Bu çalışma, Hava Kuvvetleri Bilimsel Araştırma Ofisi'nden LRIR 16RH3003J hibesi ve ABD Savunma Bakanlığı Araştırma ve Mühendislik Müsteşarlığı Ofisi'nin S&T Önceliklerinin Geliştirilmesi için Askeri Ortamlar için Sentetik Biyoloji Uygulamalı Araştırma (ARAP) programı ile desteklenmiştir.
ThyRS için plazmid vektör dizisi (pSALv-RS-GFPa1, 3.4 kb) Dr. J. Gallivan tarafından cömertçe sağlanmıştır. Bombyx mori'den ipekböceği kozaları, Tufts Üniversitesi, MA'dan Dr. D.L. Kaplan tarafından cömertçe bağışlandı.

<ol>
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Burada sunulan görüş ve düşünceler yazarların görüşleridir ve mutlaka DoD'nin veya Bileşenlerinin görüşlerini temsil etmez

Bu protokolü takiben çeşitli DNA kodlu sensör tasarımları ile yüklenen seçici geçirgen hidrojel mikrokapsüller hazırlanabilir. LbL yaklaşımının ayırt edici özelliklerinden biri, genellikle moleküler türlerin kurban şablonları üzerinde adsorpsiyonuyla başlayan aşağıdan yukarıya montaj sırasında mikrokapsüllerin karmaşıklığını uyarlama yeteneğidir. İlk bileşenlerin konsantrasyonlarını, pH koşullarını ve katman sayısını dikkatlice ayarlayarak, farklı DNA yükleme parametreleri...

