PEG modification increases thermostability and inhibitor resistance of Bst DNA polymerase.

Polyethylene glycol modification (PEGylation) is a widely used strategy to improve the physicochemical properties of various macromolecules, especially protein drugs. However, its application in enhancing the performance of enzymes for molecular biology remains underexplored. This study explored the PEGylation of Bst DNA polymerase, determining optimal modification reaction conditions. In comparison to the unmodified wild-type counterpart, the modified Bst DNA polymerase exhibited significantly improved activity, thermal stability, and inhibitor tolerance during loop-mediated isothermal amplification. When applied for the detection of Salmonella in crude samples, the modified enzyme demonstrated a notably accelerated reaction rate. Therefore, PEGylation emerges as a viable strategy for refining DNA polymerases, helping in the development of novel molecular diagnostic reagents.

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.

Enzymatic synthesis of hypermodified DNA polymers for sequence-specific display of four different hydrophobic groups.

A set of modified 2'-deoxyribonucleoside triphosphates (dNTPs) bearing a linear or branched alkane, indole or phenyl group linked through ethynyl or alkyl spacer were synthesized and used as substrates for polymerase synthesis of hypermodified DNA by primer extension (PEX). Using the alkyl-linked dNTPs, the polymerase synthesized up to 22-mer fully modified oligonucleotide (ON), whereas using the ethynyl-linked dNTPs, the enzyme was able to synthesize even long sequences of >100 modified nucleotides in a row. In PCR, the combinations of all four modified dNTPs showed only linear amplification. Asymmetric PCR or PEX with separation or digestion of the template strand can be used for synthesis of hypermodified single-stranded ONs, which are monodispersed polymers displaying four different substituents on DNA backbone in sequence-specific manner. The fully modified ONs hybridized with complementary strands and modified DNA duplexes were found to exist in B-type conformation (B- or C-DNA) according to CD spectral analysis. The modified DNA can be replicated with high fidelity to natural DNA through PCR and sequenced. Therefore, this approach has a promising potential in generation and selection of hypermodified aptamers and other functional polymers.

Synthesis of Polyanionic C5-Modified 2'-Deoxyuridine and 2'-Deoxycytidine-5'-Triphosphates and Their Properties as Substrates for DNA Polymerases.

Modified 2'-deoxyribonucleotide triphosphates (dNTPs) have widespread applications in both existing and emerging biomolecular technologies. For such applications it is an essential requirement that the modified dNTPs be substrates for DNA polymerases. To date very few examples of C5-modified dNTPs bearing negatively charged functionality have been described, despite the fact that such nucleotides might potentially be valuable in diagnostic applications using Si-nanowire-based detection systems. Herein we have synthesised C5-modified dUTP and dCTP nucleotides each of which are labelled with an dianionic reporter group. The reporter group is tethered to the nucleobase via a polyethylene glycol (PEG)-based linkers of varying length. The substrate properties of these modified dNTPs with a variety of DNA polymerases have been investigated to study the effects of varying the length and mode of attachment of the PEG linker to the nucleobase. In general, nucleotides containing the PEG linker tethered to the nucleobase via an amide rather than an ether linkage proved to be the best substrates, whilst nucleotides containing PEG linkers from PEG6 to PEG24 could all be incorporated by one or more DNA polymerase. The polymerases most able to incorporate these modified nucleotides included Klentaq, Vent(exo-) and therminator, with incorporation by Klenow(exo-) generally being very poor.

Bisubstrate Function of RNA Polymerases Triggered by Molecular Crowding Conditions.

Since the origin of life on Earth, the role of carrying genetic information has been presumably transferred from RNA to DNA. At present, cellular environments are extremely dense, packed with cosolutes and macromolecules. Hence, the preference between RNA-dependent RNA and DNA polymerization may be affected by molecular crowding. In this study, we investigated both RNA-dependent RNA and DNA polymerizations by tC9Y polymerase ribozyme, T7 RNA polymerase (T7 RNAP), and Klenow fragment DNA polymerase (KF) under different molecular crowding conditions. Poly(ethylene glycol) (PEG) of various molecular weights was used as a crowding agent and found to promote both RNA and DNA ribozyme-catalyzed polymerizations. In contrast, PEG with an average molecular weight of 200 (PEG200) reduced the level of RNA polymerization by proteinaceous T7 RNAP but simultaneously promoted DNA polymerization, without affecting the activity of KF. Thus, proteinaceous RNA polymerase might potentially display bisubstrate specificity, which can be switched in response to changes in the dielectric constant and excluded volume in crowded environments. Our findings validate the bisubstrate activity of RNA polymerase from an evolutionary perspective for the development of non-natural materials.

