Design sirna pdf


















Parallel and linear synthesis siRNA-peptide and -cholesterol conjugates. For parallel synthesis Fig. The above siRNA conjugates were reported to show silencing effects in different human cell lines.

Linear synthesis functional groups are added sequentially is also widely used for a variety of chemical conjugations to siRNA Fig. In , Letsinger and co-workers reported the synthesis of amide-linked, cholesterol-modified oligonucleotides In , cholesterol and lipid modified siRNAs were created using a pyrrolidine-based linkage These lipophilic siRNA conjugates were shown to silence apolipoprotein B through intravenous injection in mice 62 , via a lipoprotein-dependent mechanism.

Both low-density lipoprotein receptor and scavenger receptor class B type I are required for the uptake of siRNA conjugates by the liver and other tissues. A solid support was installed on the intermediate through a succinic acid linker and gave the substrate 7 that could then be used to generate the siRNA strand Fig.

Finally, the GalNAc-siRNA strand was synthesized by adding the nucleotide one by one through the solid phase-based approach as discussed above. Results from a clinical trial of this conjugate demonstrate significant reduction of serum transthyretin TTR protein for treating TTR mediated amyloidosis Cemdisiran is in phase II clinical investigation for patients with complement-mediated diseases 2 , In addition, several promising candidates including Vutrisiran indication: TTR-mediated amyloidosis , Fitusiran indication: hemophilia and rare bleeding disorders , Inclisiran indication: hypercholesterolemia , and Lumasiran indication: primary hyperoxaluria type 1 are in Phase III clinical trials 2 , In addition to the clinical advance, a wide variety of new chemical approaches were reported to optimize the GalNAc-siRNA conjugate.

Matsuda el al systematically explored the effects of GalNAc on different sites of siRNA strands and identified several potent sequences These findings provide new insights on next generation siRNA conjugates.

Nucleotides have long been utilized as building blocks to assemble a wide variety of nanoparticles A number of two-dimensional and three-dimensional nanostructures have been self-assembled through branched DNA motifs and crystalized for characterization and visualization. The Each edge is 30 base pairs long and contains a nick in the middle Fig.

In order to differentiate tumor cells from normal cells, a cancer-targeting ligand, folate, was installed on the nanoparticle surface. SNA nanostructures are determined by the shape of the cores and the shells can accommodate both single- and double-stranded nucleic acids with sequences of interest.

To construct functional SNA, three components were necessary: a particle attachment moiety, a spacer region, and a programmable recognition region In the past decade, a number of inorganic cores and nucleic acids shells have been investigated for diverse applications including diagnosis, small molecular drug delivery, and DNA and siRNA delivery Currently, this platform is in the clinical trial for treating glioblastoma.

Later on, a diverse set of RNA based nanomaterials were created for gene silencing and other applications 71 , In , Hammond and co-workers developed self-assembled RNA interference microsponges via rolling circle replication RCT , which was applied by viruses to amplify their genes To achieve this process, they constructed a linear DNA strand encoding the antisense and sense sequences.

Also, their ends were partially complementary to the T7 promoter. A single sponge contains approximately five hundred thousand copies of siRNA. Tetrahedron DNA origami. Three-way junction RNA nanoparticles. Phospholipids are natural components of cell membranes that form lipid bilayers Liposomes have been developed as drug delivery carriers using a variety of synthetic lipids They have been widely used to encapsulate small molecule drugs for treating diseases in humans, most notably Doxil for breast cancer and AmBisome for fungal infection These previous studies provide important guidance for the development of lipid-based siRNA delivery systems.

Lipid-based nanoparticles LPNs , particularly lipids with single or multiple cationic centers Fig. SNALPs are typically composed of a formulation consisting of an amine-based lipid, cholesterol, a PEG-lipid, as well as helper phospholipids DlinDMA is composed of an ionizable amino head group, a glycerol-based ether linker, and two unsaturated carbon tails Fig.

Amino lipids are central components of SNALPs, as they play a role in the assembly of the nanoparticles, by binding the siRNA through electrostatic interactions These amino groups also facilitate endosomal escape, through interaction with endosomal components during acidification The common structure of this type of lipids includes a cationic head group, a linker, and two long hydrophobic domains Fig.

