Natural DNA Polymerases Research Articles

Light-start DNA amplification using light-controlled DNA polymerase

Non-specific amplification caused by the uncontrollable activity of natural DNA polymerase greatly limits the application of DNA amplification techniques. Modification of DNA polymerase using hot-start technology is an effective way to improve the accuracy of nucleic acid detection. However, hot-start technology was only suitable for DNA polymerases working at high temperatures. Accordingly, there is an urgent need for more applicable methods of controlling DNA polymerase activity that do not rely on changes in temperature. In this work, we therefore developed a simple and novel approach to construct light-controlled DNA polymerases. These DNA polymerases are efficiently blocked by DNA aptamers and rapidly activated by UV light irradiation. Using the light-controlled DNA polymerases, we successfully developed light-start loop-mediated isothermal amplification (LAMP), light-start PCR, and light-start rolling circle amplification (RCA). Compared with hot-start technology, light-start technology improves the accuracy of DNA amplification reaction over a wider range of temperature conditions, which will further expand the application scenarios of DNA amplification techniques.

Redesigning the Genetic Polymers of Life.

Genomes can be viewed as constantly updated memory systems where information propagated in cells is refined over time by natural selection. This process, commonly known as heredity and evolution, has been the sole domain of DNA since the origin of prokaryotes. Now, some 3.5 billion years later, the pendulum of discovery has swung in a new direction, with carefully trained practitioners enabling the replication and evolution of "xeno-nucleic acids" or "XNAs"-synthetic genetic polymers in which the natural sugar found in DNA and RNA has been replaced with a different type of sugar moiety. XNAs have attracted significant attention as new polymers for synthetic biology, biotechnology, and medicine because of their unique physicochemical properties that may include increased biological stability, enhanced chemical stability, altered helical geometry, or even elevated thermodynamics of Watson-Crick base pairing.This Account describes our contribution to the field of synthetic biology, where chemical synthesis and polymerase engineering have allowed my lab and others to extend the concepts of heredity and evolution to synthetic genetic polymers with backbone structures that are distinct from those found in nature. I will begin with a discussion of α-l-threofuranosyl nucleic acid (TNA), a specific type of XNA that was chosen as a model system to represent any XNA system. I will then proceed to discuss advances in organic chemistry that were made to enable the synthesis of gram quantities of TNA phosphoramidites and nucleoside triphosphates, the monomers used for solid-phase and polymerase-mediated TNA synthesis, respectively. Next, I will recount our development of droplet-based optical sorting (DrOPS), a single-cell microfluidic technique that was established to evolve XNA polymerases in the laboratory. This section will conclude with structural insights that have been gained by solving X-ray crystal structures of a laboratory-evolved TNA polymerase and a natural DNA polymerase that functions with general reverse transcriptase activity on XNA templates.The final passage of this Account will examine the role that XNAs have played in synthetic biology by highlighting examples in which engineered polymerases have enabled the evolution of biologically stable affinity reagents (aptamers) and catalysts (XNAzymes) as well as the storage and retrieval of binary information encoded in electronic word and picture file formats. Because these examples provide only a glimpse of what the future may have in store for XNA, I will conclude the Account with my thoughts on how synthetic genetic polymers could help drive new innovations in synthetic biology and molecular medicine.

Programmed Allelic Mutagenesis of a DNA Polymerase with Single Amino Acid Resolution.

Most DNA polymerase libraries sample unknown portions of mutational space and are constrained by the limitations of random mutagenesis. Here we describe a programmed allelic mutagenesis (PAM) strategy to comprehensively evaluate all possible single-point mutations in the entire catalytic domain of a replicative DNA polymerase. By applying the PAM strategy with ultrafast high-throughput screening, we show how DNA polymerases can be mapped for allelic mutations that exhibit enhanced activity for unnatural nucleic acid substrates. We suggest that comprehensive missense mutational scans may aid the discovery of specificity determining residues that are necessary for reprogramming the biological functions of natural DNA polymerases.

The crystal structure of a natural DNA polymerase complexed with mirror DNA.

The intrinsic l-DNA binding properties of a natural DNA polymerase was discovered. The binding affinity of Dpo4 polymerase for l-DNA was comparable to that for d-DNA. The crystal structure of Dpo4/l-DNA complex revealed a dimer formed by the little finger domain that provides a binding site for l-DNA.

Crystal structures of a natural DNA polymerase that functions as an XNA reverse transcriptase

Replicative DNA polymerases are highly efficient enzymes that maintain stringent geometric control over shape and orientation of the template and incoming nucleoside triphosphate. In a surprising twist to this paradigm, a naturally occurring bacterial DNA polymerase I member isolated from Geobacillus stearothermophilus (Bst) exhibits an innate ability to reverse transcribe RNA and other synthetic congeners (XNAs) into DNA. This observation raises the interesting question of how a replicative DNA polymerase is able to recognize templates of diverse chemical composition. Here, we present crystal structures of natural Bst DNA polymerase that capture the post-translocated product of DNA synthesis on templates composed entirely of 2′-deoxy-2′-fluoro-β-d-arabino nucleic acid (FANA) and α-l-threofuranosyl nucleic acid (TNA). Analysis of the enzyme active site reveals the importance of structural plasticity as a possible mechanism for XNA-dependent DNA synthesis and provides insights into the construction of variants with improved activity.