The importance of an interaction network for proper DNA polymerase ζ heterotetramer activity.

Precisely controlled mechanisms have been evolved to rescue impeded DNA replication resulting from encountered obstacles and involve a set of low-fidelity translesion synthesis (TLS) DNA polymerases. Studies in recent years have brought new insights into those TLS polymerases, especially concerning the structure and subunit composition of DNA polymerase zeta (Pol ζ). Pol ζ is predominantly involved in induced mutagenesis as well as the bypass of noncanonical DNA structures, and it is proficient in extending from terminal mismatched nucleotides incorporated by major replicative DNA polymerases. Two active forms of Pol ζ, heterodimeric (Pol ζ2) and heterotetrameric (Pol ζ4) ones, have been identified and studied. Here, in the light of recent publications regarding induced and spontaneous mutagenesis and diverse interactions within Pol ζ holoenzyme, combined with Pol ζ binding to the TLS polymerase Rev1p, we discuss the subunit composition of Pol ζ in various cellular physiological conditions. Available data show that it is the heterotetrameric form of Pol ζ that is involved both during spontaneous and induced mutagenesis, and underline the importance of interactions within Pol ζ when an increased Pol ζ recruitment occurs. Understanding Pol ζ function in the bypass of DNA obstacles would give a significant insight into cellular tolerance of DNA damage, genetic instability and the onset of cancer progression.

Substitution-Inert Polynuclear Platinum Complexes That Inhibit the Activity of DNA Polymerase in Triplex-Forming Templates.

The formation of triple-helical DNA is implicated in the regulation of gene expression. The triplexes are, however, unstable under physiological conditions so that effective stabilizers for the triplex formation are needed. Herein, we describe a new strategy for the stabilization of such triplexes that is based on antitumor substitution-inert polynuclear platinum complexes (SI-PPCs). These compounds were previously shown to bind to DNA through the phosphate clamp-a discrete mode of DNA-ligand recognition distinct from the canonical intercalation and minor-groove binding. We have found that SI-PPCs efficiently inhibit DNA synthesis by DNA polymerase in sequences prone to the formation of pyrimidine- and purine-motif triplex DNAs. Moreover, the results suggest that SI-PPCs are able to induce the formation of triple-helical DNA between duplexes and strands that are not completely complementary to each other. Collectively, these data provide evidence that SI-PPCs are very efficient stabilizers of triple-stranded DNA that might exert their action by stabilizing higher-order structures such as triple-helical DNA.

An improved surface passivation method for single-molecule studies.

We report a surface passivation method based on dichlorodimethylsilane (DDS)-Tween-20 for in vitro single-molecule studies, which, under the conditions tested here, more efficiently prevented nonspecific binding of biomolecules than the standard poly(ethylene glycol) surface. The DDS-Tween-20 surface was simple and inexpensive to prepare and did not perturb the behavior and activities of tethered biomolecules. It can also be used for single-molecule imaging in the presence of high concentrations of labeled species in solution.

Sphingosine, a modulator of human translesion DNA polymerase activity.

Translesion (TLS) DNA polymerases are specialized, error-prone enzymes that synthesize DNA across bulky, replication-stalling DNA adducts. In so doing, they facilitate the progression of DNA synthesis and promote cell proliferation. To potentiate the effect of cancer chemotherapeutic regimens, we sought to identify inhibitors of TLS DNA polymerases. We screened five libraries of ∼ 3000 small molecules, including one comprising ∼ 600 nucleoside analogs, for their effect on primer extension activity of DNA polymerase η (Pol η). We serendipitously identified sphingosine, a lipid-signaling molecule that robustly stimulates the activity of Pol η by ∼ 100-fold at low micromolar concentrations but inhibits it at higher concentrations. This effect is specific to the Y-family DNA polymerases, Pols η, κ, and ι. The addition of a single phosphate group on sphingosine completely abrogates this effect. Likewise, the inclusion of other sphingolipids, including ceramide and sphingomyelin to extension reactions does not elicit this response. Sphingosine increases the rate of correct and incorrect nucleotide incorporation while having no effect on polymerase processivity. Endogenous Pol η activity is modulated similarly as the recombinant enzyme. Importantly, sphingosine-treated cells exhibit increased lesion bypass activity, and sphingosine tethered to membrane lipids mimics the effects of free sphingosine. Our studies have uncovered sphingosine as a modulator of TLS DNA polymerase activity; this property of sphingosine may be associated with its known role as a signaling molecule in regulating cell proliferation in response to cellular stress.

