- Email: [email protected]
Multi-Scale Structure and Conformational Dynamics of Scaffolded DNA Origami Nanoparticles
70a
Sunday, February 12, 2017
specifically compare the patterns of melting observed experimentally with those obtained from the coarse-grained simulations.
Through a combination of simulations and control experiments we demonstrate these limits, and present a collection of best-practices.
356-Pos Board B121 Role of Watson-Crick-Like Mismatches in DNA Replication Fidelity Eric S. Szymanski1, Isaac J. Kimsey2, Hashim M. Al-Hashimi1. 1 Dept. of Biochemistry, Duke University, Durham, NC, USA, 2Nymirium, Ann Arbor, MI, USA. DNA replication, transcription, and translation rely on the strict Watson-Crick base pairing rules to ensure faithful transmission of genetic information. The Watson-Crick pairing rules are determined by the predominant neutral tautomeric forms of the nucleic acid bases. Incorrect base pairing during replication, if left unrepaired, leads to transition or transversion point mutations. Spontaneous mutagenesis from replication errors is believed to be a prominent source of base substitution errors in tumor suppressor genes in multiple forms of cancer. Rare tautomeric and ionized nucleotide bases can form mismatches that conform to the Watson-Crick like geometry, subverting proof reading mechanisms. These tautomeric and anionic mismatches have long been suspected to contribute to spontaneous replication errors; they have proved difficult to visualize as the conformational changes are subtle and involve the rearmament of protons. Nuclear magnetic resonance relaxation dispersion techniques have allowed for the characterization of a highly sequencedependent kinetic network connecting the wobble dG$dT mismatch to multiple Watson-Crick-like tautomeric and anionic dG$dT mismatch ‘excited states’. We have obtained evidence in support of a kinetic model for misincorporation which introduces a rate-limiting on-pathway tautomerization or ionization step that leads to Watson-Crick-like mismatches prior to incorporation through the canonical synthesis pathway. This kinetic model can account for i) the three orders of magnitude difference seen in vitro between rates of correct and incorrect nucleotide incorporations, ii) nucleotide selectivity fidelity as low as 106, and iii) the poorly understood sequence dependence of polymerization errors.
359-Pos Board B124 Small Molecule Aptamers for Biosensing Gregory Wiedman, Yunan Zhao, David Perlin. Public Health Research Institute, Rutgers New Jersey Medical School, Newark, NJ, USA. In this work, we used modified Synthetic Evolution of Ligands through Exponential Enrichment (SELEX) to discover a DNA aptamer recognizing azole class antifungal drugs. This aptamer undergoes a secondary structural change upon binding its target molecule as shown through fluorescence anisotropy based binding measurements. Using circular dichroism spectroscopy, we found a unique double G-quadruplex structure that was essential for binding to the target: azole antifungal drugs. This type of aptamer has the potential to be used as a small molecule captor component of a device for therapeutic drug monitoring.
357-Pos Board B122 Direct Observation of Single Biopolymer Folding and Unfolding Process by Solid-State Nanopore Xin Shi, Rui Gao, Shao-Chuang Liu, Qiao Li, Yi-Tao Long. East China University of Science and Technology, Shanghai, China. Biomolecular conformation and their transition play a crucial role in various in vivo or in vitro system. The most of the practical techniques for resolving the secondary structures of biomolecules could provide quite precise structural information for their solid-state or steady state, even at atomic resolution. For example, Cryo-EM determines high-resolution structures for the frozenhydrated specimens of biomolecules. polymers, but it is still challenging to resolve the dynamic process of multiple functional conformational states for biomolecules at single-molecular scale. Here, we direct observed DNA folding and unfolding process in real-time by using sub-5 nm solid-state nanopores. In our experiments, a single-stranded DNA adhered to single monovalent streptavidin could be reversibly trapped in a solid-state nanopore. Then, the fluctuations of the blockade current could be recorded, which reveals the dynamic structural transitions among DNA secondary structures. For example, after trapping the cytosine-rich DNA strains in slightly alkaline solution, the formation of multiple unstable and semi-folded i-motif structures could be observed. More important, well time-resolved transitions between these structures could be obtained. When using slightly acidic solution, the stable structures with stable blockade current could be found. With this new approach, we can directly observe the dynamic conformational change of biomolecules at single-molecular scale, which would be of great help for resolving single molecule interactions, designing single-molecule machine and understanding the working process of biomolecular in biological system. 358-Pos Board B123 Tuning Up Tethered Particle Motion Daniel T. Kovari, Eric Weeks, David Dunlap, Laura Finzi. Physics, Emory University, Atlanta, GA, USA. Tethered Particle Motions assays are a simple but powerful tools for monitoring the effective length of individual DNA strands and other linear bio-polymers in real-time. The technique has been employed in various capacities including characterization of DNA topology, transcription factor - DNA interactions, and monitoring progress of enzymes that translocate along DNA. At its core the technique is relatively simple to implement, only requiring a researchgrade microscope and a video camera; however, it is important to note that optical resolution, frame rate, exposure time, particle size, and solution viscosity all affect the ability to discriminate different tether lengths and detect changes.
