Unnatural DNA Base Pair Research Articles

Eight-Letter DNA.

Steve Benner and collaborators have recently reported an analysis of DNA containing eight nucleotide letters, the four natural letters (dG, dC, dA, and dT) and four additional letters (dP, dZ, dS, and dB). Their analysis demonstrates that the additional letters do not perturb the structure or stability of the base pairs formed between the natural letters and, remarkably, that the new base pairs, dP-dZ and dS-dB, behave virtually identically to the natural base pairs. This unprecedented result convincingly demonstrates that the thermodynamic and structural behavior previously thought to be the purview of only natural DNA is in fact not unique and can be imparted to suitably designed synthetic components. In addition, the first evidence that the eight-letter DNA can be transcribed into RNA by a mutant RNA polymerase is presented, paving the way for the transfer of more information from one biopolymer to another. Along with others working to develop unnatural DNA base pairs for both in vitro and in vivo applications, this work represents an important step toward the expansion of the genetic alphabet, a central goal of synthetic biology, and has profound implications for our understanding of the molecules and forces that can make life possible.

Sequencing DNA for the Oxidatively Modified Base 8-Oxo-7,8-Dihydroguanine.

The DNA base guanine (G) can be oxidatively modified to 8-oxo-7,8-dihydroguanine (OG). Extraction of genomic DNA followed by nuclease digestion and mass spectrometry analysis has found OG is present at background levels of ~1 out of 106 Gs; however, this approach cannot determine the locations for the OGs in the genome. Thus, in this methods report, we outline three different methods (A, B, and C) for sequencing OG in DNA. Method A sequences OG by utilizing the base excision repair pathway to delete the OG nucleotide from the DNA that is then detected by Sanger sequencing as a deletion signature. Method B sequences OG by harnessing the base excision repair pathway to convert OG to an unnatural DNA base pair followed by Sanger sequencing to locate the unnatural base pair indicating where OG was located. Method C (i.e., OG-Seq) takes genomic DNA sheared to ~150bps followed by selectively biotinylating the OG-containing fragments for affinity purification and enrichment of the OG-modified strands. The OG-modified fragments are sequenced on a next-generation sequencing platform to locate OG on the genomic scale with a resolution of ~150bps. The methods outlined are then compared and contrasted allowing researchers to select the one that best suits their experimental goals.

Synthetic biology: New letters for life's alphabet.

The five bases found in nucleic acids define the 'alphabet' used to encode life on Earth. The construction of an organism that stably propagates an unnatural DNA base pair redefines this fundamental feature of life. See Letter p.385 The genetic code is simple: four bases that form two pairs (A–T and G–C) are used in all of life. Expansion of this code to incorporate unnatural nucleotides and base pairing has been a goal of synthetic biology, as it would open up ways to tailor organisms for directed purposes. Although this has been achieved in proof-of-principle experiments in vitro, stable propagation of an expanded code had not been demonstrated in vivo until now. Floyd Romesberg and colleagues present evidence that two hydrophobic nucleotides, d5SICSTP and dNaMTP, can be added to the medium in which Escherichia coli expressing an exogenous algal nucleotide triphosphate transporter is growing, and that these nucleotides will be incorporated in the genome and are not recognized as lesions by the repair pathway. Consequently, the unnatural-base-pair-containing DNA is replicated, without cell growth being significantly affected.

Site‐Specifically Arraying Small Molecules or Proteins on DNA Using An Expanded Genetic Alphabet

A class of replicable unnatural DNA base pairs formed between d5SICS and either dMMO2, dDMO, or dNaM were developed. To explore the use of these pairs to produce site-specifically labeled DNA, the synthesis of a variety of derivatives bearing propynyl groups, an analysis of their polymerase-mediated replication, and subsequent site-specific modification of the amplified DNA by Click chemistry is reported. With the d5SICS scaffold a propynyl ether linker is accommodated better than its aliphatic analogue, but not as well as the protected propargyl amine linker explored previously. It was also found that with the dMMO2 and dDMO analogues, the dMMO2 position para to the glycosidic linkage is best suited for linker attachment and that although aliphatic and ether-based linkers are similarly accommodated, the direct attachment of an ethynyl group to the nucleobase core is most well tolerated. To demonstrate the utility of these analogues, a variety of them were used to site-selectively attach a biotin tag to the amplified DNA. Finally, we use d5SICS(CO) -dNaM to couple one or two proteins to amplified DNA, with the double labeled product visualized by atomic force microscopy. The ability to encode the spatial relationships of arrayed molecules in PCR amplifiable DNA should have important applications, ranging from SELEX with functionalities not naturally present in DNA to the production, and perhaps "evolution" of nanomaterials.

KlenTaq polymerase replicates unnatural base pairs by inducing a Watson-Crick geometry

Many candidate unnatural DNA base pairs have been developed, but surprisingly, some of the best replicated adopt intercalated structures in free DNA that are difficult to reconcile with known mechanisms of polymerase recognition. Here we present crystal structures of KlenTaq DNA polymerase at different stages of replicating one of the more promising pairs, dNaM-d5SICS, and show that efficient replication results from the polymerase itself inducing the required natural-like structure.