Sentetik biyoloji alanı, çevrelerini ve potansiyel tehditlerini izlemek için mikroorganizmalar tarafından geliştirilen doğal mekanizmalardan yararlanarak algılama yeteneklerini geliştirmek için eşsiz fırsatlar sunmaktadır. Önemli olarak, bu algılama mekanizmaları tipik olarak bu mikroorganizmaları zararlı maruziyetten koruyan, olumsuz etkileri hafifletmek veya toksik maddelerin alımını önlemek için gen ekspresyonunu düzenleyen bir yanıtla bağlantılıdır. Bu mikroorganizmaları, bu doğal tepkilerden yararlanarak tüm hücre sensörleri oluşturmak üzere tasarlamak, ancak yeni hedefleri tanımak ve / veya nicelleştirme amacıyla (tipik olarak floresan) ölçülebilen ölçülebilir bir sinyal üretmek için yeniden yönlendirmek için önemli çabalar sarf edilmiştir1,2. Şu anda, genetiği değiştirilmiş mikroorganizmaların (GDO'lar) kullanımıyla ilgili endişeler, özellikle çevrede veya insan vücudunda salınırken, tüm hücrelerin veya genetik materyallerinin bir kısmının sızması nedeniyle, bir polimer matrisinde kapsüllenmiş olsa bile, bu algılama yaklaşımlarından yararlanmak için alternatif yollara ihtiyaç duyulduğunu göstermektedir3.
GDO'ların konuşlandırılması endişesi olmadan mikroorganizma bazlı algılamanın faydalarından yararlanmak için güçlü bir yaklaşım, in vitro transkripsiyon / translasyon (IVTT) sistemlerinin kullanılmasıdır. Pratik bir bakış açısıyla, IVTT sistemleri, hücre bileşenlerinin çoğunu, sonikasyon, boncuk dövme veya diğerleri de dahil olmak üzere hücrelerden farklı yollarla "ekstrakte edilmiş" aktif bir durumda içeren bir karışımdan oluşur4. Bu işlemin nihai ürünü, tüm hücrelerin kullanımıyla ilişkili kısıtlamalar (membran difüzyonu, transformasyon verimliliği, hücre toksisitesi, vb.) olmaksızın, farklı sensörleri "açık kap" formatında test etmek için kullanılabilecek transkripsiyon ve translasyon gerçekleştirmek için zaten optimize edilmiş bir biyokimyasal reaksiyon karışımıdır. Önemli olarak, farklı sensör bileşenleri nicel olarak eklenebilir ve etkileri, gösterdiğimiz gibi farklı optik ve spektrometrik tekniklerle incelenebilir5. IVTT sistemlerinin performansının tutarsız olabileceği fark edilmiştir; Bununla birlikte, son zamanlarda yapılan çalışmalar, sensör tasarımı6'daki performanslarını incelerken çok yardımcı olan hazırlık ve karakterizasyonlarını standartlaştırmak için yaklaşımlar göstermiştir. Son zamanlarda, kağıt matrislerindeki bileşenlerinin liyofilizasyonu yoluyla kağıt bazlı tahliller oluşturmak için kullanılan IVTT sistemlerinin birçok örneği, ağır metal iyonlarının, ilaçların, çekirdek algılama elemanlarının ve diğerlerinin tespiti de dahil olmak üzere gösterilmiştir 7,8,9. IVTT tabanlı sensörler için heyecan verici bir uygulama alanı, toprak, su ve insan vücudu dahil olmak üzere farklı ortam türlerinde algılama uygulamalarında kullanılmasıdır. Bu IVTT sistemlerini bu zorlu ortamlara dağıtmak için, IVTT bileşenlerini içermek ve bozulmaya karşı korumak için bir kapsülleme yaklaşımının uygulanması gerekir.
IVTT sistemleri için en yaygın kapsülleme yaklaşımları, lipit kapsüllerin, misellerin, polimerzomların ve diğer sıkıca kapatılmış mikro kapların kullanımını içerir10,11,12. Bu yaklaşımın bir dezavantajı, dış çevre ile iletişime izin vermek ve algılama yetenekleri sağlamak için malzemeleri konteynerlerin içine ve dışına taşımak için pasif veya aktif mekanizmaların dahil edilmesi ihtiyacıdır. Bu sorunların bazılarının üstesinden gelmek için, buradaki çalışma, IVTT sistemlerinde ifade edilecek farklı sensör tasarımları için kodlama malzemelerini kapsüllemek için basit ama etkili bir yaklaşım sağlayan bir yöntem bildirmektedir. Bu yaklaşım, korunan genetik materyalin tercih edilen IVTT'nin farklı bileşenleri ile etkileşime girmesini sağlayan, yüksek gözenekliliğe sahip içi boş mikrokapsüller oluşturmak için ilgili plazmidlerin varlığında bir biyopolimerin Katman Katman (LbL) birikiminin kullanılmasına dayanmaktadır. Çalışma, kapsüllenmiş plazmidlerin, plazmid kodlu bir aptamer ve bir riboswitch'in karşılık gelen hedeflerine tepkisiyle gösterildiği gibi, bu polimerik matris içinde aktive edildiğinde transkripsiyon ve translasyonu yönlendirebileceğini göstermiştir. Ek olarak, bu LbL kaplama, plazmidleri herhangi bir özel depolama koşulu olmadan aylarca korur.

