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.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.