Cationic head groups can be single or multiple cationic centers. Linkers span from ester, amide to ketal. Hydrophobic tails can accommodate unsaturated bonds, cholesterol, and ester groups. This chemical alteration reduces the transition temperatures of DLin-KC2-DMA and facilitates its ability to form hexagonal structures when it interacts with naturally occurring anionic phospholipids in the endosomal membrane.

This process is believed to promote endosomal release Head groups were substituted by amino groups with different size and ring structures. SAR studies of 56 amino lipids in vivo indicate that a dimethyl substitution on the amine head group was preferred to diethyl, diisopropyl, and ringed structures.

The linkers ranged from ester, amide, ketal, ether, and carbamate. Efficacy was generally retained for ester, ketal, and carbamate linkers, though the length and functional groups of the linker could significantly affect the activity.

Lipids with amide and ether linkers possessed reduced delivery performance when formulated. The authors show that pKa is an important factor for delivery efficiency and an optimum pKa of the nanoparticles was between 6.

Consistent with this report, a recent study investigated the correlation between nanoparticle properties and siRNA delivery efficiency In this study, pKa was also identified as a key determinant of nanoparticle delivery efficacy Hydrophobic tails can accommodate diverse functional groups, including unsaturated carbon bonds and small molecules Maier and coworkers report that an ester bond can be installed in the middle of hydrophobic tails Fig.

In addition to lipids with single cationic center, numerous lipid derivatives and lipid-like materials with multiple cationic centers have been developed Here, we discuss two representative examples: aminoglycoside and amino acid derivatives.

Aminoglycoside-based lipids are composed of an aminoglycosides head, an amide linker, and two unsaturated tails 89 Fig. In , an arginine based lipid, AtuFECT01 was reported, which consists of arginine derived head, an amide linker, and two different carbon tails Recently, a similar arginine based liposomal delivery system was developed for hepatic silencing The lead material was reported to have dose dependent silencing with an ED 50 of 0.

In , a library of lipid-like molecules, termed lipidoids, was developed for siRNA delivery 93 Fig. Lipidoids are composed of one or more amine centers and multiple hydrophobic tails.

Over diverse lipidoids were synthesized with a range of functional amines and acrylates or acrylamides through a one-step Michael addition reaction without the need of catalysts or solvents. The amino groups function similarly to the amino head group in the lipid nanoparticles mentioned above, by neutralizing negative charges of siRNA and facilitating cytoplasmic release of siRNA.

In general, lipidoids with amide linkages and more than two alkyl tails 8—12 carbons had the most activity when formulated with siRNA. One lipidoid termed 98N12—5 cite figure 6 in particular was investigated for its potential to deliver siRNA Thousands of lipid-like compounds were synthesized through Michael addition reactions, epoxide ring-opening reactions, reductive amination reactions and thiol-ene reactions.

Materials were screened with high throughput bioassays both in vitro and in vivo. In an effort to improve the delivery efficiency, a library of amino alcohol-based lipidoids was developed using epoxide-based chemistry A one-step ring-opening reaction between functional amines and epoxides afforded over new lipidoids. The lead material, named C12— Fig. The potency of this formulation also allows the potential for simultaneous gene silencing.

C12— formulations containing five different siRNAs at once were shown capable of silencing their target genes in mice. This material was also shown capable of silencing certain immune cells in mice and primates More recently, several combinatorial libraries have been constructed using structural information gained in these earlier studies In particular, a lipopetide material termed cKK-E12, Fig.

Meanwhile, Whitehead and co-workers made a series of biodegradable lipidoids such as O13 Fig. In , Siegwart et al applied a two-step synthetic route, Michael addition and thiol-ene reaction, to prepare degradable dendrimers Fig. More and more lipid analogs and derivatives are under development, which may facilitate siRNA or other types RNA therapeutics.

The utility of polymers to function as intracellular delivery systems for nucleic acids, including antisense oligonucleotides ASOs and plasmid DNA pDNA , has been studied for several decades , For instance, cationic polymers are able to condense nucleic acids into polyplexes via electrostatic interactions, enhancing cellular uptake and endosome escape.

Notably, the physiochemical properties of polymers can be carefully adjusted through bottom-up chemical synthesis. The eventual nano structure can also be dictated through chemical synthesis, covering a range of assemblies including block and star shaped copolymers, micelles, dendrimers, solid nanoparticles, polyplexes, polymer-siRNA conjugates, and more. A few polymer-based siRNA delivery systems have shown therapeutic potential in clinical trials Examples of major classes of polymer-based delivery systems are discussed below.

