پيشرفت مهم در طراحي تعيين توالي دقيق با روشهاي الكترونيكي 

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IMAGE: This image shows single-molecule nanopore DNA sequencing by synthesis data from a template with homopolymer sequences (http://www.pnas.org/content/early/2016/04/14/1601782113.full). view more

Credit: Jingyue Ju,Columbia Engineering

New York, NY–April 21, 2016–Researchers from Columbia University, with colleagues at Genia Technologies (Roche), Harvard University and the National Institute of Standards and Technology (NIST) report achieving real-time single molecule electronic DNA sequencing at single-base resolution using a protein nanopore array.

DNA sequencing is the key technology for personalized and precision medicine initiatives, enabling rapid discoveries in biomedical science. An individual’s complete genome sequence provides important markers and guidelines for medical diagnostics, healthcare, and maintaining a healthy life. To date, the cost and speed involved in obtaining highly accurate DNA sequences has been a major challenge. While various advancements have been made over the past decade, the high-throughput sequencing instruments widely used today depend on optics for the detection of four DNA building blocks: A, C, G and T. To explore alternative measurement capabilities, electronic sequencing of an ensemble of DNA templates has been developed for genetic analysis. Nanopore strand sequencing, wherein a single strand DNA is threaded through the nanoscale pores under an applied electrical voltage to produce electronic signals for sequence determination at single molecule level, has recently been developed; however, because the four nucleotides are very similar in their chemical structures, they cannot easily be distinguished using this method. Researchers are therefore actively pursing the research and development of an accurate single-molecule electronic DNA sequencing platform as it has the potential to produce a miniaturized DNA sequencer capable of deciphering the genome to facilitate personalized precision medicine.

A team of researchers at Columbia Engineering, headed by Jingyue Ju (Samuel Ruben-Peter G. Viele Professor of Engineering, Professor of Chemical Engineering and Pharmacology, Director of the Center for Genome Technology & Biomolecular Engineering), with colleagues at Harvard Medical School, led by George Church (Professor of Genetics); Genia Technologies, led by Stefan Roever (CEO of Genia); and John Kasianowicz, the Principal Investigator at NIST, have developed a complete system to sequence DNA in nanopores electronically at single molecule level with single-base resolution. This work, entitled, “Real-Time Single Molecule Electronic DNA Sequencing by Synthesis Using Polymer Tagged Nucleotides on a Nanopore Array,” is published in the journal, Proceedings of the National Academy of Sciences (PNAS) Early Edition: http://www. pnas. org/ content/ early/ 2016/ 04/ 14/ 1601782113. full.

Previously, researchers from the laboratories of Ju at Columbia and Kasianowicz at NIST reported the general principle of nanopore sequencing by synthesis (SBS), the feasibility of design and synthesis of polymer-tagged nucleotides as substrates for DNA polymerase, the detection and the differentiation of the polymer tags by nanopore at the single molecule level [Scientific Reports 2, 684 (2012) doi: 10.1038/srep00684; http://www. nature. com/ articles/ srep00684]. The current PNAS paper describes the construction of the complete nanopore SBS system to produce single molecule electronic sequencing data with single-base resolution. This SBS strategy accurately distinguishes four DNA bases by electronically detecting and differentiating four different polymer tags attached to the 5′-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by polymerase, a DNA-synthesizing enzyme. The researchers designed and synthesized novel nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin protein nanopore array platform. The tags on the polymer-labeled nucleotides, which were verified to be active substrates for DNA polymerase, produce different electrical current blockade levels. They constructed a nanopore array on an electronic chip bearing multiple electrodes; the array is composed of protein channels that were coupled to a DNA polymerase that was bound to a primed DNA template. Addition of distinct custom-designed polymer tagged nucleotides to the nanopore array triggers DNA synthesis. By blocking the channel’s ionic current to different levels, the distinct tags provide a readout of the template sequence in real time with single-base resolution.