360-Pos Board B125 Improved Sampling in Molecular Dynamics Studies of DNA and the B To Z[WC] To Z-DNA Transition Lam T. Nguyen, Ashutosh Rai, Micaela E. Bush, Alma Gracic, Ahsan A. Khoja, Jinhee Kim, Sunil Pun, Alexander K. Seewald, Benjamin L. Yee, Michael G. Lerner. Physics and Astronomy, Earlham College, Richmond, IN, USA. Although DNA is most commonly found in the right-handed B-DNA structure, it is known that biologically active systems also contain left-handed ZIIDNA. We used both steered and targeted molecular dynamics in combination with umbrella sampling to produce potentials of mean force for the B to ZII transition along both the direct B-ZII pathway as well as the B-Z[WC]-ZII pathway. Full pathway sampling is not feasible on smaller computer clusters, so we used Hamiltonian Replica Exchange to relax individual portions of the pathway. This technique is generalizable to larger systems and larger computer clusters. 361-Pos Board B126 Energetic Contributions of Plectoneme Tips and Tails Andrew Dittmore, Keir C. Neuman. NIH, Bethesda, MD, USA. Global DNA topology is sensed locally by enzymes that act on plectonemes in supercoiled DNA. Here we report that the formation and diffusion of plectonemes are determined by the energetic contributions of their tips and tails. First, to systematically vary the geometry and formation energy of plectoneme endloops, we introduced base-pair defect regions of variable size (1-16 bp) using a cassette based single-strand nicking template generated by PCR. Direct manipulation measurements with magnetic tweezers revealed that even a single mismatch or abasic site is sufficient to nucleate formation of a plectoneme. Presentation of the defect precisely at an extruded plectoneme tip potentially serves as a damage-sensing mechanism and may facilitate the search process of repair enzymes. Second, our measurements unexpectedly revealed that after twisted DNA abruptly buckles into an initial plectoneme loop, further plectoneme extrusion occurs through a cascade of additional buckling steps in which the torque changes by roughly half of the initial overshoot value. These discrete steps do not match any obvious scale of the system but are consistent with discontinuous feed-in of curving plectoneme tails. In light of these results, theoretical models of plectonemes should include their overall structure, including the often neglected tips and tails. 362-Pos Board B127 Multi-Scale Structure and Conformational Dynamics of Scaffolded DNA Origami Nanoparticles William Bricker, Keyao Pan, Mark Bathe. Massachusetts Institute of Technology, Cambridge, MA, USA. Synthetic DNA can be programmed into self-assembled 3D nanoparticles using a DX design motif and the principle of scaffolded DNA origami. A topdown design procedure (DAEDALUS) (Veneziano, Ratanalert, et al., Science, 2016) facilitates the design of arbitrary DNA nanoparticle geometries on the 5 to 100 nanometer scale, which we investigate in detail here using multi-scale structural modeling. While coarse-grained modeling is useful for generating equilibrium structures of DNA nanoparticles (Pan et al., Nat. Comm., 2014), only all-atom models reveal fine structural details and mechanical properties that contribute to overall structure and conformational dynamics. Here, we first use all-atom molecular dynamics (MD) to simulate two 0.5 1.0 MDa DNA polyhedral nanoparticles: a tetrahedron with 63 base pair
Sunday, February 12, 2017 (bp) edge lengths, and an octahedron with 52 bp edge lengths. Using 150 ns trajectories, we are able to elucidate subtle structural features seen in experimental cryo-EM maps, including right-handed twisting of the vertices in the octahedron and outward bowing of the edges in the tetrahedron. Next, these results are compared with all-atom MD simulations of unconstrained vertex building blocks including the tetrahedron (3-arm vertex) and octahedron (4-arm vertex). In these simulations, a notable feature is the significant out-of-plane bending angle away from the minor groove at the vertex due to the chirality of dsDNA. Finally, equilibrium solution structures of 45 DX-based DNA origami nanoparticles are predicted by implementing an updated bulge stiffness parameter for our coarse-grained FE model CanDo (Kim et al., Nucleic Acids Res., 2012). These multi-scale structural results show the interplay between coarse-grained and all-atom models in the ab initio prediction and elucidation of complex features of DNA nanoparticles seen in experimental cryo-EM maps.