Direct conjugation of siRNA to polymers offers an attractive avenue to improve stability, pharmacokinetics, cellular uptake, and delivery. PEG conjugated to siRNA via an acid-labile linker was reported to facilitate gene silencing in hepatoma cells in vitro and in a tumor model Kataoka and co-workers pioneered the use of polyion complexes PICs for drug delivery, and used this system to graft siRNA through disulfide linkages to a polymer to improve the physicochemical properties and transfection efficacy This system is currently the approach in a recruiting Phase 2 clinical trial.

Saltzman and coworkers reported using PLGA-spermidine nanoparticles gene silencing in the vaginal lumen and uterine horns of mice following topical delivery The development of Controlled Radical Polymerization CRP has made a major impact on polymer science, and these techniques have also been used to form bioconjugates , star polymers, nanogels , and other delivery materials for siRNA Lastly, in situ radical polymerization of siRNA enabled formation of siRNA nanoparticles and intracellular delivery Similarly, a poly butyl amino vinyl ether PBAVE polymer was designed with side chains of alkyl and amino groups to mediate cellular uptake and endosomal release, and GalNac groups for hepatocyte targeting.

This polymer was conjugated to siRNA, providing a single-component system with multiple functionalities for distinct purposes. Newer generation PolyConjugates, that improve on the initial design have been reported to fully silence liver genes 0. A biodegradable design based on polypeptide and poly amido amine conjugates further expanded the in vivo activity and tolerability of the siRNA conjugates.

One of the initial requirements of polymeric carriers is the ability to complex siRNA. This has most frequently been achieved using electrostatic interactions between positively charged groups on polymer chains and the negatively charged phosphates in siRNA molecules. Tertiary amines are particularly well suited for siRNA delivery because they can be charged at low pH during self-assembly with siRNA, neutral at extracellular pH, and positively charged after endocytosis to enable endosomal release.

Although efforts have been made to use other interaction parameters, such as intercalation and physical entrapment, these efforts have yielded less efficacious carriers In addition to functional groups that mediate siRNA binding, a second fundamental feature of efficacious polymers for siRNA delivery is hydrophobicity. It is understood that hydrophobic interactions can further stabilize siRNA-polymer nanoparticles above the nanoparticle pKa.

This can be accomplished by inclusion of hydrophobic molecules e. Thus, an optimal balance between pKa 6. In general, BPEI provides greater complexation ability with siRNA due to the flexible structure, an increased number of charges per volume, and more folding options. In particular, hydrophobic modifications have been shown to improve siRNA delivery , For example, lipid derived BPEI can efficiently deliver siRNA to endothelial cells and silence multiple endothelial genes in mice , Moreover, the formulation showed selective silencing in endothelial cells.

PAMAM-RNA complexes have also been incorporated into degradable polymer scaffolds to mediate controlled local release and sustained gene silencing to increase survival of mice bearing aggressive triple-negative cancer For example, Yang et al. PLGA-based block copolymer systems have also been developed by Farokhzad and coworkers , for systemic delivery of siRNA against various targets, including demonstration of efficacy in a prostate cancer model. The use of well-defined polymer architecture and precise cross-linking chemistry are also useful strategies for siRNA delivery.

Wagner and co-workers showed that the incorporation of two or three cysteine cross-links into poly amido amines was not necessary for pDNA delivery, but was absolutely critical to achieve siRNA delivery , It has also been shown that delivery via block and core-shell type architectures helps to increase electrostatic interactions by physically concentrating the cationic charges. Dimethylamine and piperazine groups, cross-linked into the cores of well-defined polymers with PEG shells, provided enhanced complexation and delivery as compared to more than other amine-based cross-linkers screened in a 1,member combinatorial library The use of endosome destabilizing agents is an additional way to enhance block copolymer-mediated delivery Overall, tertiary amines and alkyl chains have been identified as key functional groups for effective siRNA delivery across a variety of siRNA delivery materials including lipids and polymers.

Natural materials have also been used for siRNA delivery. Of note, Davis and co-workers demonstrated that cyclodextrin-based self-assembling polymeric nanoparticles can facilitate siRNA delivery, including in humans , For instance, a cyclodextrin-containing polymer CDP , a PEG stabilization agent, and human transferrin have been used in self-assembled nanoparticles where transferrin acts as a targeting ligand for transferrin receptors that are frequently overexpressed on cancer cells.

