M. Rutkauskas

Directional R-Loop Formation by the CRISPR-Cas Surveillance Complex Cascade Provides Efficient Off-Target Site Rejection

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against foreign nucleic acids. In type I CRISPR-Cas systems, invading DNA is detected by a large ribonucleoprotein surveillance complex called Cascade. The crRNA component of Cascade is used to recognize target sites in foreign DNA (protospacers) by formation of an R-loop driven by base-pairing complementarity. Using single-molecule supercoiling experiments with near base-pair resolution, we probe here the mechanism of R-loop formation and detect short-lived R-loop intermediates on off-target sites bearing single mismatches. We show that R-loops propagate directionally starting from the protospacer-adjacent motif (PAM). Upon reaching a mismatch, R-loop propagation stalls and collapses in a length-dependent manner. This unambiguously demonstrates that directional zipping of the R-loop accomplishes efficient target recognition by rapidly rejecting binding to off-target sites with PAM-proximal mutations. R-loops that reach the protospacer end become locked to license DNA degradation by the auxiliary Cas3 nuclease/helicase without further target verification.

Fabrication of micro-tube arrays in photopolymer SZ2080 by using three different methods of a direct laser polymerization technique

In this paper we demonstrate femtosecond laser fabrication of micro-tubes with a height of several tens of micrometers in the photopolymer SZ2080 by three different methods: direct laser writing, using the optical vortex beam and holographic lithography. The flexibility of direct laser writing and dramatic increase of production efficiency by applying the vortex-shaped beam and four-beam interference approaches are presented. Sample arrays of micro-tubes were successfully manufactured applying all three methods and the fabrication quality as well as efficiency of the methods is compared. The processing time of a single micro-tube with 60 ?m height and 3 ?m inner radius is reduced 400 times for the holographic lithography technique and 500 times for the optical vortex method compared with the direct laser writing technique. The processing time of a micro-tube array containing 400?micro-tubes is the shortest for the holographic lithography method but not for the optical vortex method as in the case of a single micro-tube, because the holographic lithography method does not require time for sample translation. Additionally, the holographic lithography enables manufacturing of the whole micro-tube array by a single exposure. Although point-by-point photo-structuring ensures unmatched complexity of manufactured microstructures, employing nowadays high repetition rate amplified femtosecond lasers combined with beam shaping or several beam interference can envisage industrial applications for practical demands.

Two-photon polymerization for fabrication of three-dimensional micro- and nanostructures over a large area

Two-photon polymerization has emerged as a technology for rapid fabrication of three-dimensional micro-structures with nanoscale resolution. Commonly, a Ti:Sapphire femtosecond laser (operating at 780-800 nm wavelength) working at MHz pulse repetition rate is applied as an irradiation source to photomodify the resin. We present a system for pinpoint two-photon polymerization which utilizes second harmonic (515 nm) of amplified Yb:KGW femtosecond laser working at 312.5 kHz pulse repetition rate. Shorter irradiation wavelength enables one to focus laser beam to a smaller spot. High repetition rate and high average power capacitates rapid fabrication of three-dimensional structures over a large area. Results obtained prove the highest resolution of fabrication to be up to ~100 nm, and reproducible resolution 200 nm. Some micro-structures fabricated rapidly over millimeter area and revealing the specific problems arising at high speed fabrication over large lateral dimensions are presented in this report. Results obtained show the system and technologies applied to be well suitable for future routine 3D structuring over the large area and application of such structures in photonics, micro-optics, micromechanics, microelectronics and cell growth for tissue engineering.

Femtosecond laser-induced two-photon photopolymerization for structuring of micro-optical and photonic devices

Light initiated liquid polymer quasi-instant solidification is attractive for its ultra precise spatial and temporal control of the reaction. Here we present femtosecond laser induced two-photon photopolymerization for structuring of microoptical and sample photonic devices. Due to nonlinear phenomena the fabrication resolution is not restricted to diffraction limit for the applied laser excitation wavelength but determined by the exposure dose. Furthermore, pinpoint structuring enables one to produce 3D structures of any form out of photopolymer. The smallest structural elements voxels of 200 nm lateral dimensions can be achieved reproducibly by using high numerical aperture optics. Axial resolution which is fundamentally few times worse than lateral can be controlled in few hundred nanometers precision by forming polymeric pad as an additional structure. In our work we applied commercially available and widely used hybrid zirconium-silicon based hybrid sol-gel material ORMOSIL (SZ2080) and an acrylate based AKRE37 photopolymer. Arrays of custom parameters spherical microlenses for microscopy applications have been fabricated. Their surface roughness, focal distance and imaging quality were tested. 3D custom form woodpile structures with submicron period and chain-mail structure were made as a sample photonic bandgap structures. Finally, we show some structures produced out of fluorescent dyes rhodamine 6G doped photopolymer.