Extending the Concepts of Heredity and Evolution to Artificial Genetic Polymers

The ability to synthesize and propagate genetic information encoded within the framework of a xeno-nucleic acid (XNA) polymer would inform a wide range of topics from the origins of life to molecular medicine. However, the engineering of natural polymerases to replicate artificial genetic polymers with backbone structures that are distinct from those found in nature is a challenging problem due to strong evolutionary constraints that have long specified the use of natural DNA and RNA substrates. Here, I will discuss an interdisciplinary approach to polymerase engineering that combines chemical synthesis, droplet microfluidics, and X-ray crystallography to establish an integrated technology platform for expanding the functional properties of natural DNA polymerases. This talk will focus on the challenges and successes of polymerase evolution and provide examples where Darwinian evolution has been used to isolate XNA aptamers to a desired target. This demonstration, which shows that XNA has the ability to fold into tertiary structures with sophisticated chemical functions, provides evidence that certain XNAs could have served as an ancestral genetic system. Given that some XNAs are refractory to nuclease digestion, this approach could also be used to develop a new generation of aptamers with practical applications in biotechnology and molecular medicine.

Compartmentalized Self-Tagging for In Vitro-Directed Evolution of XNA Polymerases.

Template-dependent synthesis of xenobiotic nucleic acids (XNAs) is an essential step for the development of functional XNA molecules, as it enables Darwinian evolution to be carried out with novel genetic polymers. The extraordinary substrate specificity of natural DNA polymerases greatly restricts the spectrum of XNAs available, thus making it necessary to identify DNA polymerase variants capable of incorporating a wider range of substrates. This unit summarizes compartmentalized self-tagging (CST), a directed evolution strategy developed for the selection of DNA polymerase variants capable of XNA synthesis.

Enzymatic Polymerization of Phosphonate Nucleosides

5'-O-phosphonomethyl-2'-deoxyadenosine (PMdA) proved to be a good substrate of the Therminator polymerase. In this article, we investigated whether the A, C, T and U analogues of this phosphonate nucleoside (PMdN) series can function as substrates of natural DNA polymerases. PMdT and PMdU could only be polymerized enzymatically to a limited extent. Nevertheless, PMdA and PMdC could be incorporated into a DNA duplex with complete chain elongation by all the DNA polymerases tested. A mixed sequence of four nucleotides containing modified C, T and A residues could be obtained with the Vent(exo(-)) and Therminator polymerases. The kinetic values for the incorporation of PMdA by Vent(exo(-)) polymerase were determined; a reduced K(M) value was found for the incorporation of PMdA compared to the natural substrate. Future polymerase directed evolution studies will allow us to select an enzyme with a heightened capacity to process these modified DNA building blocks into modified strands.

Recent Patents of Gene Sequences Relative to DNA Polymerases

Organisms with DNA genomes encode one or more DNA polymerases that are essential enzymes for chromosome replication, DNA repair and recombination. The ability of DNA polymerases to copy DNA templates has been exploited in a variety of in vitro reactions to sequence, amplify, mutate, label and recombine DNA, and in several other applications that are fundamental to molecular biology. Because natural DNA polymerases may have activities that interfere with in vitro applications or their substrate specificity is too narrow, DNA polymerases have been modified for specific applications. Patents are reviewed here on natural and variant DNA polymerases and their uses.

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.

Discovery, characterization, and optimization of an unnatural base pair for expansion of the genetic alphabet.

DNA is inherently limited by its four natural nucleotides. Efforts to expand the genetic alphabet, by addition of an unnatural base pair, promise to expand the biotechnological applications available for DNA as well as to be an essential first step toward expansion of the genetic code. We have conducted two independent screens of hydrophobic unnatural nucleotides to identify novel candidate base pairs that are well recognized by a natural DNA polymerase. From a pool of 3600 candidate base pairs, both screens identified the same base pair, dSICS:dMMO2, which we report here. Using a series of related analogues, we performed a detailed structure-activity relationship analysis, which allowed us to identify the essential functional groups on each nucleobase. From the results of these studies, we designed an optimized base pair, d5SICS:dMMO2, which is efficiently and selectively synthesized by Kf within the context of natural DNA.

Functional analysis of HIV-1 reverse transcriptase motif C: site-directed mutagenesis and metal cation interaction.

Motif C, present in all polymerases, has been proposed to be part of the catalytic and metal binding site of the enzyme, suggesting that polymerases have a common origin. Previously, we have shown that the metal ion manganese induces alterations in nucleotide substrate specificity in some polymerases. However, it is not known if the active site responsible for incorporation of nonspecific substrates is the same as that which incorporates specific ones. Here we show that manganese enables HIV-1 reverse transcriptase (RT) to incorporate rNTP's using RNA as a template, thus behaving as an RNA replicase. Also, we show that the mutation D186H in motif C strongly affects the natural DNA polymerase activity and that the RNA replicase activity becomes undetectable, suggesting that both activities depend on the same active site. This mutation changes the metal ion preference, with mutant RT presenting only 0.5% of the wild-type DNA polymerase activity in the presence of magnesium but 1.6% of the same activity in the presence of manganese. This variation in cation preference suggests that residue D186 is part of the metal binding site. Since residue D186 of motif C is essential for both activities and appears to be involved in the binding of an important cation needed for the specific activity, our results support the idea of a common origin for all polymerases, from an ancestral unspecified polymerase containing at least motif C.