RNA Binding 363-Pos Board B128 Shedding Light on Cas9 Target Search Viktorija Globyte1, Seung Hwan Lee1, Luuk Loeff1, Jin Soo Kim2, Chirlmin Joo1. 1 TU Delft, Delft, Netherlands, 2Seoul National University, Seoul, Korea, Republic of. The CRISPR/Cas adaptive immune system provides prokaryotes with a defensive mechanism against invading viruses and plasmids. The invading viral DNA fragments are incorporated into the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) locus in the bacterial genome and are later used to recognize and destroy the invader when it returns. In the last stage of CRISPR immunity, called the interference stage, Cas (CRISPR-associated) proteins assemble with short guide RNA molecules which are transcribed from the CRISPR locus. These guide RNA molecules can be programmed to recognize any DNA sequence. In recent years the CRISPR/Cas adaptive immune system has seen an immense growth in interest with the type II CRISPR-Cas9 system being in the center of attention. In this system, the DNA of the invading virus is recognized and cleaved by a single protein Cas9 which is guided by an RNA duplex. Due to its simplicity, CRISPRCas9 system is a promising tool in gene engineering as its guide RNA can be programmed to recognize virtually any sequence in the genome. The CRISPR-Cas9 has been demonstrated to work in a variety of organisms, however, despite the large interest in this system, the precise mechanism by which Cas9 finds and cleaves its target remains ambiguous. We utilize biophysical single-molecule techniques, namely total internal reflection fluorescence microscopy (TIRFM) together with Forster resonance energy transfer (FRET) to investigate the mechanics of Cas9 target search with nanometer sensitivity. We are probing one-dimensional diffusion of the protein along the DNA strand and investigating the effects different DNA sequences have on the mechanics of Cas9 target search. 364-Pos Board B129 To Cleave or not to Cleave: Predicting the Target Specificity of CRISPRCas Systems through Theoretical Modeling Misha Klein, Martin Depken. Kavli institute of Nanoscience, Departement of BioNanoScience, TU Delft, Delft, Netherlands. Many prokaryotes employ the CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeat – CRISPR associated) system to fend off attacks by hostile genetic elements. This adaptive immune system recognizes invaders based on their level of sequence complementarity with RNA transcribed from a library of past invasions stored at the CRISPR locus. To avoid infection, the CRISPR interference complex must be able to single out a short target sequence (20 – 40 nt) among a total of 105-106 nt in the cell, while at the same time recognizing targets that have evolved away from the record stored at the CRISPR locus. Despite the tremendous interest the CRISPR-Cas9 system has gained as a novel genome editing tool, the sequence preference of the interference complex remains poorly understood. Experiments have shown that it is not just the amount of mutations, but also their placement along the guide/target, that determine the level of interference. Through kinetic modeling we provide simple rules to assess sequence specificity based on mismatch patterns, and quantitatively explain the experimentally observed seed region over which no mismatches are permitted. Moving beyond sequence complementarity, we also quantify how changes in the conformation of the interference complex can serve to improve specificity during target recognition. Finally, by fitting our model to published experimental
71a
