Purifying DNA without Membrane Binding

Purifying DNA from natural samples or biochemical reactions is one the most frequently performed experiments in virtually all molecular biology labs. Binding DNA to silica membranes in chaotropic binding buffers is the currently prevailing method, pioneered by Qiagen. Before Qiagen columns and the similar columns from a number of companies, including Allele, there was the silica resin, mostly from Promega. Before silica, it was phenol extraction or CsCl gradient.

Silica-based technology has been around for more than a decade, and it is time for a new generation of technologies that are more convenient and efficient than silica membrane to take center stage of DNA purification, especially given the fast-paced advances in polynucleotide analysis in microarrays and deep sequencing. Solid Surface Reversible Binding (SSRB) technology should be a shining star in coming years. The process is simple: DNA or RNA molecules in a simple binding buffer bind to the surface of plastics of any size and shape (PCR tube, 2.0ml eppendorf, 96-well plates, even 15 ml or 50 ml conical tubes) that is treated by a special process, washed, and eluted in any volume of water or even downstream reaction buffers. The utmost convenience is that the downstream reaction can be performed in the same tube!

This process is different from the electroreversion type of binding and releasing that requires buffers of different and extreme pH. Allele Biotech has started marketing SurfaceBind PCR purification kits, and will roll out products that are specifically tailored for genomic DNA, mRNA, size-differentiated DNA or RNA, DNA or RNA from fixed samples, from different species, etc. The convenience and cost-efficiency of these systems will provide significant contributions to the broad scientific community.

New Product of the Week 101810-102410:

SurfaceBind PCR purification kit, questions? Please email us at oligo@allelebiotech.com for a product description and introduction quotes.

Promotion of the Week 101810-102410:

Updated: GFP-nAb products (equivalent to previously distributed GFP-trap), good for genomic DNA pull down by transcription factor-GFP fusion, RNA co-IP via RNA binding protein-GFP fusion. Or try our brand new, the brightest mFP–mNeonGreen and already available mNeonGreen-nAb, or anti-mNeonGreen nano antibody (also referred to by others as “nanobodies”).

Tags: deep sequencing, DNA purification, genomic DNA, microarrey DNA sample, miniprep, miRNA, nextgen sequencing, PCR cleanup, PFA linked, RNA purification, silica membrane, surface resersible binding

Dealing with Interferon Response When Doing RNAi

Off-target effects are a major problem when using RNA interference (RNAi) to silence genes in mammalian systems. One potential source of off-target effects, by either transfected siRNA duplexes or transcriptionally expressed shRNAs, is the inadvertent activation of the interferon response. There are several steps that can be taken to deal with this problem.

Delivery
Interferon response is more likely when high levels of siRNA are used; it is important to transfect the minimum amount of the siRNA duplex that gives rise to a specific RNAi response, as assessed by the level of expression of the target mRNA and/or protein. The level of stable shRNA expression achieved by using lentiviral or retroviral vectors is comparatively modest. Unless very high levels of shRNA expression are achieved, for example, by using highly transfectable cells and a very efficient shRNA expression plasmid, nonspecific activation of the innate immune response are less likely to be induced.

Design
Previous work has shown that the interferon response is induced by dsRNAs of ?30 bp in length and that perfect dsRNAs of as little as 11 bp in length can produce a weak induction. One possible approach to solving the problem of nonspecific activation of the cellular interferon response is to design the siRNA duplex or shRNA precursor so that it does not contain any stretches of perfect dsRNA of ?11 bp.

Detection
If activation of the interferon response remains a concern, it is possible to routinely check for this effect during the course of an RNAi experiment. Analyzing the level of expression of an interferon-response gene, such as oligoadenylate synthase-1 (OAS1), interferon-stimulated gene-54 (ISG54), and guanylate-binding protein (GBP), in the transfected or transduced cells by northern blot or RT- PCR assays are commonly used.