Proofreading dynamics of a processive DNA polymerase.

Replicative DNA polymerases present an intrinsic proofreading activity during which the DNA primer chain is transferred between the polymerization and exonuclease sites of the protein. The dynamics of this primer transfer reaction during active polymerization remain poorly understood. Here we describe a single-molecule mechanical method to investigate the conformational dynamics of the intramolecular DNA primer transfer during the processive replicative activity of the Phi 29 DNA polymerase and two of its mutants. We find that mechanical tension applied to a single polymerase-DNA complex promotes the intramolecular transfer of the primer in a similar way to the incorporation of a mismatched nucleotide. The primer transfer is achieved through two novel intermediates, one a tension-sensitive and functional polymerization conformation and a second non-active state that may work as a fidelity check point for the proofreading reaction.

Rapid high-throughput genotyping of HBV DNA using a modified hybridization-extension technique.

China has the highest incidence of hepatitis B virus (HBV) infection worldwide. HBV genotypes have variable impacts on disease pathogenesis and drug tolerance. We have developed a technically simple and accurate method for HBV genotyping that will be applicable to pre-treatment diagnosis and individualized treatment. Multiple sequence alignments of HBV genomes from GenBank were used to design primers and probes for genotyping of HBV A through H. The hybridization was carried out on nitrocellulose (NC) membranes with probes fixed in an array format, which was followed by hybrid amplification by an extension step with DNA polymerase to reinforce the double-stranded DNA hybrids on the NC membrane and subsequent visualization using an avidin-biotin system. Genotyping results were confirmed by DNA sequencing and bioinformatics analysis using the National Center for Biotechnology Information genotyping database, and compared with results from the line probe assay. The data show that multiple sequence alignment defined a 630 bp region in the HBV PreS and S regions that was suitable for genotyping. All genotyping significant single nucleotides in the region were defined. Two-hundred-and-ninety-one HBV-positive serum samples from Northwest Chinese patients were genotyped, and the genotyping rate from the new modified hybridization-extension method was 100% compared with direct sequencing. Compared with line probe assay, the newly developed method is superior, featuring reduced reaction time, lower risk of contamination, and increased accuracy for detecting single nucleotide mutation. In conclusion, a novel hybridization-extension method for HBV genotyping was established, which represents a new tool for accurate and rapid SNP detection that will benefit clinical testing.

A novel amplification strategy for genotyping with liquid chromatography-electrospray ionization mass spectrometry.

Among numerous available genotyping techniques, mass spectrometry (MS) based methods play a major role in providing high quality genotype data at reasonable costs for research and diagnostics, e.g. for pharmacogenetic applications. Ion-pair reversed-phase liquid chromatography hyphenated to electrospray ionization time-of-flight MS (ICEMS) is, for example, a powerful instrument that allows a direct characterization of complex mixtures of polymerase chain reaction (PCR) amplified DNA fragments. Current limitations of PCR-ICEMS genotyping are mainly concerned with the multiplex PCR set-up. Assay development often requires time-consuming primer design and intensive optimization of PCR conditions. To overcome this restraint, a robust amplification strategy originally combined with arrayed primer extension genotyping was transferred and adapted to ICEMS genotyping. The modifications involved limitation of the primer length, application of two universal sequences and amplification with an appropriate DNA polymerase. To demonstrate the applicability of the novel amplification strategy for ICEMS, a 23-plex pharmacogenetic genotyping assay was developed. After slight optimization steps, an efficient and quantitatively balanced amplification of all targeted markers was achieved, resulting in a convenient characterization of the multiplexed PCR fragments with ICEMS. Expenditure of time, costs and hands-on work associated with assay design and optimization was dramatically lowered compared to previous multiplex PCR-ICEMS assays. The developed 23-plex assay was applied in a pharmacogenetic study including 284 individuals (genotype call rate 99.0%). A total of 399 SNPs were retyped by Sanger sequencing (concordance rate 99.8%). The PCR-ICEMS assay turned out to be an accurate, reliable, cost-effective and a ready-to-use tool for pharmacogenetic genotyping.