data, we give estimates for the base-pairing energies within the interference complex for various CRISPR systems. 365-Pos Board B130 Conformational Dynamics of Cas9 during DNA Binding Yavuz S. Dagdas1, Janice S. Chen2, Samuel H. Sternberg3, Jennifer A. Doudna2,3, Ahmet Yildiz2,4. 1 Biophysics Graduate Group, University of California, Berkeley, Berkeley, CA, USA, 2Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA, 3Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA, 4Department of Physics, University of California, Berkeley, Berkeley, CA, USA. Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA using two conserved nuclease domains, RuvC and HNH, as part of CRISPR-Cas bacterial adaptive immune systems. Together with singleguide RNAs, Cas9 is also widely utilized as a programmable genome editing tool. DNA cleavage activity is controlled directly by the conformational state of the HNH nuclease domain, but Cas9 conformational dynamics during DNA binding remain poorly characterized. Using single-molecule FRET assays, we identified a long-lived intermediate state of S. pyogenes Cas9. Upon DNA binding, the HNH nuclease reversibly transitions between RNA-bound, DNA-bound and docked conformations, before it docks irreversibly into its catalytically active conformation for DNA cleavage. Docking of HNH to its active state requires divalent cation, and HNH remains in the docked state after cleavage of a complementary target. Increasing the number of mutations in the target region distal to the protospacer adjacent motif (PAM) prevents transitions from intermediate to the docked conformation. The results provide a structural explanation for the lack of DNA cleavage activity when Cas9 binds to off-target sites. 366-Pos Board B131 Repetitive Loop Formation by the CRISPR-Cas3 Helicase Luuk Loeff1, Stan Brouns1,2, Chirlmin Joo1. 1 Technical University Delft, Delft, Netherlands, 2Wageningen University, Wageningen, Netherlands. E. coli maintain CRISPR-Cas adaptive immune systems to protect the cell against invading genetic elements. Immunity relies on the RNA guided surveillance complex Cascade (CRISPR-associated complex for antiviral defense) and the trans-acting Cas3 protein with helicase and nuclease activities. We recently showed that Cas3 generates degradation products ranging from 30 to 150 nt that act as pre-cursors for primed spacer acquisition. However, it remains unclear which mechanism drives the generation of these fragments with a specific size. Here we employed single-molecule FRET to probe the molecular dynamics of Cas3 in real-time. Our data shows that Cas3 repeatedly generates DNA loops in the target strand whilst remaining in tight contact with Cascade. DNA loops are generated by breaking open the dsDNA helix in distinctive steps of 3 bp, arising from the RecA like folds of the helicase domain. Repetitive unwinding achieved by slipping of the helicase domain, which limits the average translocation distance to ~90 nt. Taken together, our data suggest that the inherent helicase properties of Cas3 drive the generation precursors of adequate size for primed spacer integration. 367-Pos Board B132 The Impact of DNA Topology on Target Selection by a Cytosine-Specific Cas9 Tsz Kin Martin Tsui, Travis H. Hand, Hong Li. Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, USA. Cas9 is an RNA-guided DNA cleavage enzyme being actively developed for genome editing and gene regulation. To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be 1) complementary to the Cas9-bound guide RNA and a short Cas9-specific sequence adjacent to protospacer, called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition in time and space of the protospacer- and PAM-interaction with Cas9 will enable development of versatile and safe Cas9-based technology. We report identification and biochemical characterization of Cas9 from Acidothermus cellulolyticus (AceCas9). AceCas9 depends strictly on a 50 -NNNCC-30 PAM and is more efficient in cleaving negative supercoils than relaxed DNA. We further showed that mismatches to the guide RNA on a supercoiled protospacer are tolerated by AceCas9, whereas the same mismatches on its relaxed form were not. The cytosine-specific and DNA topology-sensitive properties of the AceCas9 maybe explored for chromosome domain specific genome editing applications. Finally, our preliminary data showed that AceCas9 can disrupt target DNA in in vivo assays, demonstrating its utility as a genome editing enzyme.
