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Allison A Henry

Publications and source records attributed to Allison A Henry.

11 recordsLinked to original sources
PubMed2006

Optimization of unnatural base pair packing for polymerase recognition.

As part of an effort to expand the genetic alphabet, we have been examining the ability of predominately hydrophobic nucleobase analogues to pair in duplex DNA and during polymerase-mediated replication. We previously reported the synthesis and thermal stability of unnatural base pairs formed between nucleotides bearing simple methyl-substituted phenyl ring nucleobase analogues. Several of these pairs are virtually as stable and selective as natural base pairs in the same sequence context. Here, we report the characterization of polymerase-mediated replication of the same unnatural base pairs. We find that every facet of replication, including correct and incorrect base pair synthesis, as well as continued primer extension beyond the unnatural base pair, is sensitive to the specific methyl substitution pattern of the nucleobase analogue. The results demonstrate that neither hydrogen bonding nor large aromatic surface area is required for polymerase recognition, and that interstrand interactions between small aromatic rings may be optimized for replication. Combined with our previous results, these studies suggest that appropriately derivatized phenyl nucleobase analogues represent a promising approach toward developing a third base pair and expanding the genetic alphabet.

Base Pair Mismatch↗
PubMed2005

Directed evolution of novel polymerases.

DNA and RNA polymerases evolved to function in specific environments with specific substrates to propagate genetic information in all living organisms. The commercial availability of these polymerases has revolutionized the biotechnology industry, but for many applications native polymerases are limited by their stability or substrate recognition. Thus, there is great interest in the directed evolution of DNA and RNA polymerases to generate enzymes with novel, desired properties, such as thermal stability, resistance to inhibitors, and altered substrate specificity. Several screening and selection approaches have been developed, both in vivo and in vitro, and have been used to evolve polymerases with a variety of important activities. Both the techniques and the evolved polymerases are reviewed here, along with a comparison of the in vivo and in vitro approaches.

Animals↗
PubMed2005

The evolution of DNA polymerases with novel activities.

DNA and RNA polymerases have evolved in nature to function in specific environments with specific substrates. Thus, although the commercial availability of these enzymes has revolutionized the biotechnology industry, their applications are limited. The availability of polymerases that have unnatural properties would be of even greater utility. Towards this goal, several activity-based screening and selection approaches have been developed. Using these techniques, polymerases that synthesize a variety of different polymers, including those containing 2'-O-methyl-modified nucleotides or unnatural base pairs, have been evolved. These results suggest that polymerases tailored for any specific application could soon be available.

DNA-Directed DNA Polymerase↗
PubMed2004

Preliminary characterization of light harvesting in E. coli DNA photolyase.

E. coli DNA photolyase is a monomeric light-harvesting enzyme that utilizes a methenyltetrahydrofolate (MTHF) antenna cofactor to harvest light energy for the repair of thymine dimers in DNA. For this purpose, the enzyme evolved to bind the cofactor and red-shift its absorption maximum by 25 nm. Using the crystal structure as a guide, we mutated each protein residue that contacts the cofactor in an effort to identify the interactions responsible for this selective stabilization of the cofactor's excited state. Hydrogen bonding, packing, and electrostatic interactions were examined. Remarkably, a single residue, Glu109, appears to play an important, if not exclusive, role in inducing the observed red-shift. Thus, this protein, the simplest light-harvesting system known, appears to have evolved a remarkably simple mechanism to tune the photophysical properties of the antenna cofactor appropriately for biological function.

Deoxyribodipyrimidine Photo-Lyase↗
PubMed2004

Efforts to expand the genetic alphabet: identification of a replicable unnatural DNA self-pair.

Six unnatural nucleotides featuring fluorine-substituted phenyl nucleobase analogues have been synthesized, incorporated into DNA, and characterized in terms of the structure and replication properties of the self-pairs they form. Each unnatural self-pair is accommodated in B-form DNA without detectable structural perturbation, and all are thermally stable and selective to roughly the same degree. Furthermore, the efficiency of polymerase-mediated mispair synthesis is similar for each unnatural nucleotide in the template. In contrast, the efficiency of polymerase-mediated self-pair extension is highly dependent on the specific fluorine substitution pattern. The most promising unnatural base pair candidate of this series is the 3-fluorobenzene self-pair, which is replicated with reasonable efficiency and selectivity.