<table><tbody><tr><td>(Z)-4-(3,5-difluoro-4-hydroxybenzylidene)-2-methyl-1-(2,2,2-trifluoroethyl)-1H-imidazol-5(4 H)-one (DFHBI-1T)</td><td>Lucerna</td><td>DFHBI-1T</td><td></td></tr><tr><td>5x T4 DNA Ligase Buffer</td><td>ThermoFisher Scientific</td><td>46300-018</td><td></td></tr><tr><td>6x Blue Gel Loading Dye</td><td>New England BioLabs</td><td>B7021S</td><td></td></tr><tr><td>96-well plates, black circular</td><td>Corning</td><td>3601</td><td></td></tr><tr><td>Agarose</td><td>Sigma-Aldrich</td><td>A9539</td><td>BioReagent, for molecular biology, low EEO</td></tr><tr><td>Ampicillin sodium salt</td><td>Sigma-Aldrich</td><td>A0166</td><td>powder or crystals, BioReagent, suitable for cell culture</td></tr><tr><td>BlpI restriction enzymes</td><td>New England BioLabs</td><td>R0585S</td><td></td></tr><tr><td>Corning Disposable Vacuum Filter/Storage Systems</td><td>FisherScientific</td><td>09-761-1</td><td></td></tr><tr><td>Dimethyl sulfoxide, DMSO</td><td>Sigma-Aldrich</td><td>472301</td><td>ACS reagent, &amp;ge;99.9%</td></tr><tr><td>DNA Plasmid, pET28c-F30-2x Broccoli (5.4 kb), BrocApt.</td><td>Addgene</td><td>Plasmid #66788</td><td></td></tr><tr><td>DyLightTM550 Antibody Labeling kit (Invitrogen)</td><td>ThermoFisher Scientific</td><td>84530</td><td></td></tr><tr><td>E. coli S30 extract system for circular DNA</td><td>Promega</td><td>L1020</td><td></td></tr><tr><td>Falcon Conical centrifuge tubes, 15 mL</td><td>FisherScientific</td><td>14-959-53A</td><td></td></tr><tr><td>Falcon Conical centrifuge tubes, 50 mL</td><td></td><td>14-432-22</td><td></td></tr><tr><td>Fisherbrand Microcentrifuge tubes, 1.5 mL</td><td>FisherScientific</td><td>05-408-129</td><td></td></tr><tr><td>Hydrofluoric acid, HF</td><td>Sigma-Aldrich</td><td>695068</td><td>ACS reagent, 48%</td></tr><tr><td>Kanamycin sulfate</td><td>Sigma-Aldrich</td><td>60615</td><td>mixture of Kanamycin A (main component) and Kanamycin B and C</td></tr><tr><td>KpnI restriction enzymes</td><td>New England BioLabs</td><td>R0142S</td><td></td></tr><tr><td>LB agar plate supplemented with 100 &amp;micro;g/mL ampicillin</td><td>Sigma-Aldrich</td><td>L5667</td><td>pre-poured agar plates with 100 &amp;micro;g/mL ampicillin</td></tr><tr><td>LB agar plate supplemented with 50 &amp;micro;g/mL kanamycin</td><td>Sigma-Aldrich</td><td>L0543</td><td>pre-poured agar plates with 50 &amp;micro;g/mL kanamycin</td></tr><tr><td>LB broth (Lennox grade)</td><td>Sigma-Aldrich</td><td>L3022</td><td></td></tr><tr><td>Lithium bromide, LiBr</td><td>Sigma-Aldrich</td><td>213225</td><td>ReagentPlus, &amp;ge;99%</td></tr><tr><td>Max Efficiency DH5-&amp;alpha; competent E. coli strain</td><td>ThermoFisher Scientific</td><td>18258012</td><td></td></tr><tr><td>Methanol</td><td>MilliporeSigma</td><td>322415</td><td>anhydrous, 99.8%</td></tr><tr><td>MilliQ-water</td><td>EMD MilliPore</td><td></td><td>Milli-Q Reference Water Purification System</td></tr><tr><td>MinElute PCR Purification Kit</td><td>Qiagen</td><td>28004</td><td></td></tr><tr><td>N-(3-Dimethylaminopropyl)-N&amp;prime;-ethylcarbodiimide hydrochloride, EDC</td><td>Sigma-Aldrich</td><td>E1769</td><td></td></tr><tr><td>PBS (phosphate buffered saline)</td><td>ThermoFisher Scientific</td><td>10010023</td><td>1x PBS, pH 7.4</td></tr><tr><td>Phusion High-Fidelity DNA Polymerase</td><td>New England Biolabs</td><td>M0530S</td><td></td></tr><tr><td>Polyethylenimine, branched</td><td>Sigma-Aldrich</td><td>408727</td><td>average Mw ~25,000</td></tr><tr><td>PURExpress In Vitro Protein Synthesis Kit</td><td>New England BioLabs</td><td>E6800S</td><td></td></tr><tr><td>QIAEX II Gel Extraction Kit</td><td>Qiagen</td><td>20021</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>Qiagen</td><td>&amp;nbsp;27104</td><td></td></tr><tr><td>Quick-Load 2-Log DNA Ladder (0.1-10.0 kb)</td><td>New England BioLabs</td><td>N0469S</td><td></td></tr><tr><td>SiO? silica microspheres, 4.0 &amp;micro;m</td><td>Polysciences, Inc.</td><td>24331-15</td><td>10% aqueous solution</td></tr><tr><td>Slide-A-Lyzer G2 Dialysis Cassettes, 3.5K MWCO, 15 mL</td><td>ThermoFisher Scientific</td><td>87724</td><td></td></tr><tr><td>Sodium carbonate, Na?CO?</td><td>Sigma-Aldrich</td><td>222321</td><td>ACS reagent, anhydrous, &amp;ge;99.5%, powder</td></tr><tr><td>Spectrum Spectra/Por Float-A-Lyzer G2 Dialysis Devices</td><td>FisherScientific</td><td>08-607-008</td><td>Spectrum G235058</td></tr><tr><td>SYBR Safe DNA gel stain</td><td>ThermoFisher Scientific</td><td>S33102</td><td></td></tr><tr><td>T4 DNA Ligase (5 U/&amp;micro;L)</td><td>ThermoFisher Scientific</td><td>EL0011</td><td></td></tr><tr><td>Theophylline</td><td>Sigma-Aldrich</td><td>T1633</td><td>anhydrous, &amp;ge;99%, powder</td></tr><tr><td>Tris Acetate-EDTA buffer (TAE buffer)</td><td>Sigma-Aldrich</td><td>T6025</td><td>Contains 40 mM Tris-acetate and 1 mM EDTA, pH 8.3.</td></tr><tr><td>UltraPure DNase/RNase-Free Distilled Water</td><td>FisherScientific</td><td>10-977-023</td><td></td></tr><tr><td>ZymoPURE II Plasmid MaxiPrep kit</td><td>ZymoResearch</td><td>D4202</td><td></td></tr></tbody></table>

