, you want to get to a place where there’s some level of standardization,” she says.

send in the Clones

One barrier to standardization is the wide range of possibilities for using biological agents in medicine. Smolke’s technique, for example, might be used to genetically modify cells in a particular tissue, but she is also exploring modification of immune cells outside of the body to combat cancer. “We’re utilizing the function that [the immune cell] already does really well, and then endowing it with enhanced functions,” she says.

Chris Anderson, a professor in the department of bioengineering at the University of California, Berkeley, envisions a different strategy, one based on engineered bacteria, but admits that “it’s impossible to know what’s going to win out.” A “huge advantage” of using bacteria, Anderson says, is that the biological processes targeted by antibacterial agents are very different from those of human cells, so the engineered bacteria can be easily killed.

For bacteria to be effective, they must be able to evade the body’s immune defenses. Anderson and his colleagues have transplanted genes from other bacteria that allow their E. coli to survive for hours in the bloodstream, instead of just a few minutes. They also introduced growth-control mechanisms into the bacteria, he stressed. “They’re not able to grow without feeding something to the patient.”

maung nyan Win anD christina D. smoLke, Proc. natL. acaD. sci. 104, 14287 (2007)