The self-assembled four component formulation was delivered IV to patients with solid cancers in a Phase 1b clinical trial. Chitosan is another natural cationic polymer that has been used for siRNA delivery. It has been shown that higher MW chitosan provides better complexation and stability, whereas lower MW chitosan with specific degrees of deacetylation offer better intracellular release , Huang and co-workers showed that a liposomeprotamine-hyaluronic acid LPH nanoparticle formulation could enable silencing of CD47 in tumor tissues after IV administration This siRNA-mediated silencing of CD47 inhibited the growth of tumors in multiple models, including melanoma and lung metastasis.

Proteins have also been utilized for siRNA delivery. Synthetic amino acid-based polymers are also attractive for delivery.

A derivative of polyglutamic acid that incorporated a cell-penetrating helical structure was used for siRNA delivery and shown to cause pore formation in cell membranes, thereby enhancing delivery In the past two decades, significant advances have been made in the development of siRNA therapeutics for treating diverse diseases.

Chemists are capable of synthesizing siRNA with modified nucleotides to achieve high efficacy, high stability, and high specificity. However, in order to maximize the advantages of siRNA therapeutics in humans, there are still formidable challenges for the delivery systems.

To address the issues associated with potency, selectivity, and safety, many strategies have been applied to develop new delivery materials. Chemists often design delivery systems considering elements, bonds, and functional groups.

The key chemical properties of successful carriers are largely consistent between lipid and polymer-based systems. These include hydrophobic modifications, tertiary amines, and the ability to interact with short siRNA strands via multiple types of bonding interactions. Currently, lipid- and lipidoid-based siRNA materials are highly effective delivery systems. Extensive SAR studies have provided design criteria, as described above.

Moreover, formulation methods are also very important to the efficacy in vivo. Direct conjugation of small molecule ligands or polymers to siRNA offers the advantage of a being single component delivery system with defined composition. For example, GalNAc-siRNA conjugates not only provide an approach for ligand based cell internalization without the need of cationic materials, but also target hepatocytes specifically.

In addition, synthetic and natural materials also offer ways to tune the degradation and responsiveness of the delivery system. For efficient siRNA delivery, cationic materials that include additional stabilizing interactions e. Outside of the classical drug delivery systems, there are also new strategies to develop siRNA therapeutics.

Overall, the major challenges for siRNA therapeutics are to increase the efficacy, enhance cell-tissue specificity, and improve the safety. Future efforts may include 1 delivery using new targeting ligands and chemical probes that specifically bind to surface markers on diseased cell populations, 2 increasing efficacy particularly with regard to understanding how much siRNA enters the cytoplasm and how to better facilitate that process , , 3 widening of therapeutic window using materials with low toxicity, 4 design of materials with defined degradation products that can be metabolized important when siRNA-based drugs will be repeatedly dosed , 5 simplification of the formulation procedure, and 6 delivery to organs other than the liver.

Chemists have the ability to build materials on the molecular level and are well suited to meet these challenges to make siRNA therapeutics successful in the clinic for a broad range of diseases.

Selected examples of conjugate, linear, and branched type architectures for siRNA delivery. The authors thank Dr. Christopher Alabi for providing critical feedback and useful suggestions. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form.

Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. National Center for Biotechnology Information , U. Adv Drug Deliv Rev. Author manuscript; available in PMC May Daniel J. Daniel G. Author information Copyright and License information Disclaimer. The final manuscript was approved by all authors. Copyright notice. The publisher's final edited version of this article is available at Adv Drug Deliv Rev.

See other articles in PMC that cite the published article. Abstract Emerging therapeutics that utilize RNA interference RNAi have the potential to treat broad classes of diseases due to their ability to reversibly silence target genes.

Introduction The presence of endogenous RNA interference pathways in mammalian cells provides a powerful mechanism for the regulation of cellular signaling pathways by enabling precise modulation of gene expression 1. Open in a separate window. Synthesis and chemical modification of siRNA Chemically modified nucleotides can improve chemical stability and efficacy, increase cell specificity, reduce immunological effects, and decrease off-target effects 6. Chemical strategies for synthesis of siRNA conjugates.

Nucleotides derived nanoparticles Nucleotides have long been utilized as building blocks to assemble a wide variety of nanoparticles Nucleotides derived nanoparticles. The editors of Nature Cell Biology have recommended several controls 2. Two of these controls are: A negative control siRNA with the same nucleotide composition as your siRNA but which lacks significant sequence homology to the genome.

