Scientists have created a double helix out of six nucleotides, two more than what nature had devised. [American Chemical Society]
Scientists have created a double helix out of six nucleotides, two more than what nature had devised. [American Chemical Society]

Additions to DNA’s four-letter alphabet have been announced before, but the new letters don’t always fit comfortably into DNA’s double helix. Even slightly awkward letters can distort DNA’s overall shape and alter the molecule’s plasticity. Worse, a sequence of awkward letters that omits natural letters can amplify any structural problems, limiting new-letter-enriched synthetic DNA’s ability to interact with structural proteins and enzymes, jamming life’s molecular machinery.

A new pair of letters, however, seems to slide into DNA fairly easily. The new letters, or nucleotide bases, are called Z and P. They emulate structural and functional features of DNA’s natural nucleotide bases, C and G (cytosine and guanine) and A and T (adenine and thymine). Not only can they be interspersed with the natural nucleotide bases, they can be run adjacent to each other in a sequence, and the resulting DNA will still form a nice helix. What’s more, the resulting DNA even shows the ability to evolve.

Z stands for 6-amino-5-nitro-2(1H)-pyridone. P stands for 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)one. Z and P were introduced in a pair of papers prepared by Millie M. Georgiadis, Steven A. Benner, and colleagues from Indiana and Florida, and published in the Journal of the American Chemical Society. (Dr. Georgiadis is affiliated with Indiana University−Purdue University Indianapolis, and Dr. Benner is affiliated with the Foundation for Applied Molecular Evolution, and Firebird Biomolecular Sciences.)

The first paper, “Structural Basis for a Six Nucleotide Genetic Alphabet”—appeared on May 11. It presented crystal structures to demonstrate that the new nucleotides paired with “geometries that are similar to those displayed by standard duplex DNA.” The second paper—“Evolution of Functional Six-Nucleotide DNA”—appeared a day later. It presented results form a laboratory in vitro evolution (LIVE) experiment to show that the new nucleotide bases could be incorporated into newly evolved, selectively binding species.

The first paper emphasized three important findings:

  1. The discovery that canonical Watson–Crick pairing survives in duplexes that contain multiple and multiple adjacent Z:P pairs.
  2. ZP-containing oligonucleotides adopt canonical helical forms, B- and A-form DNA. The ability of DNA to adopt A-form enables a number of important protein–DNA interactions such as those in the polymerase active site, which requires that the nucleobase pair within the active site and the adjacent pair adopt A-form in order to appropriately position the template-primer for optimal interactions with the polymerase increasing fidelity.
  3. The Z-nitro group imparts new properties to the major groove of DNA that can potentially be exploited for recognition by proteins.

The second paper explained how the LIVE experiment involved developing a GACTZP library, which was challenged to deliver molecules that bind selectively to liver cancer cells, but not to untransformed liver cells.

“Unlike in classical in vitro selection, low levels of mutation allow this system to evolve to create binding molecules not necessarily present in the original library,” the article’s authors wrote. “Over a dozen binding species were recovered. The best had Z and/or P in their sequences. Several had multiple, nearby, and adjacent Zs and Ps. Only the weaker binders contained no Z or P at all. This suggests that this system explored much of the sequence space available to this genetic system and that GACTZP libraries are richer reservoirs of functionality than standard libraries.”

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