Creating a unique environment for selecting reactive enzymes with DNA: 'Sticky' binding of oligocation-grafted polymers to DNA.

To provide colloidally stable polyplexes formed between pDNA and cationic polymers, cationic polymers have been modified with hydrophilic polymers to form a hydrophilic shell. Block copolymers of cationic and hydrophilic polymers and cationic polymers grafted with hydrophilic polymers are representative designs of such polymers. Here, we report a new design of cationic polymers and oligocationic peptide-grafted polymers. We synthesized 15 kinds of graft copolymers by varying the number of cationic charges of the peptides and their grafting density. We found that graft copolymers with less cationic peptides and less grafting density formed colloidally stable polyplexes. Interestingly, the less cationic graft copolymers bind to excess amounts of pDNA. We also found that the graft copolymers showed selectivity toward reactive enzymes affording the reaction of pDNA with nucleases, while suppressing both the replication of DNA by DNA polymerase and gene expression. The suppression of the replication and expression is considered to result from the high capacity of the graft copolymers for binding with pDNA. The polynucleotides produced by DNA polymerase or RNA polymerase would be captured by the graft copolymers to impede these enzymatic reactions.

Synergy between DNA polymerases increases polymerase chain reaction inhibitor tolerance in forensic DNA analysis.

The success rate of diagnostic polymerase chain reaction (PCR) analysis is lowered by inhibitory substances present in the samples. Recently, we showed that tolerance to PCR inhibitors in crime scene saliva stains can be improved by replacing the standard DNA polymerase AmpliTaq Gold with alternative DNA polymerase-buffer systems (Hedman et al., BioTechniques 47 (2009) 951-958). Here we show that blending inhibitor-resistant DNA polymerase-buffer systems further increases the success rate of PCR for various types of real crime scene samples showing inhibition. For 34 of 42 "inhibited" crime scene stains, the DNA profile quality was significantly improved using a DNA polymerase blend of ExTaq Hot Start and PicoMaxx High Fidelity compared with AmpliTaq Gold. The significance of the results was confirmed by analysis of variance. The blend performed as well as, or better than, the alternative DNA polymerases used separately for all tested sample types. When used separately, the performance of the DNA polymerases varied depending on the nature of the sample. The superiority of the blend is discussed in terms of complementary effects and synergy between the DNA polymerase-buffer systems.

Polymerization fidelity of a replicative DNA polymerase from the hyperthermophilic archaeon Sulfolobus solfataricus P2.

Sulfolobus solfataricus P2 is an aerobic crenarchaeon which grows optimally at 80 degrees C and pH 2-4. This organism encodes a B-family DNA polymerase, DNA polymerase B1 (PolB1), which faithfully replicates its genome of 3 million base pairs. Using pre-steady-state kinetic methods, we estimated the fidelity of PolB1 to be in the range of 10(-6) to 10(-8), or one error per 10(6) to 10(8) nucleotide incorporations in vivo. To discern how the polymerase and 3' --> 5' exonuclease activities contribute to the high fidelity of PolB1, an exonuclease-deficient mutant of PolB1 was constructed by mutating three conserved residues at the exonuclease active site. The base substitution fidelity of this mutant was kinetically measured to be in the range of 10(-4) to 10(-6) at 37 degrees C and pH 7.5. PolB1 exhibited high fidelity due to large differences in both ground-state nucleotide binding affinity and nucleotide incorporation rates between correct and incorrect nucleotides. The kinetic partitioning between the slow mismatch extension catalyzed by the polymerase activity and the fast mismatch excision catalyzed by the 3' --> 5' exonuclease activity further lowers the error frequency of PolB1 by 14-fold. Furthermore, the base substitution error frequency of the exonuclease-deficient PolB1 increased by 5-fold as the reaction temperature increased. Interestingly, the fidelity of the exonuclease-deficient PolB1 mutant increased by 36-fold when the buffer pH was lowered from 8.5 to 6.0. A kinetic basis for these temperature and pH changes altering the fidelity of PolB1 was established. The faithful replication of genomic DNA catalyzed by PolB1 is discussed.