Base Pairing↗
PubMed2004

Expanding the substrate repertoire of a DNA polymerase by directed evolution.

Nucleic acid polymerases are the most important reagents in biotechnology. Unfortunately, their high substrate specificity severely limits their applications. Polymerases with tailored substrate repertoires would significantly expand their potential and allow enzymatic synthesis of unnatural polymers for in vivo and in vitro applications. For example, the ability to synthesize 2'-O-methyl-modified polymers would provide access to materials possessing properties that make them attractive for biotechnology and therapeutic applications, but unfortunately, no known polymerases are capable of efficiently accepting these modified substrates. To evolve such enzymes, we have developed an activity-based selection method which isolates polymerase mutants with the desired property from libraries of the enzyme displayed on phage. In this report, mutants that could efficiently synthesize an unnatural polymer from 2'-O-methyl ribonucleoside triphosphates were immobilized and isolated by means of their activity-dependent modification of a DNA oligonucleotide primer attached to the same phage particle. In each case, directed evolution resulted in relocating a critical side chain to a different position in the polypeptide, thus re-engineering the overall active site while preserving critical protein-DNA interactions. Remarkably, one evolved polymerase is shown to incorporate the modified substrates with an efficiency and fidelity equivalent to that of the wild-type enzyme with natural substrates.

Amino Acid Sequence↗
PubMed2003

Determinants of unnatural nucleobase stability and polymerase recognition.

Six new unnatural nucleobases have been synthesized and characterized in terms of stability and selectivity of self-pairing in duplex DNA and efficiency and fidelity of self-pairing during polymerase-mediated replication. Each nucleobase has a conserved ring structure but differs from the others in its specific pattern of substitution with oxygen and sulfur atoms. Heteroatom derivatization within the conserved scaffold is shown to have only moderate effects on unnatural self-pair synthesis by the polymerase; larger effects were observed on the thermal stability and polymerase-mediated extension of the self-pairs. The largest effects of heteroatom substitution were on the stability and synthesis of mispairs between the unnatural and natural bases. Certain heteroatom substitutions were found to have a general effect while others were found to have effects that were specific for a particular unnatural or natural base. The data are useful for designing stable and replicable third base pairs and for understanding the contributions of nucleobase shape, polarity, and polarizability to the stability and replication of DNA.

Base Pairing↗
PubMed2003

The effect of minor-groove hydrogen-bond acceptors and donors on the stability and replication of four unnatural base pairs.

The stability and replication of DNA containing self-pairs formed between unnatural nucleotides bearing benzofuran, benzothiophene, indole, and benzotriazole nucleobases are reported. These nucleobase analogues are based on a similar scaffold but have different hydrogen-bond donor/acceptor groups that are expected to be oriented in the duplex minor groove. The unnatural base pairs do not appear to induce major structural distortions and are accommodated within the constraints of a B-form duplex. The differences between these unnatural base pairs are manifest only in the polymerase-mediated extension step, not in base-pair stability or synthesis. The benzotriazole self-pair is extended with an efficiency that is only 200-fold less than a correct natural base pair. The data are discussed in terms of available polymerase crystal structures and imply that further modifications may result in unnatural base pairs that can be both efficiently synthesized and extended, resulting in an expanded genetic alphabet.

Base Pairing↗
PubMed2003

Beyond A, C, G and T: augmenting nature's alphabet.

Efforts to expand the genetic alphabet are predicated upon a stable and replicable third base pair. Recent progress has resulted in the development of several candidates that are both stable in duplex DNA and replicated by DNA polymerases with various degrees of efficiency and fidelity. The candidate base pairs draw upon unnatural hydrogen-bonding topologies as well as upon shape complementarity and hydrophobic forces. This review provides a critical comparison of the third base pair candidates and discusses the further work required to expand the genetic alphabet.

Base Pairing↗
PubMed2002

Stability and selectivity of unnatural DNA with five-membered-ring nucleobase analogues.

In an effort to develop an orthogonal third base pair for the storage of genetic information, thiophene and furan heterocycles have been examined as nucleobase analogues. The stability of the unnatural bases was evaluated in duplex DNA paired opposite other unnatural bases as well as opposite the natural bases. Several unnatural base pairs are identified that are both reasonably stable and strongly selective against mispairing with native bases. These results expand the potential nucleobase analogues with which the genetic alphabet may be expanded to include five-membered-ring heterocycles.

Base Pair Mismatch↗