Scientists succeed in replicating semi-synthetic DNA containing unnatural bases

May 13, 2014

Researchers at The Scripps Research Institute (TSRI) have succeeded in replicating deoxyribonucleic acid (DNA) which contained artificially inserted synthetic pair of DNA “letters,” or bases, not found in nature. It was done through an engineered E. coli bacterium which replicated the unnatural semi-synthetic DNA. Replication is the process whereby DNA makes a copy of itself before cell division.

Scientists have been working for long to identify bases other than organic that could serve as new, functional DNA bases and could code for proteins and organisms that have never existed before. Generally, organisms on earth have natural nucleoside base-pairs adenine-thymine and cytosine-guanine which forms their DNA.

This experiment was lead by TSRI Associate Professor Floyd E. Romesberg. In 2008, his lab had identified sets of nucleoside molecules that can hook up across a double-strand of DNA practically as comfortably as organic base pairs (A, T, C, G) and showed that DNA containing these unnatural base pairs can replicate in the presence of the correct enzymes. In the following year, the researchers had been able to locate enzymes that transcribe this semi-synthetic DNA into RNA. But all this was carried out in vitro (in test tube) and this time they wanted to see them working in the significantly extra complicated atmosphere of a living cell.

Latest Experiment:

This time scientists engineered a stretch of circular DNA known as a plasmid and inserted it into cells of the typical bacterium E. coli. The plasmid DNA contained all-natural T-A and C-G base pairs along with the unnatural base pair Romesberg’s lab had discovered, two molecules identified as d5SICS and dNaM. The objective was to get the E. coli cells to replicate this semi-synthetic DNA as typically as possible.

Why it is not easy to get a DNA with synthetic base pair/pairs replicated in a living cell?

There are a number of obstacles in making a living cell replicate a semi-synthetic DNA. 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.

In this experiment, as d5SICS and dNaM are not naturally in cells, the building blocks for their replication had to be provided artificially, by adding them to the fluid resolution outside the cell. To get the creating blocks, recognized as nucleoside triphosphates, into the cells, scientists had to find unique triphosphate transporter molecules.

Challenges tackled:

In a significant breakthrough, scientists found a triphosphate transporter, created by a species of microalgae, that was efficient at importing the unnatural triphosphates. Surprisingly, the semi-synthetic plasmid replicated and did not greatly hamper the development of the E. coli cells, and showed no sign of losing its unnatural base pairs to DNA repair mechanisms.

Importantly, this process can be performed in a controlled manner as it was found that when the flow of the unnatural triphosphate transporter or building blocks is stopped, the DNA reverts to normal A, T, G, C base pairs and the d5SICS and dNaM disappear from the genome.

Possible Applications:

In future this research could be useful in encoding new proteins produced from new, unnatural amino acids—which would give us higher power than ever to design protein therapeutics and diagnostics and laboratory reagents to have desired functions. Other applications, such as nano-materials, are also possible.

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