Researchers Create Artificial DNA Bases

The creation of 2 artificial DNA building blocks gives researchers the capability of increasing DNA's information potential and may make it possible to synthesize proteins with novel properties (Leconte AM et al. J Am Chem Soc. 2008;130[7]:2336-2343).

Like DNA's natural base-pair constituents, guanine pairing with cytosine and adenine with thymine, the additional DNA nucleotides pair up and could allow researchers to create unique DNA molecules with tailor-made binding or enzymatic activities for use in the laboratory and the clinic, said principal investigator and chemist Floyd Romesberg, PhD, of Scripps Research Institute in La Jolla, Calif.

Through experiments led by graduate student Aaron Leconte, Romesberg's group generated thousands of artificial nucleotides and searched for candidates that could be accurately copied by the enzymes responsible for DNA replication. From a pool of 3600 candidate base pairs, a duo rose above the rest. They then designed an optimized base pair, called d5SICS:dMMO2.

“The Romesberg lab has done a beautiful job in evolving this research,” said Eric Kool, PhD, a chemist at Stanford University in Palo Alto, Calif, who was not involved in the study. Kool and his team created the first human-designed DNA base that functions in a living cell (Kim TW et al. Proc Natl Acad Sci U S A. 2005;102[44]:15803-15808).

“With additional base pairs, you could make proteins that are much different than the natural repertoire allows us,” Kool explained. Currently, artificial oligonucleotides with unnatural base pairs are used in diagnostic tests for HIV, hepatitis B, and hepatitis C developed by Steven Benner, PhD, currently of the Foundation for Applied Molecular Evolution in Gainesville, Fla (Benner SA et al. Nucleic Acids Res Suppl. 2003;3:125-126; Sismour AM et al. Nucleic Acids Res. 2004;32[2]:728-735; Yang Z et al. Nucleic Acids Res. 2006; 34[21]:6095-6101). But constructing new nucleotides that can be efficiently incorporated into DNA, replicated, and used to produce proteins has proved difficult.

Romesberg noted that unique stretches of DNA that include d5SICS:dMMO2 pairings might be used to enhance DNA amplification, and novel DNA tags could be made that detect processes occurring within cells, not unlike technologies that use a protein that fluoresces green as a marker of gene expression and a means of tracking the location of a protein in cells.

“Right now you can label a protein and follow it in the cell, but you can't do that with RNA,” said Romesberg. “There are catalytic RNAs, small inhibitory RNAs, and all sorts of other types of RNAs,” he said, “and investigators would love to be able to label them and follow them in the cell.” This type of technology could also be used to detect defects in natural DNA, such as those that cause cancer and genetic diseases.

Some researchers are exploring strategies to modify DNA in other ways. For example, scientists have engineered DNA molecules that lack negatively charged phosphate groups or hydrogen-bonding groups, modifications that may have therapeutic benefits. For example, an uncharged DNA analog might be able to pass more easily through a cell membrane and bind to and neutralize unwanted RNA molecules (Nielsen PE. Mol Biotechnol. 2004;26[3]:233-248; Freier SM and Altmann KH. Nucleic Acids Res. 1997;25[22]:4429-4443).

Recently, researchers rebuilt the genome of the bacterium Mycoplasma genitalium from scratch, using DNA that was chemically synthesized with the genes from wild-type bacteria (Gibson DG et al. Science. doi:10.1126/science.1151721 [published online ahead of print January 24, 2008]). Scientists see potential for artificial nucleotides in such research by incorporating them randomly or at specific locations within the genome to cause desired properties.

Ultimately, Romesberg hopes to apply his work to expand the genetic code of an organism. In doing so, “you could give it increased information and then see if this has an evolutionary advantage,” he explained.

With these various technologies, “we can expect in the not-too-distant future fully artificial genetic systems that support a synthetic biology—a set of artificial chemical systems that can direct their own replication and evolve,” wrote Benner (Benner SA. Science. 2004;306[5696]:625-626).