Linear doggybone DNA vaccine induces similar immunological responses to conventional plasmid DNA independently of immune recognition by TLR9 in a pre-clinical model.

Vaccination with DNA that encodes cancer antigens is a simple and convenient way to raise immunity against cancer and has already shown promise in the clinical setting. Conventional plasmid DNA is commonly used which together with the encoded antigen also includes bacterial immunostimulatory CpG motifs to target the DNA sensor Toll-like receptor 9. Recently DNA vaccines using doggybone DNA (dbDNA™), have been developed without the use of bacteria. The cell-free process relies on the use of Phi29 DNA polymerase to amplify the template followed by protelomerase TelN to complete individual closed linear DNA. The resulting DNA contains the required antigenic sequence, a promoter and a poly A tail but lacks bacterial sequences such as an antibiotic resistance gene, prompting the question of immunogenicity. Here we compared the ability of doggybone DNA vaccine with plasmid DNA vaccine to induce adaptive immunity using clinically relevant oncotargets E6 and E7 from HPV. We demonstrate that despite the inability to trigger TLR9, doggybone DNA was able to induce similar levels of cellular and humoral immunity as plasmid DNA, with suppression of established TC-1 tumours.

A toolkit and benchmark study for FRET-restrained high-precision structural modeling.

We present a comprehensive toolkit for Förster resonance energy transfer (FRET)-restrained modeling of biomolecules and their complexes for quantitative applications in structural biology. A dramatic improvement in the precision of FRET-derived structures is achieved by explicitly considering spatial distributions of dye positions, which greatly reduces uncertainties due to flexible dye linkers. The precision and confidence levels of the models are calculated by rigorous error estimation. The accuracy of this approach is demonstrated by docking a DNA primer-template to HIV-1 reverse transcriptase. The derived model agrees with the known X-ray structure with an r.m.s. deviation of 0.5 Å. Furthermore, we introduce FRET-guided 'screening' of a large structural ensemble created by molecular dynamics simulations. We used this hybrid approach to determine the formerly unknown configuration of the flexible single-strand template overhang.

dNTP-dependent conformational transitions in the fingers subdomain of Klentaq1 DNA polymerase: insights into the role of the "nucleotide-binding" state.

BACKGROUND: Conformational selection plays a key role in the polymerase cycle. RESULTS: Klentaq1 exists in conformational equilibrium between three states (open, closed, and "nucleotide-binding") whose level of occupancy is determined by the bound substrate. CONCLUSION: The "nucleotide-binding" state plays a pivotal role in the reaction pathway. SIGNIFICANCE: Direct evidence is provided for the role of a conformationally distinct "nucleotide-binding" state during dNTP incorporation. DNA polymerases are responsible for the accurate replication of DNA. Kinetic, single-molecule, and x-ray studies show that multiple conformational states are important for DNA polymerase fidelity. Using high precision FRET measurements, we show that Klentaq1 (the Klenow fragment of Thermus aquaticus DNA polymerase 1) is in equilibrium between three structurally distinct states. In the absence of nucleotide, the enzyme is mostly open, whereas in the presence of DNA and a correctly base-pairing dNTP, it re-equilibrates to a closed state. In the presence of a dNTP alone, with DNA and an incorrect dNTP, or in elevated MgCl2 concentrations, an intermediate state termed the "nucleotide-binding" state predominates. Photon distribution and hidden Markov modeling revealed fast dynamic and slow conformational processes occurring between all three states in a complex energy landscape suggesting a mechanism in which dNTP delivery is mediated by the nucleotide-binding state. After nucleotide binding, correct dNTPs are transported to the closed state, whereas incorrect dNTPs are delivered to the open state.

Single-molecule multiparameter fluorescence spectroscopy reveals directional MutS binding to mismatched bases in DNA.