As Carl Fuller, lead author, Adjunct Senior Research Scientist in the Ju Laboratory of the Chemical Engineering Department at Columbia and Director of Chemistry at Genia, points out, “The novelty of our nanopore SBS approach begins with the design, synthesis, and selection of four different polymer-tagged nucleotides. We use a DNA polymerase covalently attached to the nanopore and the tagged nucleotides to perform SBS. During replication of the DNA bound to the polymerase, the tag of each complementary nucleotide is captured in the pore to produce a unique electrical signal. Four distinct polymer tags yielding distinct signatures that are recognized by the electronic detector in the nanopore array chip are used for sequence determination. Thus, DNA sequences are obtained for many single molecules in parallel and in real time. The four polymer tags are designed to offer much better distinctions among themselves, in contrast to the small differences among the four native DNA nucleotides, thereby overcoming the major challenge faced by other direct nanopore sequencing methods.” Moreover, the tags can be further optimized with respect to size, charge, and structure to provide optimal resolution in the nanopore SBS system.

“This exciting project brings together scientists and engineers from both academia and industry with combined expertise in molecular engineering, nanotechnology, genomics, electronics and data science to produce revolutionary, cost-effective genetic diagnostic platforms with unprecedented potential for precision medicine,” says Ju. “We are extremely grateful for the generous support from the NIH that enabled us to make rapid progress in the research and development of the nanopore SBS technology, and the outstanding contributions from all the members of our research consortium.”

According to Ju, the researchers have already pushed beyond what was demonstrated in the PNAS study where the sequencing data was obtained on an early prototype sequencer based on nanopore SBS. The throughput and performance of the current sequencer has progressed beyond what was reported in the PNAS paper. The feasibility of reaching read-lengths of over 1000 bases of DNA has recently been achieved. Going forward, the collaborative research team will continue to optimize the tags by tweaking the linkers, structure, and charge at the molecular level, and fine tuning the polymerase and the electronics for the nanopore SBS system with an aim to accurately sequence an entire human genome rapidly and at low cost, thereby enabling it to be used in routine medical diagnoses.

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This research was funded by a grant from National Institutes of Health (NIH).

The PNAS paper is titled: “Real-Time Single Molecule Electronic DNA Sequencing by Synthesis Using Polymer Tagged Nucleotides on a Nanopore Array.” (http://www. pnas. org/ content/ early/ 2016/ 04/ 14/ 1601782113. full) C. W. Fuller, S. Kumar, M. Porel, M. Chien, A. Bibillo, P. B. Stranges, M. Dorwart, C. Tao, Z. Li, W. Guo, S. Shi, D. Korenblum, A. Trans, A. Aguirre, E. Liu, E. T. Harada, J. Pollard, A. Bhat, C. Cech, A. Yang, C. Arnold, M. Palla, J. S. Hovis, R. Chen, I. Morozova, S. Kalachikov, J. J. Russo, J. Kasianowicz, R. Davis, S. Roever, G. M. Church, and J. Ju.

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روش نوين درمان انزيمي با كمك مغناطيس

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IMAGE: Self-stabilizing magnetic colloid. view more

Credit: ITMO University

Researchers from ITMO University, the Hebrew University of Jerusalem and Cyril and Methodius University in Skopje have fabricated a new magnetically controlled material composed of enzymes entrapped directly within magnetite particles. Combined with water, it forms a stable solution that can be used for safe intravenous injection for medical purposes, in particular, for targeted treatment of cancer and thrombosis. Previously, the synthesis of similar materials involved using additional components that impaired the magnetic response and enzymatic activity as well as created obstacles for intravenous injection into the human body. The results of the study were published in the Chemistry of Materials magazine.

The researchers are going to apply this approach to the treatment of thrombosis. Targeted magnetic introduction of enzymes that dissolve the clot seems to be a very promising approach in terms of fighting the disease. “Using a magnetic field, the particles can be condensed on blood clots; moreover, such systems will work for quite a long time until the enzyme is completely oxidized”, says Andrey Drozdov, research associate at the International laboratory of Solution Chemistry of Advanced Materials and Technologies (SCAMT).

What makes the difference with the new material is that particles of magnetite with entrapped enzymes are extremely resistant to sedimentation in water. This was made possible thanks to a new method of magnetic hydrosols synthesis devised by ITMO University researchers.

Thus, the resulting system stabilizes itself without any need for additional stabilizers that may attenuate its magnetic properties, reduce the enzymatic activity and increase potential toxic effect for humans. The nanocomposite is absolutely biocompatible and harmless for injection into the human body. “Separately, both magnetite and therapeutic enzymes have medical approval for intravenous Injection. Therefore, to approve their joint use should not be difficult. The body already knows what to do with these substances and how to incorporate them into the process of metabolism,” adds Andrey Drozdov.