Can there be any more convenient alternative method for checking interferon response? One potentially useful product could be HiTiter™ pre-packaged lentiviruses that would have a fluorescent protein (mTFP1, mWasabi, or the brightest FP in lanYFP) under the control of an ISRE (IFN-stimulated response element) or GAS (IFN gamma-activating sequence)*. This could be another group of Product-on-Demand type of reagents, meaning that we will have the design ready, but only to produce them upon ordering. This way the cost to us and the price to customers can be kept at minimum.

*The expression of the interferon-stimulated genes (ISGs) is induced by the type I interferons IFN-alpha and IFN-beta. A cis-acting element (TAGTTTCACTTTCCC, nucleotides -101 to -87) has been identified in its promoter of one of these genes, ISG54. This element is responsible for the inducible expression of the ISG54 gene and is referred to as IFN-stimulated response element. The human guanylate-binding (GBP) gene is induced by INF-gamma in fibroblasts within 15 minutes of treatment. An IFN gamma-activating sequence (GAS) has been identified in the GBP promoter (nucleotides -123 to -103). To create the interferon reporters, we would insert five direct repeats of this ISRE and/or four direct repeats of this GAS upstream of the basic promoter element (TATA box) and mWasabi GFP gene of the Allele’s patented pLico lentiviral plasmid backbone.

It should be noted, however, that simple transfection of cells with expression plasmids can induce low-level activation of the interferon response, presumably owing to the presence of cryptic convergent promoters that cause the expression of low levels of dsRNA. In general, very low-level activation of the interferon response, that is, activation that exerts a global inhibitory effect on protein translation of less than twofold, is unlikely to be a problem as long as the specificity of any observed phenotype is fully confirmed.

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The most photostable photoconvertible fluorescent protein mClavGR, email FP@allelebiotech.com for details.

Promotion of the Week 092010-092710:

Buy any two Allele or Orbigen brand antibodies, get the 3rd one of equal or less value free! Put in orders by Friday. Follow us on Facebook and get the weekly promotion early.

Tags: dsRNA, expressed shRNA, interferon, miRNA, off-target, RNA interference, RNAi, RNAi toxicity, siRNA

RNAi Therapy Mediated by Linear DNA Cassettes

RNA interference (RNAi) has been demonstrated to be a powerful tool to silence gene expression. Therapies based on RNAi are being developed in numerous application areas at fast paces. Although in basic research both expressed and synthetic double-stranded RNA molecules are broadly used to induce gene silencing, synthetic small interfering RNAs (siRNAs) are deemed easier to deliver in preclinical and clinical studies. Compared to synthetic siRNAs, DNA cassettes that express small hairpin RNA (shRNA), microRNA (miRNA), or strands of siRNAs have advantages of prolonged effects.

RNAi-expressing DNA cassettes have been incorporated into viral and non-viral vectors for delivery. Viral vectors for RNAi carry the same risks as those for gene therapies, and are currently not the method of choice for human therapies. Non-viral DNA molecules, often in the form of plasmids, can be easily created and reproduced, but their efficacy is hindered by delivery barriers at the tissue, cell, and the nucleus levels. These difficulties are in part due to the plasmids’ large size, presence of antibiotic resistance genes, and immuresponse-generating CpG islands created in bacteria during propagation.

One way to alleviate these difficulties with non-viral DNA vectors for RNAi is to use linear DNA cassettes. Linear DNAs traverse nucleopores efficiently. The DNA molecules can be conveniently produced by PCR reactions without going through production in bacteria, avoiding DNA modifications such as CpG motifs and the need for replication origin or drug-resistance genes. Linear DNA encompassing a promoter, coding region, and poly(A) signals has been used for protein production. Similarly, by incorporating a miRNA cassette into linear transcription unit driven by a Pol II promoter was used to express RNAi for inhibiting HBV (Chattopadhyay et al. (2009). There are now available technologies and commercial services (e.g. Vandalia Research, Inc.) to produce therapeutic grade linear DNA by specialized PCR reactions.

Allele Biotech’s patents on DNA-expressed RNAi provide a platform for highly express shRNA or siRNA from a DNA molecule as short as fewer than 200 basepairs, potentially more suitable for large scale production, and even more efficient transduction trough tissue, cell membrane, and nuclear pores than the large linear cassettes used by Chattopadhyay et al. A set of experiments similar to the cited HBV studies could quickly lead to the validation of a possibly the most effect way yet for RNAi therapeutics.

