Enzymes stitch together non-natural DNA

Guided evolution and HIV help create man-made stuff of life.

The four letters of life are being added to. Credit: © Photodisc

Researchers have found new ways to string together artificial DNA bases. The techniques could aid the creation of altered genetic material for applications in medicine and technology.

Floyd Romesberg and co-workers at the Scripps Research Institute in La Jolla, California, have harnessed the principles of evolution to find an enzyme capable of assembling non-standard DNA1.

In a second study, Steven Benner of the University of Florida in Gainesville and colleagues used an enzyme made by HIV, the virus that causes AIDS, to do the same job. Benner's enzyme is even capable of making multiple copies of non-natural DNA, opening up the possibility that the code could eventually evolve on its own2.

All natural DNA is made up of just four bases, but researchers have created non-natural bases. These can be used to make forms of DNA that are more robust than the natural kind and do not break apart when exposed to high temperatures. Such super-DNA could be useful in a wide range of medical and technological applications.

Stitched up

The tricky part is stitching these artificial bases into a chain, and getting the chains to replicate as natural DNA does. An enzyme called DNA polymerase does the job in nature.

DNA polymerase works its way along a template strand of DNA, reading each base and collecting a matching base to create a new, copied strand. But the enzyme turns up its nose at artificial bases.

By systematically tinkering with the structure of DNA polymerase at one or two specific locations, researchers can make enzymes that work with artificial bases. But this technique, called 'rational design', is a tedious and unpredictable process.

"Going the full distance is very difficult to do in a rational way," says Rui Sousa of the University of Texas Health Science Center at San Antonio. Sousa has designed polymerases that are being used commercially.

Romesberg and his co-workers tried a different approach called 'directed evolution'. They made millions of mutant polymerases by randomly scrambling part of the natural enzyme's chemical structure. Most of the mutants were useless, but some were quite good at stitching together non-standard bases. They plucked these useful mutants out of the crowd, and repeated the mutation and selection process to fine-tune their abilities.

After four rounds of selection, they found several mutants capable of doing the job. One was particularly good: it was able to copy the sequence of a template molecule into the modified form of DNA as efficiently and faithfully as DNA polymerase working with natural bases.

Carbon copies

But the mutant is not perfect. Like most modified polymerases, Romesberg's top candidate runs out of steam and stops working after adding five artificial bases to a growing chain.

Benner's team created a more successful polymerase by starting with a different enzyme altogether - a reverse transcriptase (RT) that is made by the HIV-1 form of the virus that causes AIDS. This RT mutates when the virus is hit with anti-HIV drugs. Benner's group checked mutated forms to find a modified RT capable of stitching two non-standard bases into strings of DNA. They fine-tuned this enzyme using rational design.

Their enzyme even works in a process called the polymerase chain reaction (PCR) , which is often used in biotechnology to make copies of short strands of DNA. Most constructed polymerases fail when researchers try to make multiple copies of artificial DNA using PCR; after several rounds of copying, imperfect polymerases start to weed out non-standard DNA. But Benner's enzyme does not seem to have this problem.

References

  1. 1

    Fa, M., Radeghieri, A., Henry, A. A. & Romesberg, F. E. Expanding the substrate repertoire of a DNA polymerase by directed evolution. Journal of the American Chemical Society, 126, 1748 - 1754, doi:10.1021/ja038950i (2004).

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  2. 2

    Sismour, A. M et al. PCR amplification of DNA containing non-standard base pairs by variants of reverse transcriptase from Human Immunodeficiency Virus-1. Nucleic Acids Research, 32, 728 - 735, (2004).

    Article  Google Scholar 

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Ball, P. Enzymes stitch together non-natural DNA. Nature (2004). https://doi.org/10.1038/news040223-2

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