DNA polymerase θ specializes in incorporating synthetic expanded-size (xDNA) nucleotides.

DNA polymerase θ (Polθ) is a unique A-family polymerase that is essential for alternative end-joining (alt-EJ) of double-strand breaks (DSBs) and performs translesion synthesis. Because Polθ is highly expressed in cancer cells, confers resistance to ionizing radiation and chemotherapy agents, and promotes the survival of homologous recombination (HR) deficient cells, it represents a promising new cancer drug target. As a result, identifying substrates that are selective for this enzyme is a priority. Here, we demonstrate that Polθ efficiently and selectively incorporates into DNA large benzo-expanded nucleotide analogs (dxAMP, dxGMP, dxTMP, dxAMP) which exhibit canonical base-pairing and enhanced base stacking. In contrast, functionally related Y-family translesion polymerases exhibit a severely reduced ability to incorporate dxNMPs, and all other human polymerases tested from the X, B and A families fail to incorporate them under the same conditions as Polθ. We further find that Polθ is inhibited after multiple dxGMP incorporation events, and that Polθ efficiency for dxGMP incorporation approaches that of native dGMP. These data demonstrate a unique function for Polθ in incorporating synthetic large-sized nucleotides and suggest the future possibility of the use of dxG nucleoside or related prodrug analogs as selective inhibitors of Polθ activity.

Covalent Modification of Synthetic Hydrogels with Bioactive Proteins via Sortase-Mediated Ligation.

Synthetic extracellular matrices are widely used in regenerative medicine and as tools in building in vitro physiological culture models. Synthetic hydrogels display advantageous physical properties, but are challenging to modify with large peptides or proteins. Here, a facile, mild enzymatic postgrafting approach is presented. Sortase-mediated ligation was used to conjugate human epidermal growth factor fused to a GGG ligation motif (GGG-EGF) to poly(ethylene glycol) (PEG) hydrogels containing the sortase LPRTG substrate. The reversibility of the sortase reaction was then exploited to cleave tethered EGF from the hydrogels for analysis. Analyses of the reaction supernatant and the postligation hydrogels showed that the amount of tethered EGF increases with increasing LPRTG in the hydrogel or GGG-EGF in the supernatant. Sortase-tethered EGF was biologically active, as demonstrated by stimulation of DNA synthesis in primary human hepatocytes and endometrial epithelial cells. The simplicity, specificity, and reversibility of sortase-mediated ligation and cleavage reactions make it an attractive approach for modification of hydrogels.

Tailoring chimeric ligands for studying and biasing ErbB receptor family interactions.

Described is the development and application of a versatile semisynthetic strategy, based on a combination of sortase-mediated coupling and tetrazine ligation chemistry, which can be exploited for the efficient incorporation of tunable functionality into chimeric recombinant proteins. To demonstrate the scope of the method, the assembly of a set of bivalent ligands, which integrate members of the epidermal growth factor (EGF) ligand family, is described. By using a series of bivalent EGFs with variable intraligand spacing, the differences in structure were correlated with the ability to bias signaling in the ErbB receptor family in a cell motility assay. Biasing away from EGFR-HER2 dimerization with a bivalent EGF was observed to reduce cell motility in an intraligand distance-dependent fashion, thus demonstrating the utility of the approach for acutely perturbing receptor-mediated cell signaling pathways.

Fluorescent amino acids: modular building blocks for the assembly of new tools for chemical biology.

Fluorescence spectroscopy is a powerful tool for probing complex biological processes. The ubiquity of peptide-protein and protein-protein interactions in these processes has made them important targets for fluorescence labeling, and to allow sensitive readout of information concerning location, interactions with other biomolecules, and macromolecular dynamics. This review describes recent advances in design, properties and applications in the area of fluorescent amino acids (FlAAs). The ability to site-selectively incorporate fluorescent amino acid building blocks into a protein or peptide of interest provides the advantage of closely retaining native function and appearance. The development of an array of fluorescent amino acids with a variety of properties, such as environment sensitivity, chelation-enhanced fluorescence, and profluorescence, has allowed researchers to gain insights into biological processes, including protein conformational changes, binding events, enzyme activities, and protein trafficking and localization.

Encoding phenotype in bacteria with an alternative genetic set.