Sunday, February 12, 2017
specifically compare the patterns of melting observed experimentally with those obtained from the coarse-grained simulations.
Through a combination of simulations and control experiments we demonstrate these limits, and present a collection of best-practices.
356-Pos Board B121 Role of Watson-Crick-Like Mismatches in DNA Replication Fidelity Eric S. Szymanski1, Isaac J. Kimsey2, Hashim M. Al-Hashimi1. 1 Dept. of Biochemistry, Duke University, Durham, NC, USA, 2Nymirium, Ann Arbor, MI, USA. DNA replication, transcription, and translation rely on the strict Watson-Crick base pairing rules to ensure faithful transmission of genetic information. The Watson-Crick pairing rules are determined by the predominant neutral tautomeric forms of the nucleic acid bases. Incorrect base pairing during replication, if left unrepaired, leads to transition or transversion point mutations. Spontaneous mutagenesis from replication errors is believed to be a prominent source of base substitution errors in tumor suppressor genes in multiple forms of cancer. Rare tautomeric and ionized nucleotide bases can form mismatches that conform to the Watson-Crick like geometry, subverting proof reading mechanisms. These tautomeric and anionic mismatches have long been suspected to contribute to spontaneous replication errors; they have proved difficult to visualize as the conformational changes are subtle and involve the rearmament of protons. Nuclear magnetic resonance relaxation dispersion techniques have allowed for the characterization of a highly sequencedependent kinetic network connecting the wobble dG$dT mismatch to multiple Watson-Crick-like tautomeric and anionic dG$dT mismatch ‘excited states’. We have obtained evidence in support of a kinetic model for misincorporation which introduces a rate-limiting on-pathway tautomerization or ionization step that leads to Watson-Crick-like mismatches prior to incorporation through the canonical synthesis pathway. This kinetic model can account for i) the three orders of magnitude difference seen in vitro between rates of correct and incorrect nucleotide incorporations, ii) nucleotide selectivity fidelity as low as 106, and iii) the poorly understood sequence dependence of polymerization errors.
359-Pos Board B124 Small Molecule Aptamers for Biosensing Gregory Wiedman, Yunan Zhao, David Perlin. Public Health Research Institute, Rutgers New Jersey Medical School, Newark, NJ, USA. In this work, we used modified Synthetic Evolution of Ligands through Exponential Enrichment (SELEX) to discover a DNA aptamer recognizing azole class antifungal drugs. This aptamer undergoes a secondary structural change upon binding its target molecule as shown through fluorescence anisotropy based binding measurements. Using circular dichroism spectroscopy, we found a unique double G-quadruplex structure that was essential for binding to the target: azole antifungal drugs. This type of aptamer has the potential to be used as a small molecule captor component of a device for therapeutic drug monitoring.
357-Pos Board B122 Direct Observation of Single Biopolymer Folding and Unfolding Process by Solid-State Nanopore Xin Shi, Rui Gao, Shao-Chuang Liu, Qiao Li, Yi-Tao Long. East China University of Science and Technology, Shanghai, China. Biomolecular conformation and their transition play a crucial role in various in vivo or in vitro system. The most of the practical techniques for resolving the secondary structures of biomolecules could provide quite precise structural information for their solid-state or steady state, even at atomic resolution. For example, Cryo-EM determines high-resolution structures for the frozenhydrated specimens of biomolecules. polymers, but it is still challenging to resolve the dynamic process of multiple functional conformational states for biomolecules at single-molecular scale. Here, we direct observed DNA folding and unfolding process in real-time by using sub-5 nm solid-state nanopores. In our experiments, a single-stranded DNA adhered to single monovalent streptavidin could be reversibly trapped in a solid-state nanopore. Then, the fluctuations of the blockade current could be recorded, which reveals the dynamic structural transitions among DNA secondary structures. For example, after trapping the cytosine-rich DNA strains in slightly alkaline solution, the formation of multiple unstable and semi-folded i-motif structures could be observed. More important, well time-resolved transitions between these structures could be obtained. When using slightly acidic solution, the stable structures with stable blockade current could be found. With this new approach, we can directly observe the dynamic conformational change of biomolecules at single-molecular scale, which would be of great help for resolving single molecule interactions, designing single-molecule machine and understanding the working process of biomolecular in biological system. 358-Pos Board B123 Tuning Up Tethered Particle Motion Daniel T. Kovari, Eric Weeks, David Dunlap, Laura Finzi. Physics, Emory University, Atlanta, GA, USA. Tethered Particle Motions assays are a simple but powerful tools for monitoring the effective length of individual DNA strands and other linear bio-polymers in real-time. The technique has been employed in various capacities including characterization of DNA topology, transcription factor - DNA interactions, and monitoring progress of enzymes that translocate along DNA. At its core the technique is relatively simple to implement, only requiring a researchgrade microscope and a video camera; however, it is important to note that optical resolution, frame rate, exposure time, particle size, and solution viscosity all affect the ability to discriminate different tether lengths and detect changes.