Femtosecond laser fabrication of hybrid micro-optical elements and their integration on the fiber tip

Femtosecond laser photo-polymerization of zirconium-silicon based sol-gel photopolymer SZ2080 is used to fabricate micro-optical elements with a single and hybrid optical functions. We demonstrate photo-polymerization of the solid immersion and Fresnel lenses. Gratings can be added onto the surface of lenses. The effective refractive index of polymerized structures can be controlled via the volume fraction of polymer. We used woodpile structure with volume fraction of 0.65-0.8. Tailoring of dispersion properties of micro-optical elements by changing filling ratio of polymer are discussed. Direct write approach is used to form such structures on a cover glass and on the tip of an optical fiber. Close matching of refractive indices between the polymer and substrate in visible and near infra red spectral regions (nSZ2080 = 1.504, nglass = 1.52) is favorable for such integration. The surface roughness of laser-polymerized resits was ~30 nm (min-max value), which is acceptable for optical applications in the visible range. For the bulk micro-optical elements the efficiency of 3D laser polymerization is increased by a factor ~ (2 - 4) × 102 times (depends on the design) by the shell-formation polymerization: (i) contour scanning for definition of shell-surface, (ii) development for removal of nonfunctional resist, and (iii) UV exposure for the final volumetric polymerization of an enclosed volume.

Large Scale Laser Two-Photon Polymerization Structuring for Fabrication of Artificial Polymeric Scaffolds for Regenerative Medicine

We present a femtosecond Laser Two‐Photon Polymerization (LTPP) system of large scale three‐dimensional structuring for applications in tissue engineering. The direct laser writing system enables fabrication of artificial polymeric scaffolds over a large area (up to cm in lateral size) with sub‐micrometer resolution which could find practical applications in biomedicine and surgery. Yb:KGW femtosecond laser oscillator (Pharos, Light Conversion. Co. Ltd.) is used as an irradiation source (75 fs, 515 nm (frequency doubled), 80 MHz). The sample is mounted on wide range linear motor driven stages having 10 nm sample positioning resolution (XY—ALS130‐100, Z—ALS130‐50, Aerotech, Inc.). These stages guarantee an overall travelling range of 100 mm into X and Y directions and 50 mm in Z direction and support the linear scanning speed up to 300 mm/s. By moving the sample three‐dimensionally the position of laser focus in the photopolymer is changed and one is able to write complex 3D (three‐dimensional) structures. An illumination system and CMOS camera enables online process monitoring. Control of all equipment is automated via custom made computer software “3D‐Poli” specially designed for LTPP applications. Structures can be imported from computer aided design STereoLihography (stl) files or programmed directly. It can be used for rapid LTPP structuring in various photopolymers (SZ2080, AKRE19, PEG‐DA‐258) which are known to be suitable for bio‐applications. Microstructured scaffolds can be produced on different substrates like glass, plastic and metal. In this paper, we present microfabricated polymeric scaffolds over a large area and growing of adult rabbit myogenic stem cells on them. Obtained results show the polymeric scaffolds to be applicable for cell growth practice. It exhibit potential to use it for artificial pericardium in the experimental model in the future.We present a femtosecond Laser Two‐Photon Polymerization (LTPP) system of large scale three‐dimensional structuring for applications in tissue engineering. The direct laser writing system enables fabrication of artificial polymeric scaffolds over a large area (up to cm in lateral size) with sub‐micrometer resolution which could find practical applications in biomedicine and surgery. Yb:KGW femtosecond laser oscillator (Pharos, Light Conversion. Co. Ltd.) is used as an irradiation source (75 fs, 515 nm (frequency doubled), 80 MHz). The sample is mounted on wide range linear motor driven stages having 10 nm sample positioning resolution (XY—ALS130‐100, Z—ALS130‐50, Aerotech, Inc.). These stages guarantee an overall travelling range of 100 mm into X and Y directions and 50 mm in Z direction and support the linear scanning speed up to 300 mm/s. By moving the sample three‐dimensionally the position of laser focus in the photopolymer is changed and one is able to write complex 3D (three‐dimensional) structures....