Mismatch repair (MMR) corrects replication errors such as mismatched bases and loops in DNA. The evolutionarily conserved dimeric MMR protein MutS recognizes mismatches by stacking a phenylalanine of one subunit against one base of the mismatched pair. In all crystal structures of G:T mismatch-bound MutS, phenylalanine is stacked against thymine. To explore whether these structures reflect directional mismatch recognition by MutS, we monitored the orientation of Escherichia coli MutS binding to mismatches by FRET and anisotropy with steady state, pre-steady state and single-molecule multiparameter fluorescence measurements in a solution. The results confirm that specifically bound MutS bends DNA at the mismatch. We found additional MutS-mismatch complexes with distinct conformations that may have functional relevance in MMR. The analysis of individual binding events reveal significant bias in MutS orientation on asymmetric mismatches (G:T versus T:G, A:C versus C:A), but not on symmetric mismatches (G:G). When MutS is blocked from binding a mismatch in the preferred orientation by positioning asymmetric mismatches near the ends of linear DNA substrates, its ability to authorize subsequent steps of MMR, such as MutH endonuclease activation, is almost abolished. These findings shed light on prerequisites for MutS interactions with other MMR proteins for repairing the appropriate DNA strand.

dbDNA™: An advanced platform for genetic medicines.

The supply of plasmid DNA has become a major bottle neck in the ever-expanding genetic medicine sector. Therefore, the development of new, scalable, faster DNA production technologies is vital for this sector going forward.

An intramolecular FRET system monitors fingers subdomain opening in Klentaq1.

A major goal of polymerase research is to determine the mechanism through which a nucleotide complementary to a templating DNA base is selected and delivered to the polymerase active site. Structural evidence suggests a large open-to-closed conformational change affecting the fingers subdomain as being crucial to the process. We previously designed a FRET system capable of measuring the rate of fingers subdomain closure in the presence of correct nucleotide. However, this FRET system was limited in that it could not directly measure the rate of fingers subdomain opening by FRET after polymerization or in the absence of DNA. Here we report the development of a new system capable of measuring both fingers subdomain closure and reopening by FRET, and show that the rate of fingers subdomain opening is limited only by the rate of polymerization. We anticipate that this system will scale down to the single molecule level, allowing measurement of fingers subdomain movements in the presence of incorrect nucleotide and in the absence of DNA.

Structure and mechanism of DNA polymerases.

DNA polymerases are molecular motors directing the synthesis of DNA from nucleotides. All polymerases have a common architectural framework consisting of three canonical subdomains termed the fingers, palm, and thumb subdomains. Kinetically, they cycle through various states corresponding to conformational transitions, which may or may not generate force. In this review, we present and discuss the kinetic, structural, and single-molecule works that have contributed to our understanding of DNA polymerase function.

Novel synthetic plasmid and Doggybone DNA vaccines induce neutralizing antibodies and provide protection from lethal influenza challenge in mice.

Nucleic acid-based vaccines (NAVs) are a promising alternative to conventional influenza vaccines with the potential to increase influenza vaccine availability due to their simplicity in design and rapid speed of production. NAVs can also target multiple influenza antigens and control flu variants. Traditionally NAVs have been DNA plasmids however, we are continuing to explore new methods that may enhance vaccine efficacy. Recently new focus has been on RNA cassettes as NAVs. RNA vaccines combine conceptual advantages in that they focus on delivery of only the coding cassette. However, RNA vaccines have a short half-life and cause interferon-induced fevers. Here we describe a new NAV approach where we study delivery of a linear DNA cassette [Doggybone linear closed DNA [(dbDNA)] produced by an enzymatic process that yields an antigen expression cassette comprising a promoter, DNA antigen, poly A tail, and telomeric ends. This focused approach has many of the advantages of plasmid DNA as well as a minimal cassette size similar to RNA strategies. For this study, we characterized the specific CD4(+) and CD8(+) T cell responses and determined the hemagglutination inhibition (HI) titers induced by dbDNA and compared the responses with those of an optimized plasmid DNA (pDNA) vaccine encoding the same H1N1 influenza A/PR/8/34 HA gene. Immunizations with the constructs resulted in similar humoral and cellular immune responses. Both constructs induced high-titer HI antibodies and fully protected animals from lethal viral challenge. The data obtained from this study provides important validation for further development of novel vector approaches.

