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Back to top. Get to Know Us. Aptamer-based therapeutics rely on direct binding of an aptamer to a therapeutic target to modulate downstream signaling.
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Leading examples include those that target receptor tyrosine kinases e. Ribozyme-based therapeutic strategies have emerged as powerful tools for the treatment of cancer and viral infections. Ribozymes are RNA motifs that possess catalytic properties much like those of proteins. Ribozymes targeting mRNA show promise for gene therapy of breast cancer and hepatocellular carcinoma in animal models as they display similar effectiveness as RNAi, while exhibiting less off-target effects.
Ribozyme sequences are fused with RNA scaffolds creating a targeting platform to specifically deliver ribozymes. Riboswitches offer therapeutic avenues for addressing a wide range of diseases by regulating gene expression. They are widespread in numerous evolutionarily distant bacteria, with counterparts in archaea, plants, fungi, and algae.
Riboswitches are composed of two domains, a sensor portion a natural aptamer for ligand recognition and an expression platform that can adopt two mutually exclusive conformations based on ligand binding for controlling gene expression without the need for protein cofactors Figure 5 D. Since riboswitches control vital metabolic and virulence genes in pathogenic species, they represent an attractive strategy for therapeutic intervention without inflicting side effects on the eukaryotic host.
Microfluidic Preparation of Polymer-Nucleic Acid Nanocomplexes Improves Nonviral Gene Transfer
Riboswitches can also be used to engineer artificial genetic circuits controlled by introducing synthetic ligands in human cells. Based on simplistic design principles, RNA nanotechnology can be applied not only to deliver the RNA switches in vivo but also to rationally engineer synthetic riboswitches to repress or activate gene expression in a ligand-dependent fashion. The thermodynamic property displayed by different types of nucleic acids can be programmed into RNA nanoparticles to trigger the release or activation of functional modules.
As discussed before, nucleotide selection provides tunable nanoparticle stability and thus controllable release.
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Strategies utilizing differences in nucleic acid thermodynamic stability can be used to tune the release of drugs from the particle, whether the drugs are intercalated or conjugated to the vertices of the particles. Additionally, functionalities can be split among different helices, and upon toe-hold interaction, their functions are triggered. To monitor the release, each piece of the split-functionality is labeled with a FRET pair molecule; therefore, upon association of the functionalities, FRET signal drastically increases Figure 5 E.
The discovery of the RNA interference RNAi pathway in created an avenue of treatments for many diseases, including cancer. RNAi is the process by which the expression of a gene is modulated by short double-stranded RNA sequences, typically 21—23 nucleotides in length. BRCAA1 knockdown down-regulated the expression of antiapoptotic Bcl-2 gene and up-regulated the expression of pro-apoptotic Rb and Bax genes, thereby regressing the growth of the tumors. Another study demonstrated that pRNA-3WJ nanoparticles harboring folate and luciferase siRNA can efficiently silence the expression of the luciferase gene after systemic injection in intracranial glioblastoma xenograft-bearing mice.
Luciferase expression was successfully silenced in subcutaneous ovarian cancer xenograft-bearing mice. To circumvent limited efficacy, multivalent siRNA delivery can be implemented.
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The versatility of RNA nanoparticles allows sequence integration of immune system stimulating motifs, such as CpG, acting as immuno-adjuvants. For example, introduction of a miRNA-like nonpairing uridine bulge in the passenger strand of siRNA that can induce immunostimulatory activity on human immune cells. Recently, aptamers were developed to target proteins involved in cancer immunotherapy preventing tumor cells from escaping the host immune system. Aptamers are available for secretory targets such as cytokines and chemokines e.
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Dual aptamers can form a bridge to enable simultaneous binding to two cell surface markers or recruit T-cells to the proximity of tumor cells, leading to destruction of diseased cells. RNA nanotechnology can help overcome challenges associated with developing bispecific aptamers such as refolding, instability, loss of binding specificity after fusing two different aptamers into one RNA sequence, and size modulation to enable binding to both targets simultaneously.
Small molecule chemotherapy drugs are routinely used for the treatment of cancers. However, chemotherapeutic drug delivery traditionally relies on passive targeting via EPR effects. Small molecule drugs pass kidneys and blood vessels quickly due to their low molecular weight. After systemic administration, however, distribution is typically observed throughout the whole body with poor accumulation at tumor sites, thus producing suboptimal antitumor potency while increasing off-target effects.
RNA nanotechnology is an attractive strategy to improve all aspects of traditional chemotherapy, including enhanced circulation time and blood plasma concentration, reduction in the administered dose, lowering of off-target effects by delivering payloads specifically to diseased cells and tissues, and enabling release mechanisms to decrease systemic toxicity. Many well-established conjugation methods are available for linking chemical drugs to chemically synthesized end-labeled RNA nanoparticles in high yield including thiourea linkage, click chemistry, amide linkage through NHS-ester chemistry, and thioether linkage, among others Figure 7 B.
Controlled release of drug conjugates is one approach to minimize off-target toxicity. Many factors present in cancer cells, but not healthy cells, can be exploited for controlled release mechanisms. For example, high esterase activity in some cancer cell types has been exploited by synthesizing a drug linker sensitive to esterase degradation. Incorporation of an acid-labile linker, where low pH causes cleavage, releases the drugs so that they can diffuse out of the endosomes.
Intercalation serves as a viable alternative to drug conjugation, as long as minute amounts of drugs are released in the circulation prior to reaching the tumor microenvironment. Encapsulation of drugs in 3D RNA architectures is another feasible alternative without changing intrinsic drug properties.