preparation of multifunctional silk-based microcapsules loaded with dna plasmids encoding rna aptamers and riboswitches

DNA yüklü ipek fibroin mikrokapsüllerinin, kurban küresel çekirdekler üzerinde Katman Katman (LbL) montaj yöntemiyle hazırlanması için bir protokol sunuyoruz. Bir asal tabakanın ve DNA plazmidlerinin adsorpsiyonunu takiben, tek bir ipek tabakasının akut dehidrasyonu sırasında ipek sekonder yapıda β-tabakaların indüklenmesiyle sağlam mikrokapsüllerin oluşumu kolaylaştırılmıştır. Bu nedenle, katmanlama çoklu hidrojen bağı ve hidrofobik etkileşimler yoluyla gerçekleşti. Çok katmanlı kabukların adsorpsiyonu üzerine, çekirdek-kabuk yapıları, uzaktan algılama ve / veya hedeflenen teslimat için kullanılacak altın nanopartiküller (AuNP'ler) ve / veya antikorlar (IgG) ile daha da işlevselleştirilebilir. Bir polimer astarının varlığı, DNA ve ipek proteininin konsantrasyonu ve bir dizi adsorbe edilmiş katmanın yanı sıra bir dizi adsorbe edilmiş katman gibi anahtar makromoleküllerin silika çekirdekleri üzerinde sıralı birikimi sırasında birkaç anahtar parametrenin ayarlanması, değişken geçirgenlik ve DNA yüklemelerine sahip biyouyumlu, DNA yüklü mikrokapsüllerle sonuçlandı. Silika çekirdeklerinin çözünmesi üzerine, protokol, kapsül zarının iç yüzeyine hareketsiz hale getirilmiş DNA plazmidleri ile içi boş ve sağlam mikrokapsüllerin oluşumunu gösterdi. DNA plazmidleri ve dış ortam arasında seçici olarak geçirgen biyouyumlu bir zar oluşturmak, uzun süreli depolama sırasında DNA'yı korudu ve mekansal olarak sınırlandırılmış plazmidlerden gelen gelişmiş çıktı tepkisinde önemli bir rol oynadı. DNA şablonlarının aktivitesi ve erişilebilirliği, in vitro transkripsiyon ve translasyon reaksiyonları (hücresiz sistemler) sırasında test edildi. RNA ışıklandırma aptamerlerini ve riboswitchlerini kodlayan DNA plazmidleri, floresan olarak etiketlenmiş RNA transkriptlerinin veya GFPa1 proteininin kabuk zarlarında lokalizasyonu sırasında görselleştirildiği gibi, karşılık gelen analitlerle başarıyla aktive edildi.