New Product of the Week 091310-091910:

LoxP-GFP Human T Cells as a reporter cell line for Cre activity. ABP-RP-TGFPLOX, 1 vial $295.

Promotion of the Week 091310-091910:

Validated GFP Expression Lentiviral Particles, ABP-RP-TLCGFPS $225.00, $25 off this week only, with an option to receive concentrated virus (10e10 TU/ml, 10 ul volume) at no additional charge! Follow us on Facebook and get the weekly promotion early.

Tags: Allele RNAi, gene silencing, miRNA, non viral vector, PCR DNA manufacture, pol II promoter, pol III promtoer, RNA interference, RNAi patent, RNAi therapeutics, RNAi therapy, shRNA, siRNA, viral vector

Take Advantage of Allele’s Essential Virus Grant Program, Have High Titer Virus for Your Research on Essential Human Genes

The number of Allele products in the form of viral particles is increasing quickly. Most of these products are also listed under product groups categorized according to their respective functions (e.g. iPS production under “Induced Pluripotent Stem Cells”, shRNA under “RNA Interference”, cytokines and enzymes under “Recombinant Proteins”). Additional background information, as well as related products, may be found on the landing pages of each product group.

The increase in scientific publications reporting the conversion of “dormant” genes into “active” ones has resulted in a significant demand for cDNA clones, antibodies, expression vectors, etc., all in short order. Providing reagents for the expansion of research on such genes is scientifically important and potentially rewarding if the market catches up. Allele Biotech works diligently to supply the most up-to-date and active researchers with pre-validated viral particles for expressing many of these “hot” genes, (e.g. iPS factors, light-activated ion channels, factors that induce neuronal cells from other cell types). Allele Biotech produces custom-packaged high titer lentivirus and retrovirus using unique technologies, as described under “Services by Category”-> “Viral Packaging”.
Under the Essential Virus Grant Program, Allele Biotech accepts custom viral packaging orders, including cDNA of shRNAs against recently established critical mammalian factors. The process begins with an evaluation of your one page application explaining why the factor can potentially be in demand by other researchers. If the application is accepted, Allele Biotech will waive the charges for the project and consider it an R&D effort. Allele Biotech will also offer it as a shelf product at a much lower cost than custom projects. Interested researchers please submit your applications with your order; our accounting department will promptly forward your request to our review group.

New Product of the Week 05-03-10 to 05-09-10: Essential Virus Grant Program

Promotion of the Week 05-03-10 to 05-09-10: for a limited time, pre-packaged, validated IL2 and IL15 lentiviruses. http://www.allelebiotech.com/shopcart/index.php?c=206&sc=0

Tags: cDNA, essential genes, lentivirus, miRNA, promoter reporter, virus packaging

Allele Received Broad Patent on DNA-Expressed RNAi in China

Allele Biotechnology & Pharmaceuticals, a San Diego based private company with associate offices and laboratories in China and distribution channels in 30 countries, was granted a major landmark patent in China in the field of RNA interference (RNAi). The patent CN02828345.7, issued on January 20, 2010, covers compositions of DNA molecules that can be transcribed into RNAi-mediating RNA molecules, including the commonly used shRNA and miRNA-like designs. The patent also grants Allele Biotech rights to the process of introducing such DNA molecules into cells. To induce gene silencing by RNA interference, researchers often bring DNA molecules that encode interfering RNAs into cells via plasmid or viral vectors. The rights to use related technologies for the purposes of completely or partially abolishing gene functions through the mechanism of RNAi are granted to Allele Biotech.