There is a significant interest in developing image-guided vectors for detecting RNA trafficking, endogenous gene expression, and silencing in vivo. Molecular beacons to image intracellular RNA expression in vitro by competitive hybridization methods is a popular detection method. Initially, the fluorophore and quencher are in close proximity, resulting in little to no fluorescence Figure 8 A. RNA nanotechnology can significantly advance the development of molecular-beacon-based RNA nanoparticles, especially in regards to improving their thermodynamic and chemical stability for efficient competitive hybridization to the mRNA targets.
For more detailed information on the design and application of molecular beacons, readers are encouraged to refer to articles referenced in this review. Importantly, survival prognosis increases with early detection. To achieve early detection, a system needs to detect as low amounts as possible of diseases markers, such as a small molecule or protein indicators.
Aptamer-based biosensors aptasensors have emerged as powerful alternatives to traditional antibodies and enzymes, especially with the advent of nanotechnology.
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These platforms can be integrated with microarrays to quantify thousands of targets. These properties of aptamers coupled with methods in signal generation e. RNA nanotechnology could facilitate further development of aptasensors given that the tunable size and shape, optical properties, surface properties, switchable modules, ease of labeling, and catalytic activities of RNA nanoparticles can be useful not only for signal generation but also for signal amplification.
For more detailed information on RNA based in vitro diagnostics, readers are encouraged to refer to articles referenced in this review. Many researchers are looking to use computation to analyze nucleic acids. The first DNA logic gate was designed in , followed by the beginning of molecular scale computing in when a DNA-based computational design was used to solve a seven-city Hamiltonian math equation.
Since RNA displays many different structural motifs compared to DNA, it could be advantageous to employ the structural diversity and complexity of RNA in nucleic acid computation. In addition, RNA can act as an insulator for biomemory applications. Challenges and Outlook. RNA nanotechnology for drug delivery has garnered much attention recently as it has tremendous potential to help treat many diseases, especially cancer. Certainly, improvements are necessary to push this innovative platform toward clinical trials and bring the product to the market.
Some of the challenges and possible solutions are discussed below. Currently, RNA nanoparticles have limited small molecule drug loading capacity. Single drugs can be labeled onto the terminal ends of RNA strands with exquisite drug release mechanisms. Whole chain labeling methods can increase chemical drug payload but can cause misfolding of RNA nanoparticles due to steric hindrance and also compromise the release of the drugs. Computational approaches can help to identify locations where drugs can be introduced without disrupting the folding of the nanoparticle.
Ensuring the drugs are orientated out of plane from the RNA nanostructure can also minimize any structural defects in the scaffold. Intercalation is another viable approach to increase drug loading capacity, but drug release profiles need to be closely evaluated as premature release of the drug leads to nonspecific side effects. For instance, if the pRNA-3WJ nanoparticle is used as a delivery vector for targeting solid tumors, the projected half-life of the intercalated drug release should be more than 4 h, as extensive biodistribution studies revealed that the vast majority of the systemically administered pRNA-3WJ nanoparticles localize in the tumor site within 1—4 h.
Recently, RNA dendrimers have been constructed up to generation More recently, 3D RNA polygons have been developed that can potentially serve as RNA cages or containers to encapsulate hundreds of small molecule drugs. However, like chemical drug conjugation, RNA nanoparticles are limited in the number of RNAi molecules that can be delivered to cells. However, this limitation may not be an issue as studies report that there are only about 10 3 —10 5 Dicer molecules per cell, and oversaturation of therapeutic RNAs can lead to nonspecific binding and off-target gene knockdown.
RNA microsponges have the potential for delivering high payloads of siRNAs, 5 and their utility in broader preclinical studies will be improved if the particle size can be reduced to avoid organ accumulation. Similar to most nanoparticle platforms, RNA nanoparticles enter cells through receptor-mediated endocytosis.
Thus, intracellular RNA nanoparticle trafficking becomes the next challenge. Early endosomal vesicles are the first destination of RNA nanoparticles. Once sorted, RNA nanoparticles are transferred to late endosomes and lysosomes, where they are trapped without reaching their intended target. Fortunately, endosomal escape using small 8 nt anti-miRNA LNA fragments in RNA nanoparticle delivery was successful and cancer regression efficient, 60 but the efficacy of endosomal escape of siRNA in RNA nanoparticles is still unknown as cancer regression after siRNA delivery via receptor-mediated endocytosis is relatively low.
Nevertheless, there are well-characterized tools available to enhance endosome escape such as chemical functional groups including acid-cleavable linkers such as acetal, hydrazone, and maleic amides, or other acid protonating groups such as amino esters and sulfonamide. As discussed earlier, a wide range of chemical conjugation strategies are available for site-specific incorporation of the endosome disrupting chemicals to RNA nanoparticles.
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One of the major bottlenecks for future clinical applications is the large-scale production, large-scale purification, and cost of RNA nanoparticle production. Typically, RNA nanoparticles are designed to be modular composed of multiple short strands that are well within the limits of chemical synthesis maximum of 80 nt.
However, large-scale purification remains a challenge. HPLC and gel electrophoresis have limited capabilities with somewhat low yields. Due to the special nature of RNA nanotechnology, the size of the assembled nanoparticle is significantly different from its building blocks, thus, preparative ultracentrifugation has recently been employed and looks to be a promising approach for purification of fully assembled RNA nanoparticles with high yield.
While chemical modifications offer many advantages concerning serum stability, thermodynamic stability, and immunogenicity, in some cases they do affect the folding of RNA motifs. Rational modification is a case by case issue and is RNA-sequence- and structure-dependent. Specific patterns of chemical modifications can work well with one system but may not for another.