The approach allows scientists to create magnetically controlled solutions of practically any enzyme that has some industrial or therapeutic value. To demonstrate this versatility, the researchers encased five different enzymes in such a magnetite “armour”.

Enzymes are added directly in magnetite hydrosol. Magnetite particles surround the enzymes and after drying out form a firm porous structure, whereof the enzyme cannot escape anymore. “This method was tailored to be used with enzymes,” explains Vladimir Vinogradov, head of International laboratory of Solution Chemistry of Advanced Materials and Technologies, “We have selected the material so that after packing, it could physically hold the enzyme inside. At the same time the enzyme is able to carry out its function through the pores in the material.”

In addition, the researchers discovered that entrapped enzymes were characterized by increased thermal stability. According to the experiments, protected enzymes remained functional at temperatures exceeding the temperature of their thermal decomposition in free form by more than 20 degrees Celsius. This phenomenon can facilitate the extension of the application range of enzymes in a variety of environments that are sensitive to temperature conditions.

The scientists suggest that magnetite particles protect the enzyme from denaturation (i.e. the process of losing its native structure because of increasing temperature or changing pH) physically by applying pressure on the enzyme from the outside. Thus, the magnetite plays a role of a solid framework.

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‘نانو لوله ها ابزار جديد در مهندسي ژنتيك

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Researchers have developed a new and highly efficient method for gene transfer. The technique, which involves culturing and transfecting cells with genetic material on an array of carbon nanotubes, appears to overcome the limitations of other gene editing technologies.

The device, which is described in a study published today in the journal Small, is the product of a collaboration between researchers at the University of Rochester Medical Center (URMC) and the Rochester Institute of Technology (RIT).

“This platform holds the potential to make the gene transfer process more robust and decrease toxic effects, while increasing amount and diversity of genetic cargo we can deliver into cells,” said Ian Dickerson, Ph.D., an associate professor in the Department of Neuroscience at the URMC and co-author of the paper.

“This represents a very simple, inexpensive, and efficient process that is well-tolerated by cells and can successfully deliver DNA into tens of thousands of cells simultaneously,” said Michael Schrlau, Ph.D., an assistant professor in the Kate Gleason College of Engineering at RIT and co-author of the paper.

Gene transfer therapies have long held great promise in medicine. New gene editing techniques, such as CRISPR-Cas9, now enable researchers to precisely target segments of genetic code giving rise to a range of potential scientific and medical applications from fixing genetic defects, to manipulating stem cells, to reengineering immune cells to fight infection and cancer.

Scientists currently employ several different methods to insert new genetic instructions into cells, including creating small holes in the cell membrane using electrical pulses, injecting DNA into cells using a device called a “gene gun,” and employing viruses to “infect” cells with new genetic code.

However, all of these methods tend to suffer from two fundamental problems. First, these processes can be highly toxic, leaving scientists with too few healthy cells to work with. And second, these methods are restricted in the amount of genetic information – or “payload” – they can deliver into the cells, limiting their application. These techniques can also be time consuming and expensive.

The new device described in the study was fabricated in the Schrlau Nano-Bio Interface Laboratory at RIT by Masoud Golshadi, Ph.D. Using a process called chemical vapor deposition, the researchers created a structure akin to a honeycomb consisting of millions of densely packed carbo nanotubes with openings on both sides of a thin disk shaped membrane.

The device was employed in the Dickerson Lab at URMC to culture a series of different human and animal cells. After 48 hours, the cells were bathed in a medium that contained liquid DNA. The carbon nanotubes acted as conduits drawing the genetic material into the cells. Using this method, the researchers observed that 98 percent of the cells survived and 85 percent were successfully transfected with the new genetic material.

The mechanism of DNA transfer is still under investigation, but the researchers suspect it may be via a process called enhanced endocytosis, a method by which cells transfer bundles of proteins back and forth through the cell membrane.

The device has also shown the ability to successfully culture a wide range of cell types, including cells that are typically difficult to grow and keep alive, such as immune cells, stem cells, and neurons.

The researchers are now optimizing the technology in hopes that the device – which is inexpensive to produce – can be made available to researchers and, ultimately, used to develop new treatments for a range of diseases.

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Additional co-authors of the study include Golshadi and Leslie Wright with RIT. The research was supported with funding from the Schmitt Program on Integrative Brain Research, the American-German Partnership to Advance Biomedical and Energy Applications of Nanocarbon, Texas Instruments, the Feinberg Foundation, and the Weizmann Institute of Science.

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