To design a negative control siRNA, scramble the nucleotide sequence of the gene-specific siRNA and conduct a search to make sure it lacks homology to any other gene.

Perhaps the best way to ensure confidence in RNAi data is to perform experiments, using a single siRNA at a time, with two or more different siRNAs targeting the same gene. Prior to these experiments, each siRNA should be tested to ensure that it reduces target gene expression by comparable levels. Most of the designs had two inverted repeats separated by a short spacer sequence and ended with a string of T's that served as a transcription termination site. The selection of siRNA target sequence, the length of the inverted repeats that encode the stem of a putative hairpin, the order of the inverted repeats, the length and composition of the spacer sequence that encodes the loop of the hairpin, and the presence or absence of 5'-overhangs, vary among different reports Ambion will synthesize a complementary pair of siRNA oligonucleotides according to your sequence.

If you wish, you can choose UU or other overhangs. Note: the 3' dTdT of the sense strand does not have to be complementary to the target gene. To order a pre-designed siRNA, search our siRNA database for your gene of interest, choose the design s you'd like to purchase, add them to your cart, and transfer the relevant information about each to our online oligo order form. Elbashir, et al. EMBO J Nat Cell Biol. Brown, D. Ambion TechNotes 9 1 : Sui, G. The effect of target secondary structure and RNA interference was extensively studied using different datasets ranging from siRNA targeting three genes, to 3, siRNAs targeting 82 genes, showing correlation between secondary structure and interference efficiency [22] — [26].

Several siRNA sequence features affect structural accessibility, such as GC-rich regions and palindrome regions that lead to the formation of stable intra-molecular structures [27]. The summation of these two energies is defined as the total interaction energy. There is evidence of the correlation between siRNA inhibition efficiency and siRNA-mRNA binding energy [28] that strengthens the findings of Ladunga, in which target accessibility information was found to provide the most predictive feature among the features studied and improve the prediction of highly efficient siRNA [21].

Upon testing siRNAs against gradually less accessible target sites, it showed that there was correlation between the target accessibility and the siRNA efficiency [28] , [29]. The third aspect is off-target filtration, as single siRNA could be targeting several mRNA targets by either sense or antisense [30]. The innate immunity effect is caused either by cytosolic double-stranded RNA dsRNA immunorecognition that could be avoided by using siRNA with length less than 30 nts [32] , or triggered via Toll-like receptor 7 sequence-dependent immunorecognition.

Although siRNAs with length less than 30 nts avoid Cytosolic dsRNA immunorecognistion they are capable of triggering Toll-like receptor 7 recognition [18]. As per Homology-based off-targets, it is very common for siRNA to have multi-targets due to their relatively small length. In fact, both sense and antisense are known to have an off-target effect with several mRNA transcripts [30] , [33].

This type of off-target could be subclassified into two subtypes. Therefore, siRNA having off-target effects may be considered undesirable [31] , [11]. Several studies have examined the use of chemical modifications to mask siRNA off-target effects, as summarized in [35] , [27].

Here, we introduce an automated tool capable of designing siRNA which takes into account multiple transcripts filtration, target accessibility and off-target filtration evaluation in a desktop application named MysiRNA-Designer. This is combined with a unique multiple score filtration and efficiency prediction using our specially designed filtration layer.

We subsequently applied our strict filtration step on whole human mRNA to demonstrate the practical usage of the tool against experiment datasets and human mRNA. It passes through seven phases and filtration steps in order to design double stranded ds siRNA with high potential to induce the desired silencing effect. First, the desired gene is targeted via selection of one of its transcripts. Next, sequence space is assigned by examining the targeted mRNA sequence, selection regions that are conserved among the mRNA other transcripts if any.

It is essential to ensure they are free from any single nucleotide polymorphisms SNPs. Thirdly, all possible siRNAs are designed with the length of 19 nt via one nucleotide shift through the sequence space selected earlier. All these are then subjected to an evaluation step that predict their efficiency using ten state of the art models see below as the fourth filtration step. A cut-off score is determined for each of these tools to accept or reject the siRNAs candidates.

MysiRNA-Designer takes the intersection between all of these tools to increase the specificity and reduce the number of false positive as much as possible. The fifth step considers the evaluation of target accessibility including both secondary structure evaluation and energetic calculation between siRNA strands and siRNA-mRNA.

