Synthetic biologists and nanobiologists are re-purposing DNA to build nanofactories, drug-delivering nanostructures, and molecular devices that can sense their environment. However, current methods for autonomously synthesizing arbitrary single-stranded DNA are limited and impeding progress.
Peng Yin, Ph.D., at Harvard's Wyss Institute for Biologically Inspired Engineering presents a solution to this problem in a Nature Chemistry paper published today. Yin and his team developed a method that allows pre-designed sequences of DNA to autonomously grow and concatenate along specific assembly routes. Putting their Primer Exchange Reaction (PER) cascades to the test, they successfully engineered a set of devices with diverse functions, such as self-building DNA-origami and DNA nanostructures that sense, amplify, record, or logically evaluate environmental signals.
"The autonomous and programmable features that PER cascades offer could engender an entirely new generation of programmable molecular devices and applications and close gaps in design efforts, for which many moving parts already exist," said Yin, who led the study and is also professor of systems biology at Harvard Medical School. "We provide proof-of-concept data for PER in a diverse range of state-of-the-art synthetic biology applications that clearly highlight the technology's broad potential."
To start the PER cascade, two basic components are needed. One is called a catalytic DNA hairpin mediator, which is a single-stranded DNA molecule that partially pairs up with itself to form a hairpin structure with a short overhanging single strand. This overhang is designed to capture the PER cascades' second component, the primer, which contains a region that is complementary to the overhang. Through a series of elongation and displacement reactions, the primer is extended with a sequence provided by the catalytic hairpin mediator and then expelled. This frees up the catalytic hairpin mediator to cascade the next round of the process, either by capturing a new starting primer or the already elongated primer.
"The approach gives us tremendous creative freedom: we can not only synthesize the same piece of DNA again and again as new additions of a growing sequence, but we can also vary the types of DNA sequences to be appended simply by changing the composition of catalytic hairpin DNAs and primers in the mix while the assembly is ongoing. This allows us to have the synthesis branch off into different directions and to intricately pattern the composition of the final DNA transcript," said the study's first author Jocelyn Kishi, who as an NSF Graduate Research Fellow at HMS works on Yin's Wyss Institute team. "We are now working toward implementing PER cascades for a variety of applications, including molecular recorders, sophisticated diagnostics, and tissue imaging. We also hope that someday these systems can be used in living cells as devices that can record events or re-program cell behavior in specific ways."
Image: Primer Exchange Reaction (PER) cascades enable the autonomous growth of single-stranded DNAs. On the top, a 'catalytic PER hairpin' binds a first 'primer' (shown as a short grey strand), triggers its elongation with a sequence encoded by the hairpin itself, and releases it to start another cycle with the already extended primer, and so forth, until a long transcript is generated. Image courtesy of Wyss Institute at Harvard University.