Nature Protocols最新文献

Successful delivery of nucleic acid therapeutics to diseased sites would present a pivotal advancement in cancer treatment. However, progress has been hindered by the lack of efficient tumor-selective vectors via clinical systemic routes, the blood-brain barrier for brain tumors and problems with repeated administrations. We present a new generation of M13 phage-based vectors termed transmorphic phage/adeno-associated virus (AAV) (TPA), wherein the phage genome has been excised to facilitate exclusive packaging of human AAV DNA by phage coat proteins. Here we provide a detailed protocol for the molecular cloning of DNA into the TPA construct, display of disease-specific ligands on the helper phage capsid for cell targeting and entry, and packaging of TPA DNA by helper phage coat proteins in a bacterial host. Furthermore, we provide methods for mammalian cell transduction and assessment of transgene expression in vitro as well as in vivo application of TPA particles in tumor-bearing mice. Unlike other similar methods, our protocol enables high-yield production and control of helper phage quantity in TPA preparations. Moreover, compared with existing M13 phage vectors, TPA particles can accommodate large size transgene inserts, despite being considerably more compact, providing superior gene delivery through enhanced diffusion across the extracellular matrix, improved cellular binding and entry and increased vector DNA accumulation in the nucleus. The protocol encompasses a timeline of 4-5 months, including construction and production of TPA particles with transgene and targeted ligand and in vitro/in vivo testing. This protocol can be conducted by researchers trained in basic molecular biology/bacteriology research techniques.

Diffusion magnetic resonance imaging (dMRI) is a versatile imaging technique that has gained popularity thanks to its sensitive ability to measure displacement of water molecules within a living tissue on a micrometer scale. Although dMRI has been around since the early 1990s, its applications are constantly evolving, primarily regarding the inference of structural connectomics from nerve fiber trajectories. However, these applications require expertise in image processing and statistics, and it can be difficult for a newcomer to choose an appropriate pipeline to fit their research needs, not least because dMRI is such a flexible methodology that dozens of acquisition and analysis pipelines have been developed over the years. This introductory guide is designed for graduate students and researchers in the neuroscience community who are interested in integrating this new methodology regardless of their background in neuroimaging and computational tools. The guide provides a brief overview of the basic dMRI methodologies but focuses on its applications in neuroplasticity and connectomics. The guide starts with dMRI experimental designs and a complete step-by-step pipeline for structural connectomics. The following section covers the basics of dMRI, including parameters and clinical applications (apparent diffusion coefficient, mean diffusivity, fractional anisotropy and microscopic fractional anisotropy), as well as different approaches and models. The final section focuses on structural connectomics, covering subjects from fiber tracking (techniques, evaluation and limitations) to structural networks (constructing, analyzing and visualizing a network).

As microRNAs (miRNA) regulate almost all physiopathological activities in the human body, miRNA therapeutics that deliver miRNA regulators have attracted considerable attention in the field of nucleic acid drug development. The use of tetrahedral DNA nanostructures to deliver miRNA regulators is promising because of their simple fabrication, enhanced cell entry, effective tissue penetration, biocompatibility and functional editability. This protocol extension builds on our previous protocol for the use of tetrahedral DNA nanostructures and was designed to establish an updated bioswitchable delivery system (BDS) for achieving controlled cargo loading and release. A ribonuclease H-sensitive sequence is designed as a bioswitchable apparatus for the targeted release of the miRNA regulator. The functional sequence of the miRNA regulator and minimal secondary structure formation tendency during annealing are two key points in cargo design. We provide two BDS design strategies; BDS-A comprises an intact DNA tetrahedron with the RNA cargo hanging outside, offering the merits of lower cost, simplicity, and more direct structural design. In the BDS-B design, the RNA regulators are embedded into the DNA tetrahedron, which is beneficial for dermal tissue permeation applications. Following sequence design in Oligo 7 and Tiamat, the BDS assembly is completed and then ribonuclease H achieves controlled release of the miRNA regulator by triggering the bioswitchable apparatus. This is verified via polyacrylamide and agarose gel electrophoresis or fluorophore modifications. Both BDSs show promising cellular membrane permeability, tissue permeability and target inhibition in vitro and in vivo. The assembly and characterization of the BDS can be completed in 4 d, and the validation time for biostability and biological applications will depend on the specific use.