An unnatural base-pair architecture with base pairs 2.4 Å larger than the natural DNA-based genetic system (xDNA) is evaluated for its ability to function like DNA, encoding amino acids in the context of living cells. xDNA bases are structurally analogous to natural bases but homologated by the width of a benzene ring, increasing their sizes and resulting in a duplex that is wider than native B-DNA. Plasmids encoding green fluorescent protein were constructed to contain single and multiple xDNA bases (as many as eight) in both strands and were transformed into Escherichia coli. Although they yielded fewer colonies than the natural control plasmid, in all cases in which a modified plasmid (containing one, two, three, or four consecutive size-expanded base pairs) was used, the correct codon bases were substituted, yielding green colonies. All four xDNA bases (xA, xC, xG, and xT) were found to encode the correct partners in the replicated plasmid DNA, both alone and in longer segments of xDNA. Controls with mutant cell lines having repair functions deleted were found to express the gene correctly, ruling out repair of xDNA and confirming polymerase reading of the unnatural bases. Preliminary experiments with polymerase deletion mutants suggested combined roles of replicative and lesion-bypass polymerases in inserting correct bases opposite xDNA bases and in bypassing the xDNA segments. These experiments demonstrate a biologically functioning synthetic genetic set with larger-than-natural architecture.

Fluorescent xDNA nucleotides as efficient substrates for a template-independent polymerase.

Template independent polymerases, and terminal deoxynucleotidyl transferase (TdT) in particular, have been widely used in enzymatic labeling of DNA 3'-ends, yielding fluorescently-labeled polymers. The majority of fluorescent nucleotides used as TdT substrates contain tethered fluorophores attached to a natural nucleotide, and can be hindered by undesired fluorescence characteristics such as self-quenching. We previously documented the inherent fluorescence of a set of four benzo-expanded deoxynucleoside analogs (xDNA) that maintain Watson-Crick base pairing and base stacking ability; however, their substrate abilities for standard template-dependent polymerases were hampered by their large size. However, it seemed possible that a template-independent enzyme, due to lowered geometric constraints, might be less restrictive of nucleobase size. Here, we report the synthesis and study of xDNA nucleoside triphosphates, and studies of their substrate abilities with TdT. We find that this polymerase can incorporate each of the four xDNA monomers with kinetic efficiencies that are nearly the same as those of natural nucleotides, as measured by steady-state methods. As many as 30 consecutive monomers could be incorporated. Fluorescence changes over time could be observed in solution during the enzymatic incorporation of expanded adenine (dxATP) and cytosine (dxCTP) analogs, and after incorporation, when attached to a glass solid support. For (dxA)(n) polymers, monomer emission quenching and long-wavelength excimer emission was observed. For (dxC)(n), fluorescence enhancement was observed in the polymer. TdT-mediated synthesis may be a useful approach for creating xDNA labels or tags on DNA, making use of the fluorescence and strong hybridization properties of the xDNA.

Toward a designed genetic system with biochemical function: polymerase synthesis of single and multiple size-expanded DNA base pairs.

The development of alternative architectures for genetic information-encoding systems offers the possibility of new biotechnological tools as well as basic insights into the function of the natural system. In order to examine the potential of benzo-expanded DNA (xDNA) to encode and transfer biochemical information, we carried out a study of the processing of single xDNA pairs by DNA Polymerase I Klenow fragment (Kf, an A-family sterically rigid enzyme) and by the Sulfolobus solfataricus polymerase Dpo4 (a flexible Y-family polymerase). Steady-state kinetics were measured and compared for enzymatic synthesis of the four correct xDNA pairs and twelve mismatched pairs, by incorporation of dNTPs opposite single xDNA bases. Results showed that, like Kf, Dpo4 in most cases selected the correctly paired partner for each xDNA base, but with efficiency lowered by the enlarged pair size. We also evaluated kinetics for extension by these polymerases beyond xDNA pairs and mismatches, and for exonuclease editing by the Klenow exo+ polymerase. Interestingly, the two enzymes were markedly different: Dpo4 extended pairs with relatively high efficiencies (within 18-200-fold of natural DNA), whereas Kf essentially failed at extension. The favorable extension by Dpo4 was tested further by stepwise synthesis of up to four successive xDNA pairs on an xDNA template.

Redesigning the architecture of the base pair: toward biochemical and biological function of new genetic sets.