Comparative purification strategies for Drosophila and human mitochondrial DNA replication proteins: DNA polymerase gamma and mitochondrial single-stranded DNA-binding protein.

The mitochondrion is the eukaryotic organelle that carries out oxidative phosphorylation, fulfilling cellular requirements for ATP production. Disruption of mitochondrial energy metabolism can occur by genetic and biochemical mechanisms involving nuclear-encoded proteins that are required at the mitochondrial DNA replication fork, which often leads to human disorders and to animal lethality during development. DNA polymerase gamma (pol gamma), the mitochondrial replicase, and the mitochondrial single-stranded DNA-binding protein (mtSSB) have been the focus of study in our lab for a number of years. Here we describe the purification strategies that we developed for obtaining the recombinant forms of pol gamma and mtSSB from both Drosophila melanogaster and humans. Despite the fact that similar approaches can be used for purifying the homologous proteins, we have observed that there are differences in the behavior of the proteins in some specific steps that may reflect differences in their structural and biochemical properties. Their purification in homogeneous, active form represents the first step toward our long-term goal to understand their biochemistry, biology, and functions at the mitochondrial DNA replication fork.

Sensitive and sustained imaging of intracellular microRNA in living cells by a high biocompatible liposomal vehicle introduced isothermal symmetric exponential amplification reaction.

A high biocompatible liposomal vehicle was used to deliver enzymes and oligonucleotide substances of the isothermal symmetric exponential amplification reaction into living cells, allowing sensitive and sustained imaging of microRNA in living cells for more than 24 h without apoptosis.

A novel random mutagenesis approach using human mutagenic DNA polymerases to generate enzyme variant libraries.

The in vitro MutaGen procedure is a new random mutagenesis method based on the use of low-fidelity DNA polymerases. In the present study, this technique was applied on a 2 kb gene encoding amylosucrase, an attractive enzyme for the industrial synthesis of amylose-like polymers. Mutations were first introduced during a single replicating step performed by mutagenic polymerases pol beta and pol eta. Three large libraries (>10(5) independent clones) were generated (one with pol beta and two with pol eta). The sequence analysis of randomly chosen clones confirmed the potential of this strategy for the generation of diversity. Variants generated by pol beta were 4-7-fold less mutated than those created with pol eta, indicating that our approach enables mutation rate control following the DNA polymerase employed for mutagenesis. Moreover, pol beta and pol eta provide different and complementary mutation spectra, allowing a wider sequence space exploration than error-prone PCR protocols employing Taq polymerase. Interestingly, some of the variants generated by pol eta displayed unusual modifications, including combinations of base substitutions and codon deletions which are rarely generated using other methods. By taking advantage of the mutation bias of naturally highly error-prone DNA polymerases, MutaGen thus appears as a very useful tool for gene and protein randomisation.

Single-Molecule FRET Analysis of Replicative Helicases.

Over the recent years single-molecule fluorescence resonance energy transfer (smFRET) technique has proven to be one of the most powerful tools for revealing mechanistic insights into helicase activities. Here we describe details of single-molecule FRET assays for probing DNA unwinding activities as well as functional dynamics by replicative helicases in real time. The ability of smFRET to measure the behavior of biomolecules at a nanometer scale enabled us to address how the leading and lagging strand synthesis are coordinated during DNA replication, to resolve DNA unwinding steps of Bacteriophage T7 helicase, and to observe heterogeneous unwinding patterns modulated by the DNA binding domain of E1 helicase. These single-molecule FRET assays are generally applicable to other replicative and nonreplicative hexameric helicases.

Self-replication of DNA by its encoded proteins in liposome-based synthetic cells.

Replication of DNA-encoded information and its conversion into functional proteins are universal properties of life. In an effort toward the construction of a synthetic minimal cell, we implement here the DNA replication machinery of the Φ29 virus in a cell-free gene expression system. Amplification of a linear DNA template by self-encoded, de novo synthesized Φ29 proteins is demonstrated. Complete information transfer is confirmed as the copied DNA can serve as a functional template for gene expression, which can be seen as an autocatalytic DNA replication cycle. These results show how the central dogma of molecular biology can be reconstituted and form a cycle in vitro. Finally, coupled DNA replication and gene expression is compartmentalized inside phospholipid vesicles providing the chassis for evolving functions in a prospective synthetic cell relying on the extant biology.