360-Pos Board B125 Improved Sampling in Molecular Dynamics Studies of DNA and the B To Z[WC] To Z-DNA Transition Lam T. Nguyen, Ashutosh Rai, Micaela E. Bush, Alma Gracic, Ahsan A. Khoja, Jinhee Kim, Sunil Pun, Alexander K. Seewald, Benjamin L. Yee, Michael G. Lerner. Physics and Astronomy, Earlham College, Richmond, IN, USA. Although DNA is most commonly found in the right-handed B-DNA structure, it is known that biologically active systems also contain left-handed ZIIDNA. We used both steered and targeted molecular dynamics in combination with umbrella sampling to produce potentials of mean force for the B to ZII transition along both the direct B-ZII pathway as well as the B-Z[WC]-ZII pathway. Full pathway sampling is not feasible on smaller computer clusters, so we used Hamiltonian Replica Exchange to relax individual portions of the pathway. This technique is generalizable to larger systems and larger computer clusters. 361-Pos Board B126 Energetic Contributions of Plectoneme Tips and Tails Andrew Dittmore, Keir C. Neuman. NIH, Bethesda, MD, USA. Global DNA topology is sensed locally by enzymes that act on plectonemes in supercoiled DNA. Here we report that the formation and diffusion of plectonemes are determined by the energetic contributions of their tips and tails. First, to systematically vary the geometry and formation energy of plectoneme endloops, we introduced base-pair defect regions of variable size (1-16 bp) using a cassette based single-strand nicking template generated by PCR. Direct manipulation measurements with magnetic tweezers revealed that even a single mismatch or abasic site is sufficient to nucleate formation of a plectoneme. Presentation of the defect precisely at an extruded plectoneme tip potentially serves as a damage-sensing mechanism and may facilitate the search process of repair enzymes. Second, our measurements unexpectedly revealed that after twisted DNA abruptly buckles into an initial plectoneme loop, further plectoneme extrusion occurs through a cascade of additional buckling steps in which the torque changes by roughly half of the initial overshoot value. These discrete steps do not match any obvious scale of the system but are consistent with discontinuous feed-in of curving plectoneme tails. In light of these results, theoretical models of plectonemes should include their overall structure, including the often neglected tips and tails. 362-Pos Board B127 Multi-Scale Structure and Conformational Dynamics of Scaffolded DNA Origami Nanoparticles William Bricker, Keyao Pan, Mark Bathe. Massachusetts Institute of Technology, Cambridge, MA, USA. Synthetic DNA can be programmed into self-assembled 3D nanoparticles using a DX design motif and the principle of scaffolded DNA origami. A topdown design procedure (DAEDALUS) (Veneziano, Ratanalert, et al., Science, 2016) facilitates the design of arbitrary DNA nanoparticle geometries on the 5 to 100 nanometer scale, which we investigate in detail here using multi-scale structural modeling. While coarse-grained modeling is useful for generating equilibrium structures of DNA nanoparticles (Pan et al., Nat. Comm., 2014), only all-atom models reveal fine structural details and mechanical properties that contribute to overall structure and conformational dynamics. Here, we first use all-atom molecular dynamics (MD) to simulate two 0.5 1.0 MDa DNA polyhedral nanoparticles: a tetrahedron with 63 base pair
Sunday, February 12, 2017 (bp) edge lengths, and an octahedron with 52 bp edge lengths. Using 150 ns trajectories, we are able to elucidate subtle structural features seen in experimental cryo-EM maps, including right-handed twisting of the vertices in the octahedron and outward bowing of the edges in the tetrahedron. Next, these results are compared with all-atom MD simulations of unconstrained vertex building blocks including the tetrahedron (3-arm vertex) and octahedron (4-arm vertex). In these simulations, a notable feature is the significant out-of-plane bending angle away from the minor groove at the vertex due to the chirality of dsDNA. Finally, equilibrium solution structures of 45 DX-based DNA origami nanoparticles are predicted by implementing an updated bulge stiffness parameter for our coarse-grained FE model CanDo (Kim et al., Nucleic Acids Res., 2012). These multi-scale structural results show the interplay between coarse-grained and all-atom models in the ab initio prediction and elucidation of complex features of DNA nanoparticles seen in experimental cryo-EM maps.