A quantum chemistry study of Ds–Pa unnatural DNA base pair

AbstractWe have performed density functional theory and MP2 calculations of the unnatural DNA base pair, Ds–Pa, (7‐(2‐thienyl)‐imidazo[4,5‐b]pyridine (Ds) and pyrrole‐2‐carbaldehyde (Pa)) in the gas phase. By calculating the interaction energy curve of Ds–Pa pair as a function of intermolecular distance, we have found that the interaction energy between Ds and Pa molecules is very weak. The detailed analysis of the structural properties of the optimized Ds–Pa pair has been also used and compared with those of A–T and G–C natural base pairs. These results clearly show that the Ds–Pa pair does not have any hydrogen bonds and suggest that the pair selectivity in the Ds–Pa pair mainly comes from the interaction of Ds or Pa molecules with other parts in DNA or RNA, such as upper or lower natural bases, sugar phosphate backbones, and water molecules. © 2012 Wiley Periodicals, Inc.

The effects of unnatural base pairs and mispairs on DNA duplex stability and solvation

In an effort to develop unnatural DNA base pairs we examined six pyridine-based nucleotides, d3MPy, d4MPy, d5MPy, d34DMPy, d35DMPy and d45DMPy. Each bears a pyridyl nucleobase scaffold but they are differentiated by methyl substitution, and were designed to vary both inter- and intra-strand packing within duplex DNA. The effects of the unnatural base pairs on duplex stability demonstrate that the pyridine scaffold may be optimized for stable and selective pairing, and identify one self pair, the pair formed between two d34DMPy nucleotides, which is virtually as stable as a dA:dT base pair in the same sequence context. In addition, we found that the incorporation of either the d34DMPy self pair or a single d34DMPy paired opposite a natural dA significantly increases oligonucleotide hybridization fidelity at other positions within the duplex. Hypersensitization of the duplex to mispairing appears to result from global and interdependent solvation effects mediated by the unnatural nucleotide(s) and the mispair. The results have important implications for our efforts to develop unnatural base pairs and suggest that the unnatural nucleotides might be developed as novel biotechnological tools, diagnostics, or therapeutics for applications where hybridization stringency is important.

Polymerase amplification, cloning, and gene expression of benzo-homologous "yDNA" base pairs.

A widened DNA base-pair architecture is studied in an effort to explore the possibility of whether new genetic system designs might possess some of the functions of natural DNA. In the "yDNA" system, pairs are homologated by addition of a benzene ring, which yields (in the present study) benzopyrimidines that are correctly paired with purines. Here we report initial tests of ability of the benzopyrimidines yT and yC to store and transfer biochemical and biological information in vitro and in bacterial cells. In vitro primer extension studies with two polymerases showed that the enzymes could insert the correct nucleotides opposite these yDNA bases, but with low selectivity. PCR amplifications with a thermostable polymerase resulted in correct pairings in 15-20 % of the cases, and more successfully when yT or yC were situated within the primers. Segments of DNA containing one or two yDNA bases were then ligated into a plasmid and tested for their ability to successfully lead the expression of an active protein in vivo. Although active at only a fraction of the activity of fully natural DNA, the unnatural bases encoded the correct codon bases in the majority of cases when singly substituted, and yielded functioning green fluorescent protein. Although the activities with native polymerases are modest with these large base pairs, this is the first example of encoding protein in vivo by an unnatural DNA base pair architecture.

Synthesis of C-Aryl-Nucleosides and O-Aryl-Glycosides via Cuprate Glycosylation

2'-Deoxy-C-aryl-nucleosides have found increasing importance in studying DNA-DNA and DNA-protein interactions, as unnatural DNA-base pairs, and as oligonucleotide based fluorophores. Access to the required C-aryl-nucleosides is provided by several glycosylation methods. Though useful in many cases, these methods often have drawbacks such as low yields or low economic efficiency. The necessity for intensive optimization of the reaction conditions and/or the use of hazardous and toxic reagents can render C-glycosylation reactions cumbersome. Herein we describe a robust and highly efficient C-glycosylation method. It is shown that aryl cuprates of the Normant-type reliably react with Hoffer's chlorosugar to deliver C-aryl-nucleosides in up to 93% yield. This method may substitute for the previously employed coupling with organocadmium or -zinc species. Peculiar reactivities are reported for C-glycosylation of Gilman-type aryl cuprates which required substantial arene-specific optimization. Interestingly, the glycosylation of Gilman cuprates was found to provide access not only to C-aryl-nucleosides but also to O-aryl-glycosides. The reactions of Gilman cuprates with Hoffer's chlorosugar 1 in the presence of oxygen provided the coresponding O-aryl-2'-deoxyribosides in up to 87% yield without concomitant C-glycosylation.

Optimization of Interstrand Hydrophobic Packing Interactions within Unnatural DNA Base Pairs

As part of an effort to expand the genetic alphabet, we have evaluated a large number of predominantly hydrophobic unnatural base pairs. We now report the synthesis and stability of unnatural base pairs formed between simple phenyl rings modified at different positions with methyl groups. Surprisingly, several of the unnatural base pairs are virtually as stable as a natural base pair in the same sequence context. The results show that neither hydrogen-bonding nor large aromatic surface area are required for base pair stability within duplex DNA and that interstrand interactions between small aromatic rings may be optimized for both stability and selectivity. These smaller nucleobases are not expected to induce the distortions in duplex DNA or at the primer terminus that seem to limit replication of larger unnatural base pairs, and they therefore represent a promising approach to the expansion of the genetic alphabet.

A novel silver(i)-mediated DNA base pair.

We have generated a novel silver(I)-mediated unnatural DNA base pair consisting of two 2,6-bis(ethylthiomethyl)pyridine nucleobases SPy. This metallo-base pair has a remarkably high pairing stability and selectivity which rivals that of the natural base pairs dA:dT and dC:dG. UV-melting experiments revealed that the dSPy:dSPy self-pair can replace natural base pairs at multiple sites and still form stable DNA duplexes.