Additional claims include methods of studying gene functions using DNA-encoded RNAi agents, or modifying gene expression profile by introducing gene expression-altering DNA molecules that will induce RNAi. The patent further protects the use of DNA-mediated RNAi in creating cell, animal models, and for curing human diseases. According to a Nov 2009 CreditSuisse analysis on the pharmaceutical market in China (and a number of other reports by JP Morgan as well as Morgan Stanley research, etc.), the drug market in China will double by 2015 and the expected revenues for major pharmaceutical companies are in the billion US dollar range each. Many large drug developers have opened research centers in China. For instance, Novartis just announced a 1.25 billion US dollar investment in Chinese R&D centers, making Shanghai one of its top three global research centers. Roche, Pfizer, JNJ, AZN, Bayer, and LLY also have substantial investments in R&D there. Some of their research teams have plans to use the virus-carried shRNA technologies in oncology and other areas, either as screening/validation tools or as therapeutic candidates. Such activities in China are now under the Allele’s recently granted RNAi patent.

The Contract Research Organization (CRO) industry in Shanghai, Suzhou, and Beijing has seen significant growth in the past few years, benefiting from R&D cost cutting in Western countries and the flow of Western-trained researchers back into China. The focus of the CRO business also shifted from chemical synthesis towards one-stop service, including functional screening and animal testing. The clarification of the RNAi patent landscape by the current granting should make the relevant CRO applications of RNAi more mature. It should also provide both the service and the customer companies with a clear route to licensing and/or collaboration.

Most major biomedical research tool and reagent companies have established themselves in the Chinese market and seen fast-growing revenues due to large funding increases to biomedical research in China. For example, Life Technologies, Promega, Millipore, Thermo Scientific, and Sigma-Aldrich all sell RNAi kits that use DNA template for expressing shRNA in mammalian cells, either by viral infection or DNA transfection. In addition, there are many local companies in China that provide reagent kits as well as services.

The Allele patent specifically states claims on reagent kits that contain shRNA-encoding DNA molecules. While being the first in China’s RNAi market, Allele Biotech manufactures in the United States and sells world-wide a set of RNAi kits in the form of retroviral or lentiviral vectors, plasmids, and linear DNA—all of which have superior design for precise shRNA production. As a matter of fact, Allele Biotech helped introduce the RNAi concept through a series of workshops in major universities in China for 3 consecutive years since 2002, at a time when most biologists had just heard of RNAi.

Allele Biotech intends to fully realize the value of this broad patent by providing opportunities to R&D centers, service providers, and reagent sellers to license at reasonable fees, so that this great technology will continue to be widely used and further developed through original research and investment. Allele Biotech intends to set licensing fees on a sliding scale in several aspects:
–the closer a drug gets to market, the higher the fees;
–the smaller the company, the lower the fees;
–the earlier the license is negotiated within an industry sector, the lower the fees.
Allele’s attorneys in China have already been contacted to start drafting plans for licensing deals and patent rights execution. “While stressing wide access, limiting the number of licenses in China is not completely out of the question. In general we want to grant all-application, non-exclusive, low-cost licenses to many companies to keep the costs affordable.” says Dr. Jiwu Wang, Allele’s CEO and the inventor of the patents. “However, if a dominant player in a particular application area is more interested in some exclusivity, a co-exclusive or conditional exclusive license may be negotiated”.

A brief background about RNAi patents:
–The original Fire and Mello patent claimed double-stranded RNAs longer than 25, eliminating use in most mammalian cells.
–The few other RNAi patents granted in the US, Europe, Japan and other markets so far mostly concern chemically synthesized siRNAs.
–The Tuschl I and II patents, with the latter being frequently mentioned in the news because it has generated hundreds of millions of dollars in licensing fees, concern siRNAs suitable for mammalian cells, but they are either chemically synthesized or processed in cell lysate.
–The Allele patent family includes 3 issued US patents on using RNA polymerase III promoter (e.g., commonly used U6 promoter) for generating RNAi. The core of the Allele patents describes making siRNAs that can be of 19 to 25 basepairs long, which are not covered by the Fire and Mello patent. Further, these transcribed siRNA are not chemically synthesized; therefore, they do not conflict with the Tuschl patents. The Allele patent in China has an even broader field of granted rights, covering any DNA-based gene silencing using double-stranded RNA as intermediates.

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Tags: Alnylam patent, Benitec, gene silencing, ISIS patents, Max Planck, miRNA, RNAi, RNAi patent, shRNA, siRNA, Tuschl patent, UMass Ownership