Toward an Expanded Genome: Structural and Computational Characterization of an Artificially Expanded Genetic Information System.

Although the fundamental properties of DNA as first proposed by Watson and Crick in 1953 provided a basic understanding of how duplex DNA was organized and might be replicated, it was not until the first crystal structures of DNA (Z-DNA in 1979, B-DNA in 1980, and A-DNA in 1982) that the true complexity of the molecule began to be appreciated. Many crystal structures of oligonucleotides have since shed light on the helical forms that "Watson-Crick" DNA can adopt, their associated groove widths, and the properties of the nucleobase pairs and their interactions in all three helical forms. Additional understanding of the properties of Watson-Crick DNA has been provided by computational studies employing a variety of theoretical methods. Together with these studies devoted to understanding Watson-Crick DNA, recent efforts to expand the genetic alphabet have founded a new field in synthetic biology. One of these efforts, the artificially expanded genetic information system (AEGIS) developed by Steven Benner and co-workers, takes advantage of orthogonal hydrogen bonding to produce DNA comprised of six nucleobase pairs, of which the most extensively studied is referred to as P:Z with P being 2-amino-imidazo[1,2-a]-1,3,5-triazin-4(8H)-one) and Z being 6-amino-5-nitro-2(1H)-pyridone. P:Z forms three edge-on hydrogen bonds that differ from standard Watson-Crick pairs in the arrangement of acceptors and donor groups; P presents acceptor, acceptor, donor, and Z presents donor, donor, acceptor. Z is unique among the AEGIS nucleobases in having a nitro group present in the major groove. PZ-containing DNA has been exploited in a number of clinical applications and is being used to develop receptors and catalysts. Ultimately, the grand challenge will be to create a semisynthetic organism with an expanded genome. Furthermore, just as our understanding of the properties of natural DNA have benefited from structural and computational characterization, so too will our understanding of artificial DNA. This Account focuses on the structural and biophysical properties of AEGIS DNA containing P:Z pairs. We begin with the fundamental properties of P:Z nucleobase pairs, including their electrostatic potential and hydrogen-bonding energies, as elucidated by quantum mechanical calculations. We then examine the impact of including multiple consecutive P:Z pairs into duplex DNA providing an opportunity to investigate stacking interactions between P:Z pairs. The self-complementary 5'-CTTATPPTAZZATAAG was crystallized in B-form using the host-guest system along with analogous natural sequences including Gs or As. Use of the host-guest system to characterize B-DNA obviates a number of limitations on the structural characterization of sequences of interest; these include the ability to crystallize the desired sequences and to distinguish structural effects imparted by the lattice constraints from those inherent in the sequence itself. On the other hand, 3/6ZP, 5'-CTTATPPPZZZATAAG, was crystallized in A-form in a DNA-only lattice allowing a comparative analysis of P:Z pairs in two of the biologically relevant helical forms: A- and B-DNA. Computational studies on the 3/6ZP sequence starting in A-form provide additional evidence for a more energetically favorable stacking interaction, which we term the "slide" conformer, observed in the A-form crystal structure; this unusual stacking interaction plays a major role in altering the conformational dynamics observed for the PZ-containing duplex as compared to a GC-containing "control" duplex in long time scale molecular dynamics simulations. This combined use of structural and computational strategies paves the way for obtaining a detailed description of artificial DNA, both in how it differs from Watson-Crick DNA and in the rational discovery of proteins, such as endonucleases, transcription factors, and polymerases, which can specifically manipulate DNA containing AEGIS nucleobase pairs.