Burada, çalışma, ipek protein kapsüllerinde kapsüllemeden sonra farklı sensör tasarımlarını (iki tip RNA tarafından düzenlenmiş transkripsiyon / translasyon elemanı) kodlayan DNA şablonlarının işlevselliğini ele almaktadır. Mikrokapsüller, anahtar bileşenlerin şablonlu Katman Katman (LbL) montajı ile hazırlandı: Bir asal katman, sensör tasarımlarını kodlayan DNA plazmidleri ve ipek fibroin biyopolimeri (Şekil 2). Makromoleküllerin katmanlı bir şekilde birikm...

Watch this Scientific Journal Video about Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches at JoVE.com

Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches

Protokol, sağlam ve biyouyumlu DNA yüklü mikrokapsüllerin oluşumunu, birkaç ligandı izleyebilen çoklanmış in vitro biyosensörler olarak tanımlar.

RNA Aptamerleri ve Riboswitch'leri kodlayan DNA Plazmidleri ile Yüklü Çok Fonksiyonlu İpek Bazlı Mikrokapsüllerin Hazırlanması

We introduce a protocol for the preparation of DNA-laden silk fibroin microcapsules via the Layer-by-Layer (LbL) assembly method on sacrificial spherical ...

preparation-multifunctional-silk-based-microcapsules-loaded-with-dna

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

1.3K Görüntüleme.  UES Inc.. Protokol, sağlam ve biyouyumlu DNA yüklü mikrokapsüllerin oluşumunu, birkaç ligandı izleyebilen çoklanmış in vitro biyosensörler olarak tanımlar.

Video: Preparation of Multifunctional Silk-Based Microcapsules Loaded with DNA Plasmids Encoding RNA Aptamers and Riboswitches

Folding and Characterization of a Bio-responsive Robot from DNA Origami

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both stimuli and cargo can be tailored according to specific needs. Here we describe the DNA nanorobot fabrication protocol, with the use of the DNA origami technique. The procedure initiates by mixing short single-strand DNA staples into a stock mixture which is then added to a long, circular, single-strand DNA scaffold in presence of a folding buffer. A standard thermo cycler is programmed to gradually lower the mixing reaction temperature to facilitate the staples-to-scaffold annealing, which is the guiding force behind the folding of the nanorobot. Once the 60 hr folding reaction is complete, excess staples are discarded using a centrifugal filter, followed by visualization via agarose-gel electrophoresis (AGE). Finally, successful fabrication of the nanorobot is verified by transmission electron microscopy (TEM), with the use of uranyl-formate as negative stain.

DNA origami is a powerful method for fabricating precise nanoscale objects by programming the self-assembly of DNA molecules. Here we describe a protocol for the folding of a bio-responsive robot from DNA origami, its purification and negative staining for transmission electron microscopic imaging (TEM).