Recognition of the nucleic acid bases within the DNA scaffold comprises the basis for transmission of genetic information, dictating protein and cell assembly, organismal development, and evolution. Driven in part by the need to test our current understanding of this information transfer, chemists have begun to design and synthesize nonnatural bases and base pair structures to mimic the function of DNA without relying on Nature's purine-pyrimidine base pair scaffold. Multiple examples have been recently described that self-assemble stably and sequence specifically in vitro, and some isolated unnatural base pairs can be replicated in vitro as well. Moreover, recent experiments with unnatural bases in bacterial cells have demonstrated surprisingly efficient reading of the chemical information. This suggests the future possibility of redesigning and replacing the chemical information of an evolving cell while retaining biological function.

Towards the replication of xDNA, a size-expanded unnatural genetic system.

Here we study the viability of an unnatural genetic system with size-expanded geometry (xDNA). xDNA contains base pairs 2.4 A larger than those of natural DNA. The expanded geometry is expected to be problematic for the natural high-fidelity replication machinery required to process genetic information. However, initial studies with a variety of DNA polymerases are promising in demonstrating replication of these unnatural bases. The results suggest the future possible viability of fully functional unnatural genetic systems, and give insight into the steric limits of some natural DNA polymerases.

Fluorescence of size-expanded DNA bases: reporting on DNA sequence and structure with an unnatural genetic set.

We recently described the synthesis and helix assembly properties of expanded DNA (xDNA), which contains base pairs 2.4 A larger than natural DNA pairs. This designed genetic set is under study with the goals of mimicking the functions of the natural DNA-based genetic system and of developing useful research tools. Here, we study the fluorescence properties of the four expanded bases of xDNA (xA, xC, xG, xT) and evaluate how their emission varies with changes in oligomer length, composition, and hybridization. Experiments were carried out with short oligomers of xDNA nucleosides conjugated to a DNA oligonucleotide, and we investigated the effects of hybridizing these fluorescent oligomers to short complementary DNAs with varied bases opposite the xDNA bases. As monomer nucleosides, the xDNA bases absorb light in two bands: one at approximately 260 nm (similar to DNA) and one at longer wavelength ( approximately 330 nm). All are efficient violet-blue fluorophores with emission maxima at approximately 380-410 nm and quantum yields (Phifl) of 0.30-0.52. Short homo-oligomers of the xDNA bases (length 1-4 monomers) showed moderate self-quenching except xC, which showed enhancement of Phifl with increasing length. Interestingly, multimers of xA emitted at longer wavelengths (520 nm) as an apparent excimer. Hybridization of an oligonucleotide to the DNA adjacent to the xDNA bases (with the xDNA portion overhanging) resulted in no change in fluorescence. However, addition of one, two, or more DNA bases in these duplexes opposite the xDNA portion resulted in a number of significant fluorescence responses, including wavelength shifts, enhancements, or quenching. The strongest responses were the enhancement of (xG)n emission by hybridization of one or more adenines opposite them, and the quenching of (xT)n and (xC)n emission by guanines opposite. The data suggest multiple ways in which the xDNA bases, both alone and in oligomers, may be useful as tools in biophysical analysis and biotechnological applications.

Model systems for understanding DNA base pairing.

The fact that nucleic acid bases recognize each other to form pairs is a canonical part of the dogma of biology. However, they do not recognize each other well enough in water to account for the selectivity and efficiency that is needed in the transmission of biological information through a cell. Thus proteins assist in this recognition in multiple ways, and recent data suggest that these mechanisms of recognition can vary widely with context. To probe how the chemical differences of the four nucleobases are defined in various biological contexts, chemists and biochemists have developed modified versions that differ in their polarity, shape, size, and functional groups. This brief review covers recent advances in this field of research.

Synthesis and properties of size-expanded DNAs: toward designed, functional genetic systems.

We describe the design, synthesis, and properties of DNA-like molecules in which the base pairs are expanded by benzo homologation. The resulting size-expanded genetic helices are called xDNA ("expanded DNA") and yDNA ("wide DNA"). The large component bases are fluorescent, and they display high stacking affinity. When singly substituted into natural DNA, they are destabilizing because the benzo-expanded base pair size is too large for the natural helix. However, when all base pairs are expanded, xDNA and yDNA form highly stable, sequence-selective double helices. The size-expanded DNAs are candidates for components of new, functioning genetic systems. In addition, the fluorescence of expanded DNA bases makes them potentially useful in probing nucleic acids.