The energetically favoured, target accessible siRNAs pass to the following step where off-target filtration starts, rejecting siRNA that lacks specificity by having off-targeted mRNA s either homology based or seed matching based, following a state of the art protocol for off-target evaluation.

Finally, all siRNAs that pass these filtration steps are evaluated according to the MysiRNA-model, an artificial neural network model previously described by the authors capable of predicting siRNA efficiency with improved efficiency and sensitivity.

We used this model to re-evaluate the siRNA candidates and provide the user with the ability to select the siRNAs passing a specified score level. These steps will be discussed in more detail below. There are seven distinguished phases for siRNA design: 1 st choosing the targeted gene for silencing. Various preprocessing techniques were combined to refine the targeted sequence and locate the most representative and conserved region s within it.

Then, these strict refining constraints were validated. In order to rationally refine the target sequence space, two preprocessing steps were proposed as a modification of the Birmingham guidelines, [27] [ Figure 2 ]:. Sequence space should be free from unstable regions black color and SNPs green color occurrence, which is conserved among different gene transcripts red color which are later, used as a template for siRNA design.

The un-gapped consensus is later calculated in order to Design siRNA targeting the desired sequence space. After sequence space preprocessing, the proposed multi-score filtration was used to evaluate each potential siRNA and filter them. Only siRNAs that passed this multi-score filtration are considered to be active, so that off-target evaluation could be conducted.

There are several methods for scoring and predicting the designed siRNA activity, some more accurate than others. However, they are generally classified into two groups Ichihara et al. The first generation tools depend on differential end GC content evaluation and base pair preferences.

These rules have been implemented in models such as Reynolds [2] , Amarzguioui [3] , Takasaki [4] , Katoh [5] , Ui-Tei [6] and Hsieh [7]. The second generation tools, on the other hand, developed via extensive examination of the Huesken dataset, comprise models such as Biopredsi [9] , DSIR [11] , ThermoComposition21 [10] and i-Score [8].

In MysiRNA-Designer , a filtration stage is implemented which takes into account high accuracy models, both first and second generation. For the first generation models, the Huesken dataset [9] was examined and active siRNAs were isolated. Then we used these experimentally verified siRNAs to assign threshold scores for each of the first generation models. The data was subjected to a normalization step to remove outliers, using a standard deviation calculation [ Table 1 ].

The second generation tools were handled differently, knowing that these tools aim to predict the siRNA inhibition efficiency rather than providing scores reflecting rules compliance as in the first generation. By taking the intersection between all these models, we developed our multi-scores filtration stage that enables identification of siRNAs capable of producing the silencing efficiency desired.

This multi-scores filtration phase reduces the incidence of false positive i. The efficiency of our proposed multi-scores filtration was demonstrated in a comparative analysis against each of the first and second generation tools, as discussed below. Target accessibility evaluation is a crucial step that affects siRNA inhibition efficiency, as it reflects where the mRNA is more likely to be accessed by short oligomers such as siRNAs.

As discussed previously, energetic calculations are required on two occasions, firstly, duplex energy hybridization energy and, secondly, opening energy that should be calculated for both siRNA duplex ds-siRNA and targeted mRNA. In addition to the total binding energy, RNA secondary structure evaluation should also be taken into account [36]. Several programs are used to calculate the binding energy, such as RNAduplex , RNAplfold and RNAup , which are capable of calculating the binding energy partially or in total [28].

RNAxs provided two major advantages: a time reduction and a single-phased process. This process is considered the rate-limiting step as it is time consuming to search and evaluate the siRNAs candidates. As the default parameters are improper for siRNA blasting, it is very important to adjust blast search parameters as recommended in the work of [27] , see supplementary data for detailed Blastn parameters Table S1.

In case where off-target has been found, it check whether it is a complete homology with one or two mismatch , where it is be rejected. If the siRNA free from both complete homology and seed matching homology it is considered as off-target free and hence pass this filtration step. The successful candidates from all of the previous steps are finally re-evaluated using the MysiRNA model. It acts as second layer neural network combining the whole stacking energy together with two best performing algorithms, enhancing both specificity and sensitivity when compared to other models as described elsewhere.

This strict filtration step was able to boost the specificity of the program without significantly affecting the sensitivity see Results. The software accepts either accession or a list of accessions and offers the user the capability of selecting the MysiRNA-model high specificity threshold. The software designs of all possible siRNAs within the consensus, using one nucleotide frame shift.



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