Primed CRISPR adaptation in Escherichia coli cells does not depend on conformational changes in the Cascade effector complex detected in Vitro

Abstract In type I CRISPR–Cas systems, primed adaptation of new spacers into CRISPR arrays occurs when the effector Cascade–crRNA complex recognizes imperfectly matched targets that are not subject to efficient CRISPR interference. Thus, primed adaptation allows cells to acquire additional protection against mobile genetic elements that managed to escape interference. Biochemical and biophysical studies suggested that Cascade–crRNA complexes formed on fully matching targets (subject to efficient interference) and on partially mismatched targets that promote primed adaption are structurally different. Here, we probed Escherichia coli Cascade–crRNA complexes bound to matched and mismatched DNA targets using a magnetic tweezers assay. Significant differences in complex stabilities were observed consistent with the presence of at least two distinct conformations. Surprisingly, in vivo analysis demonstrated that all mismatched targets stimulated robust primed adaptation irrespective of conformational states observed in vitro. Our results suggest that primed adaptation is a direct consequence of a reduced interference efficiency and/or rate and is not a consequence of distinct effector complex conformations on target DNA.

Single-Molecule Insight Into Target Recognition by CRISPR–Cas Complexes

Ribonucleoprotein (RNP) complexes from CRISPR-Cas systems have attracted enormous interest since they can be easily and flexibly reprogrammed to target any desired locus for genome engineering and gene regulation applications. Basis for the programmability is a short RNA (crRNA) inside these complexes that recognizes the target nucleic acid by base pairing. For CRISPR-Cas systems that target double-stranded DNA this results in local DNA unwinding and formation of a so-called R-loop structure. Here we provide an overview how this target recognition mechanism can be dissected in great detail at the level of a single molecule. Specifically, we demonstrate how magnetic tweezers are applied to measure the local DNA unwinding at the target in real time. To this end we introduce the technique and the measurement principle. By studying modifications of the consensus target sequence, we show how different sequence elements contribute to the target recognition mechanism. From these data, a unified target recognition mechanism can be concluded for the RNPs Cascade and Cas9 from types I and II CRISPR-Cas systems. R-loop formation is hereby initiated on the target at an upstream element, called protospacer adjacent motif (PAM), from which the R-loop structure zips directionally toward the PAM-distal end of the target. At mismatch positions, the R-loop propagation stalls and further propagation competes with collapse of the structure. Upon full R-loop zipping conformational changes within the RNPs trigger degradation of the DNA target. This represents a shared labor mechanism in which zipping between nucleic acid strands is the actual target recognition mechanism while sensing of the R-loop arrival at the PAM-distal end just verifies the success of the full zipping.

Functional significance of protein assemblies predicted by the crystal structure of the restriction endonuclease BsaWI

Type II restriction endonuclease BsaWI recognizes a degenerated sequence 5′-W/CCGGW-3′ (W stands for A or T, ‘/’ denotes the cleavage site). It belongs to a large family of restriction enzymes that contain a conserved CCGG tetranucleotide in their target sites. These enzymes are arranged as dimers or tetramers, and require binding of one, two or three DNA targets for their optimal catalytic activity. Here, we present a crystal structure and biochemical characterization of the restriction endonuclease BsaWI. BsaWI is arranged as an ‘open’ configuration dimer and binds a single DNA copy through a minor groove contacts. In the crystal primary BsaWI dimers form an indefinite linear chain via the C-terminal domain contacts implying possible higher order aggregates. We show that in solution BsaWI protein exists in a dimer-tetramer-oligomer equilibrium, but in the presence of specific DNA forms a tetramer bound to two target sites. Site-directed mutagenesis and kinetic experiments show that BsaWI is active as a tetramer and requires two target sites for optimal activity. We propose BsaWI mechanism that shares common features both with dimeric Ecl18kI/SgrAI and bona fide tetrameric NgoMIV/SfiI enzymes.

Collimation and imaging behind a woodpile photonic crystal

We investigate different linear effects that appear in the light beam propagation behind a three-dimensional woodpile photonic crystal. On one hand we report and analyse experimental observation of narrow and well collimated laser beam formation behind the photonic crystal. We show that this collimation depends on the input laser beam focusing conditions. On the other hand we also report the first theoretical and numerical observation double imaging formation behind the photonic crystal. That conclusion comes from the idea that the spatial dispersion possess multiple branches (Bloch branches), generally of different curvatures, for a fixed frequency.