DNA Origami bir Bio-duyarlı Robot katlanması ve Karakterizasyonu

The DNA nanorobot is a hollow hexagonal nanometric device, designed to open in response to specific stimuli and present cargo sequestered inside. Both ...

Kimya

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Sağlam, mikroakışkan Hücre içi Delivery Platform olarak hücre Sıkma

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Biyomühendislik

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

SiRNA Therapeutics teslimi için Gözenekli Silikon Mikropartiküller

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Gene-therapy Inspired Polycation Coating for Protection of DNA Origami Nanostructures

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in physiological fluids induce denaturation and degradation of self-assembled DNA nanostructures. In non-viral gene delivery, enzymatic degradation of DNA is overcome by the encapsulation of the negatively charged DNA in a cationic shell. Herein, inspired by gene delivery advancements, a simple, one-step and robust methodology is presented for the stabilization of DNA origami nanostructures by coating them with chitosan and linear polyethyleneimine. The polycation coating efficiently protects DNA origami nanostructures in Mg-depleted and nuclease-rich media. This method also preserves the full addressability of enzyme- and aptamer-based functionalization of DNA nanostructures.

Here, a protocol for the protection of DNA origami nanostructures in Mg-depleted and nuclease-rich media using natural cationic polysaccharide chitosan and synthetic linear polyethyleneimine (LPEI) coatings is presented.

Gen terapisi Polycation kaplama DNA Origami nanoyapıların korunması için ilham.

DNA origami nanostructures hold an immense potential to be used for biological and medical applications. However, low-salt conditions and nucleases in ...

Synthesis, Functionalization, and Characterization of Fusogenic Porous Silicon Nanoparticles for Oligonucleotide Delivery

With the advent of gene therapy, the development of an effective in vivo nucleotide-payload delivery system has become of parallel import. Fusogenic porous silicon nanoparticles (F-pSiNPs) have recently demonstrated high in vivo gene silencing efficacy due to its high oligonucleotide loading capacity and unique cellular uptake pathway that avoids endocytosis. The synthesis of F-pSiNPs is a multi-step process that includes: (1) loading and sealing of oligonucleotide payloads in the silicon pores; (2) simultaneous coating and sizing of fusogenic lipids around the porous silicon cores; and (3) conjugation of targeting peptides and washing to remove excess oligonucleotide, silicon debris, and peptide. The particle&#8217;s size uniformity is characterized by dynamic light scattering, and its core-shell structure may be verified by transmission electron microscopy. The fusogenic uptake is validated by loading a lipophilic dye, 1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate (DiI), into the fusogenic lipid bilayer and treating it to cells in vitro to observe for plasma membrane staining versus endocytic localizations. The targeting and in vivo gene silencing efficacies were previously quantified in a mouse model of Staphylococcus aureus pneumonia, in which the targeting peptide is expected to help the F-pSiNPs to home to the site of infection. Beyond its application in S. aureus infection, the F-pSiNP system may be used to deliver any oligonucleotide for gene therapy of a wide range of diseases, including viral infections, cancer, and autoimmune diseases.

We demonstrate the synthesis of fusogenic porous silicon nanoparticles for effective in vitro and in vivo oligonucleotide delivery. Porous silicon nanoparticles are loaded with siRNA to form the core, which is coated by fusogenic lipids through extrusion to form the shell. Targeting moiety functionalization and particle characterization are included.

Functionalization, malzemelerin ve Fusogenic gözenekli Silisyum nano tanecikleri'Oligonükleotid dır

With the advent of gene therapy, the development of an effective in vivo nucleotide-payload delivery system has become of parallel import. Fusogenic ...

Mühendislik

Assembly and Characterization of Polyelectrolyte Complex Micelles

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful platform for exploring the physics of polyelectrolyte interactions and also offer a promising solution to the pressing problem of delivering therapeutic oligonucleotides in vivo. Developing predictive structure-property relationships for PCMs has proven difficult, in part due to the presence of strong kinetic traps during nanoparticle self-assembly. This article discusses criteria for choosing polymers for PCM construction and provides protocols based on salt annealing that enable assembly of repeatable, low-polydispersity nanoparticles. We also discuss PCM characterization using light scattering, small-angle X-ray scattering, and electron microscopy.