Accurate single-molecule FRET studies using multiparameter fluorescence detection.

In the recent decade, single-molecule (sm) spectroscopy has come of age and is providing important insight into how biological molecules function. So far our view of protein function is formed, to a significant extent, by traditional structure determination showing many beautiful static protein structures. Recent experiments by single-molecule and other techniques have questioned the idea that proteins and other biomolecules are static structures. In particular, Förster resonance energy transfer (FRET) studies of single molecules have shown that biomolecules may adopt many conformations as they perform their function. Despite the success of sm-studies, interpretation of smFRET data are challenging since they can be complicated due to many artifacts arising from the complex photophysical behavior of fluorophores, dynamics, and motion of fluorophores, as well as from small amounts of contaminants. We demonstrate that the simultaneous acquisition of a maximum of fluorescence parameters by multiparameter fluorescence detection (MFD) allows for a robust assessment of all possible artifacts arising from smFRET and offers unsurpassed capabilities regarding the identification and analysis of individual species present in a population of molecules. After a short introduction, the data analysis procedure is described in detail together with some experimental considerations. The merits of MFD are highlighted further with the presentation of some applications to proteins and nucleic acids, including accurate structure determination based on FRET. A toolbox is introduced in order to demonstrate how complications originating from orientation, mobility, and position of fluorophores have to be taken into account when determining FRET-related distances with high accuracy. Furthermore, the broad time resolution (picoseconds to hours) of MFD allows for kinetic studies that resolve interconversion events between various subpopulations as a biomolecule of interest explores its structural energy landscape.

A pre-equilibrium before nucleotide binding limits fingers subdomain closure by Klentaq1.

Numerous studies have been undertaken to establish the mechanism of dNTP binding and template-directed incorporation by DNA polymerases. It has been established by kinetic experiments that a rate-limiting step, crucial for dNTP selection, occurs before chemical bond formation. Crystallographic studies indicated that this step may be due to a large open-to-closed conformational transition affecting the fingers subdomain. In previous studies, we established a fluorescence resonance energy transfer system to monitor the open-to-closed transition in the fingers subdomain of Klentaq1. By comparing the rates of the fingers subdomain closure with that of the rate-limiting step for Klentaq1, we showed that fingers subdomain motion was significantly faster than the rate-limiting step. We have now used this system to characterize DNA binding as well as to complete a more extensive characterization of incorporation of all four dNTPs. The data indicate that DNA binding occurs by a two-step association and that dissociation of the DNA is significantly slower in the case of the closed ternary complex. The data for nucleotide incorporation indicate a step occurring before dNTP binding, which differs for all four nucleotides. As the only difference between the (E x p/t) complexes is the templating base, it would suggest an important role for the templating base in initial ground state selection.

Motions of the fingers subdomain of klentaq1 are fast and not rate limiting: implications for the molecular basis of fidelity in DNA polymerases.

Various kinetic studies on nucleotide incorporation by DNA polymerases have established that a rate-limiting step occurs that is crucial in the mechanism of discrimination between correct versus incorrect nucleotide. Crystallographic studies have indicated that this step may be due to a large open-to-closed conformational transition affecting the fingers subdomain. However, there is no direct evidence to support this hypothesis. In order to investigate whether or not the open-to-closed conformational transition affecting the fingers subdomain is rate limiting, we have developed a fluorescence resonance energy transfer (FRET) system, which monitors motions of the fingers subdomain. We establish that the closing of the fingers subdomain is significantly faster than the kinetically determined rate-limiting step. We propose that the rate-limiting step occurs after the closing of the fingers subdomain and is caused by local reorganization events in the active site.