RNA Binding 363-Pos Board B128 Shedding Light on Cas9 Target Search Viktorija Globyte1, Seung Hwan Lee1, Luuk Loeff1, Jin Soo Kim2, Chirlmin Joo1. 1 TU Delft, Delft, Netherlands, 2Seoul National University, Seoul, Korea, Republic of. The CRISPR/Cas adaptive immune system provides prokaryotes with a defensive mechanism against invading viruses and plasmids. The invading viral DNA fragments are incorporated into the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) locus in the bacterial genome and are later used to recognize and destroy the invader when it returns. In the last stage of CRISPR immunity, called the interference stage, Cas (CRISPR-associated) proteins assemble with short guide RNA molecules which are transcribed from the CRISPR locus. These guide RNA molecules can be programmed to recognize any DNA sequence. In recent years the CRISPR/Cas adaptive immune system has seen an immense growth in interest with the type II CRISPR-Cas9 system being in the center of attention. In this system, the DNA of the invading virus is recognized and cleaved by a single protein Cas9 which is guided by an RNA duplex. Due to its simplicity, CRISPRCas9 system is a promising tool in gene engineering as its guide RNA can be programmed to recognize virtually any sequence in the genome. The CRISPR-Cas9 has been demonstrated to work in a variety of organisms, however, despite the large interest in this system, the precise mechanism by which Cas9 finds and cleaves its target remains ambiguous. We utilize biophysical single-molecule techniques, namely total internal reflection fluorescence microscopy (TIRFM) together with Forster resonance energy transfer (FRET) to investigate the mechanics of Cas9 target search with nanometer sensitivity. We are probing one-dimensional diffusion of the protein along the DNA strand and investigating the effects different DNA sequences have on the mechanics of Cas9 target search. 364-Pos Board B129 To Cleave or not to Cleave: Predicting the Target Specificity of CRISPRCas Systems through Theoretical Modeling Misha Klein, Martin Depken. Kavli institute of Nanoscience, Departement of BioNanoScience, TU Delft, Delft, Netherlands. Many prokaryotes employ the CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeat – CRISPR associated) system to fend off attacks by hostile genetic elements. This adaptive immune system recognizes invaders based on their level of sequence complementarity with RNA transcribed from a library of past invasions stored at the CRISPR locus. To avoid infection, the CRISPR interference complex must be able to single out a short target sequence (20 – 40 nt) among a total of 105-106 nt in the cell, while at the same time recognizing targets that have evolved away from the record stored at the CRISPR locus. Despite the tremendous interest the CRISPR-Cas9 system has gained as a novel genome editing tool, the sequence preference of the interference complex remains poorly understood. Experiments have shown that it is not just the amount of mutations, but also their placement along the guide/target, that determine the level of interference. Through kinetic modeling we provide simple rules to assess sequence specificity based on mismatch patterns, and quantitatively explain the experimentally observed seed region over which no mismatches are permitted. Moving beyond sequence complementarity, we also quantify how changes in the conformation of the interference complex can serve to improve specificity during target recognition. Finally, by fitting our model to published experimental
71a