We provide protocols and representative data for designing, assembling, and characterizing polyelectrolyte complex micelles, core-shell nanoparticles formed by polyelectrolytes and hydrophilic charged-uncharged block copolymers.

Polielektrolit Kompleksi Misellerinin Montajı ve Karakterizasyonu

Polyelectrolyte complex micelles (PCMs), core-shell nanoparticles formed by self-assembly of charged polymers in aqueous solution, provide a powerful ...

Predicting Gene Silencing Through the Spatiotemporal Control of siRNA Release from Photo-responsive Polymeric Nanocarriers

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise regulation of gene activity. In particular, novel stimuli-responsive materials with improved spatiotemporal control over gene expression would unlock translatable platforms in drug discovery and regenerative medicine technologies. Furthermore, an enhanced ability to control nucleic acid release from materials would enable the development of streamlined methods to predict nanocarrier efficacy a priori, leading to expedited screening of delivery vehicles. Herein, we present a protocol for predicting gene silencing efficiencies and achieving spatiotemporal control over gene expression through a modular photo-responsive nanocarrier system. Small interfering RNA (siRNA) is complexed with mPEG-b-poly(5-(3-(amino)propoxy)-2-nitrobenzyl methacrylate) (mPEG-b-P(APNBMA)) polymers to form stable nanocarriers that can be controlled with light to facilitate tunable, on/off siRNA release. We outline two complementary assays employing fluorescence correlation spectroscopy and gel electrophoresis for the accurate quantification of siRNA release from solutions mimicking intracellular environments. Information gained from these assays was incorporated into a simple RNA interference (RNAi) kinetic model to predict the dynamic silencing responses to various photo-stimulus conditions. In turn, these optimized irradiation conditions allowed refinement of a new protocol for spatiotemporally controlling gene silencing. This method can generate cellular patterns in gene expression with cell-to-cell resolution and no detectable off-target effects. Taken together, our approach offers an easy-to-use method for predicting dynamic changes in gene expression and precisely controlling siRNA activity in space and time. This set of assays can be readily adapted to test a wide variety of other stimuli-responsive systems in order to address key challenges pertinent to a multitude of applications in biomedical research and medicine.

We present a novel method that uses photo-responsive block copolymers for more efficient spatiotemporal control of gene silencing with no detectable off-target effects. Additionally, changes in gene expression can be predicted using straightforward siRNA release assays and simple kinetic modeling.

Fotoğrafa Duyarlı Polimerik Nanokarbonlardan SiRNA Salınımının Spatiotemporal Kontrolü ile Gen Silenceresinin Öngörülmesi

New materials and methods are needed to better control the binding vs. release of nucleic acids for a wide range of applications that require the precise ...

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive properties of in vitro transcribed (IVT) mRNA. With the help of IVT mRNA, the de novo synthesis of a desired protein can be induced without changing the physiological state of the target cell. Moreover, protein biosynthesis can be precisely controlled due to the transient effect of IVT mRNA.
For the efficient transfection of cells, nanoliposomes (NLps) may represent a safe and efficient delivery vehicle for therapeutic mRNA. This study describes a protocol to generate safe and efficient cationic NLps consisting of DC-cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as a delivery vector for IVT mRNA. NLps having a defined size, a homogeneous distribution, and a high complexation capacity, and can be produced using the dry-film method. Moreover, we present different test systems to analyze their complexation and transfection efficacies using synthetic enhanced green fluorescent protein (eGFP) mRNA, as well as their effect on cell viability. Overall, the presented protocol provides an effective and safe approach for mRNA complexation, which may advance and improve the administration of therapeutic mRNA.

Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA

Here we describe a protocol for the generation of cationic nanoliposomes, which is based on the dry-film method and can be used for the safe and efficient delivery of in vitro transcribed messenger RNA.

Vitro verimli bir şekilde teslim için katyonik Nanoliposomes nesil mesajcı RNA transkripsiyonu

The development of messenger RNA (mRNA)-based therapeutics for the treatment of various diseases becomes more and more important because of the positive ...

DNA-Tethered RNA Polymerase for Programmable In vitro Transcription and Molecular Computation

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work demonstrates that concepts from DNA nanotechnology can be used to program the enzymatic activity of the phage-derived T7 RNA polymerase (RNAP) and build scalable synthetic gene regulatory networks. First, an oligonucleotide-tethered T7 RNAP is engineered via expression of an N-terminally SNAP-tagged RNAP and subsequent chemical coupling of the SNAP-tag with a benzylguanine (BG)-modified oligonucleotide. Next, nucleic-acid strand displacement is used to program polymerase transcription on-demand. In addition, auxiliary nucleic acid assemblies can be used as &#34;artificial transcription factors&#34; to regulate the interactions between the DNA-programmed T7 RNAP with its DNA templates. This in vitro transcription regulatory mechanism can implement a variety of circuit behaviors such as digital logic, feedback, cascading, and multiplexing. The composability of this gene regulatory architecture facilitates design abstraction, standardization, and scaling. These features will enable the rapid prototyping of in vitro&#160;genetic devices for applications such as bio-sensing, disease detection, and data storage.

We describe the engineering of a novel DNA-tethered T7 RNA polymerase to regulate in vitro transcription reactions. We discuss the steps for protein synthesis and characterization, validate proof-of-concept transcriptional regulation, and discuss its applications in molecular computing, diagnostics, and molecular information processing.

Programlanabilir In vitro Transkripsiyon ve Moleküler Hesaplama için DNA-Bağlı RNA Polimeraz

DNA nanotechnology enables programmable self-assembly of nucleic acids into user-prescribed shapes and dynamics for diverse applications. This work ...

Amfifilik DNA Nanoyıldızlarından veya C-Yıldızlarından Çok Yönlü Biyomoleküler Kondensatlar

Synthetic Condensates and Cell-Like Architectures from Amphiphilic DNA Nanostructures

Synthetic droplets and condensates are becoming increasingly common constituents of advanced biomimetic systems and synthetic cells, where they can be used to establish compartmentalization and sustain life-like responses. Synthetic DNA nanostructures have demonstrated significant potential as condensate-forming building blocks owing to their programmable shape, chemical functionalization, and self-assembly behavior. We have recently demonstrated that amphiphilic DNA "nanostars", obtained by labeling DNA junctions with hydrophobic moieties, constitute a particularly robust and versatile solution. The resulting amphiphilic DNA condensates can be programmed to display complex, multi-compartment internal architectures, structurally respond to various external stimuli, synthesize macromolecules, capture and release payloads, undergo morphological transformations, and interact with live cells. Here, we demonstrate protocols for preparing amphiphilic DNA condensates starting from constituent DNA oligonucleotides. We will address (i) single-component systems forming uniform condensates, (ii) two-component systems forming core-shell condensates, and (iii) systems in which the condensates are modified to support in vitro transcription of RNA nanostructures.

Yazar Spotlight: Programlanabilir Amfifilik DNA Nanoyapılarından Sentetik Hücreler Geliştirme

We present a protocol for preparing synthetic biomolecular condensates consisting of amphiphilic DNA nanostars starting from their constituent DNA oligonucleotides. Condensates are produced from either a single nanostar component or two components and are modified to sustain in vitro transcription of RNA from an embedded DNA template.

Amfifilik DNA Nanoyapılarından Sentetik Kondensatlar ve Hücre Benzeri Mimariler

Synthetic droplets and condensates are becoming increasingly common constituents of advanced biomimetic systems and synthetic cells, where they can be ...