data, we give estimates for the base-pairing energies within the interference complex for various CRISPR systems. 365-Pos Board B130 Conformational Dynamics of Cas9 during DNA Binding Yavuz S. Dagdas1, Janice S. Chen2, Samuel H. Sternberg3, Jennifer A. Doudna2,3, Ahmet Yildiz2,4. 1 Biophysics Graduate Group, University of California, Berkeley, Berkeley, CA, USA, 2Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA, 3Department of Chemistry, University of California, Berkeley, Berkeley, CA, USA, 4Department of Physics, University of California, Berkeley, Berkeley, CA, USA. Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA using two conserved nuclease domains, RuvC and HNH, as part of CRISPR-Cas bacterial adaptive immune systems. Together with singleguide RNAs, Cas9 is also widely utilized as a programmable genome editing tool. DNA cleavage activity is controlled directly by the conformational state of the HNH nuclease domain, but Cas9 conformational dynamics during DNA binding remain poorly characterized. Using single-molecule FRET assays, we identified a long-lived intermediate state of S. pyogenes Cas9. Upon DNA binding, the HNH nuclease reversibly transitions between RNA-bound, DNA-bound and docked conformations, before it docks irreversibly into its catalytically active conformation for DNA cleavage. Docking of HNH to its active state requires divalent cation, and HNH remains in the docked state after cleavage of a complementary target. Increasing the number of mutations in the target region distal to the protospacer adjacent motif (PAM) prevents transitions from intermediate to the docked conformation. The results provide a structural explanation for the lack of DNA cleavage activity when Cas9 binds to off-target sites. 366-Pos Board B131 Repetitive Loop Formation by the CRISPR-Cas3 Helicase Luuk Loeff1, Stan Brouns1,2, Chirlmin Joo1. 1 Technical University Delft, Delft, Netherlands, 2Wageningen University, Wageningen, Netherlands. E. coli maintain CRISPR-Cas adaptive immune systems to protect the cell against invading genetic elements. Immunity relies on the RNA guided surveillance complex Cascade (CRISPR-associated complex for antiviral defense) and the trans-acting Cas3 protein with helicase and nuclease activities. We recently showed that Cas3 generates degradation products ranging from 30 to 150 nt that act as pre-cursors for primed spacer acquisition. However, it remains unclear which mechanism drives the generation of these fragments with a specific size. Here we employed single-molecule FRET to probe the molecular dynamics of Cas3 in real-time. Our data shows that Cas3 repeatedly generates DNA loops in the target strand whilst remaining in tight contact with Cascade. DNA loops are generated by breaking open the dsDNA helix in distinctive steps of 3 bp, arising from the RecA like folds of the helicase domain. Repetitive unwinding achieved by slipping of the helicase domain, which limits the average translocation distance to ~90 nt. Taken together, our data suggest that the inherent helicase properties of Cas3 drive the generation precursors of adequate size for primed spacer integration. 367-Pos Board B132 The Impact of DNA Topology on Target Selection by a Cytosine-Specific Cas9 Tsz Kin Martin Tsui, Travis H. Hand, Hong Li. Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, USA. Cas9 is an RNA-guided DNA cleavage enzyme being actively developed for genome editing and gene regulation. To be cleaved by Cas9, a double stranded DNA, or the protospacer, must be 1) complementary to the Cas9-bound guide RNA and a short Cas9-specific sequence adjacent to protospacer, called Protospacer Adjacent Motif (PAM). Understanding the correct juxtaposition in time and space of the protospacer- and PAM-interaction with Cas9 will enable development of versatile and safe Cas9-based technology. We report identification and biochemical characterization of Cas9 from Acidothermus cellulolyticus (AceCas9). AceCas9 depends strictly on a 50 -NNNCC-30 PAM and is more efficient in cleaving negative supercoils than relaxed DNA. We further showed that mismatches to the guide RNA on a supercoiled protospacer are tolerated by AceCas9, whereas the same mismatches on its relaxed form were not. The cytosine-specific and DNA topology-sensitive properties of the AceCas9 maybe explored for chromosome domain specific genome editing applications. Finally, our preliminary data showed that AceCas9 can disrupt target DNA in in vivo assays, demonstrating its utility as a genome editing enzyme.