About Our Products: Onco-Morpholinos and Endo-Porter

We are pleased to offer our customers a pre-designed selection of high-potency, precision-targeting antisense products and delivery reagents.  To better understand our products, this page provides and links to general information about our Onco-Morpholino products - their structure and properties, data from demonstration studies of their potency, how they are designed, and procedures for their handling and use.  For additional information about the unconjugated Morpholino, see the wiki page and our manufacturer’s website at Gene Tools LLC.  A description of our delivery reagent Endo-Porter will follow, and more information can be found here.

 


What is an Onco-Morpholino?

Our Morpholino oligos are chains of genetic bases typically 21-25 Morpholino subunits in length.  Each subunit is comprised of a nucleic acid base, a morpholine ring and a non-ionic phosphorodiamidate inter-subunit linkage.  Morpholino oligos bind to complementary RNA and get in the way of processes; they can knock down gene expression, modify RNA splicing or inhibit miRNA activity and maturation.  Morpholinos sterically block RNA activity via an RNAse H-independent mechanism.  With their requirement for greater complementarity with their target RNAs, Morpholinos are free of the widespread off-target expression modulation predicted and observed for other antisense technologies that rely on RISC or RNase-H activity.  For a lengthy discussion on this latter topic, see the section on our website that talks about Antisense Oligos.

Our manufacturer Gene Tools LLC has useful topic pages that discuss the remarkable properties of Morpholinos, including:

Onco-Tools LLC sells Onco-Morpholinos, a delivery-enhanced version of the standard Morpholino molecule with a sequence targeted against RNA splice isoforms of cancer-associated genes.  Our Morpholino molecule contains an alkyl chain conjugated to the 3’ end.  This alkyl chain functions as a “lipid anchor” to substantially increase Morpholino binding to cell membranes.  Through cellular uptake via endocytosis, Onco-Morpholinos will then accumulate into vesicles of the endo-lysosomal system.  When combined with an acidic vesicle-permeabilizing release agent, such as Endo-Porter, Onco-Morpholinos escape from lysosomes and enter the cytosol where they can bind to their mRNA target and knock down expression.  Their size and chemical properties also allow them to freely enter the cell nucleus, where they can bind to a complementary pre-mRNA target and modulate splicing.

Whiteboard1_delivery_2018_0.jpg


Enhanced Morpholino Delivery

Improvements in Morpholino delivery can be validated using the Kole functional splice-correction assay [Kang et al., Biochemistry 1998].  Splice correction offers a positive readout of Morpholino activity, reducing misinterpretations of experimental outcomes caused by non-specific targeting effects. The ON 705 HeLa cell line stably expresses a firefly Luciferase gene containing a thalassemic intron of the human beta-globin gene.  The inserted intron has a point mutation which creates a 5’ splice site and activates a cryptic 3’ splice site upstream.  Aberrant splicing retains the intron and generates an early stop codon, preventing translation of the full Luciferase transcript.   Morpholino treatment corrects the engineered splicing defect in the pre-mRNA to generate the full Luciferase protein translational frame and increase functional protein.  Thus, light units resulting from Luciferase chemiluminescent reaction provides a quantitative readout of splice-correction generated by the Morpholino.

ON 705 HeLa cell treatment with a splice-modifying Onco-Morpholino in combined treatment with Endo-Porter increases Luciferase activity substantially compared to unconjugated Morpholino (Image 1, see our Endo-Porter protocol below).  In the absence of a release agent, 100 nM standard Morpholino and Onco-Morpholino both generated Luciferase activity equivalent to no treatment (background).  When co-treated with 18 µM Endo-Porter release agent, the Onco-Morpholino produced more than 10-fold increase in activity compared to standard Morpholinos and an astonishing 50-fold increase compared to treatment without a release agent.  This result demonstrates the substantial potency from antisense activity – and by extension cellular delivery – afforded by Onco-Morpholinos when combined with a release agent for cell culture applications.

 


How we design Onco-Morpholinos

An Onco-Morpholino is also a classification of Morpholino based on its relationship to cancer genes.  Gene products, such as RNA and protein, from cancer-associated genes are expressed in abnormal amounts or uniquely present in tumors and often serve a role in facilitating the progression of cancer.  We begin with an initial product catalog of Onco-Morpholinos that target 500 cancer-associated genes chosen as a starting point to represent the several so-called ‘hallmarks of cancer’ proposed by Hanahan and Weinberg [“Hallmarks of cancer: the next generation” 2011. Cell 144, 646-674]. You can search for your genes of interest by browsing cancer hallmarks, or simply alphabetically.  The majority of genes are, as expected, protein coding but we have also included cancer-related non-coding RNAs (microRNAs, lncRNAs etc). 

As steric blocking oligos, Morpholinos can be used not only to block translation but also to alter mRNA splicing, to bind miRNAs or to block binding of miRNA or regulatory proteins to RNA targets.  We designate two product types – Translation Blocker and Splice Modifier – which reflect each Onco-Morpholino’s design strategy.  Translation blockers target the 5’ untranslated region (UTR) up to the first ~25 nucleotides past a start codon of an RNA transcript to suppress RNA translation to protein.  Most of our initial offering of Translation Blocker Onco-Morpholinos were designed so that a single Onco-Morpholino will target as many splice isoforms of a cancer-associated gene as possible through selection of a consensus start codon amongst known splice isoforms.  In addition, we label oligos that target miRNAs as “Other” to reflect the variety of mechanisms affected when non-coding RNAs are blocked.

A splice modifier Onco-Morpholino targets sequence near the splice junctions of a pre-mRNA.  This allows researchers to delete targeted exons and analyze specific splice-forms of a cancer-associated gene that has multiple splice variants.  Onco-Morpholino oligos can target a pri-miRNA or a pre-miRNA to inhibit miRNA maturation or target a miRNA or one of its targets to inhibit miRNA activity.  In addition, binding of a splice-regulatory protein can be prevented by protecting its binding site with an oligo.  Importantly, over 94% of human genes are alternatively spliced and often splice isoforms have very different and even opposing attributes: e.g. pro- or anti-apoptotic; pro- or anti-angiogenic etc. Therefore, where splice isoforms have been shown to be functionally important in the literature, we will design splice-switching Onco-Morpholinos that bind across 5’ or 3’ splice sites causing exons to be skipped.  In some cases, the splice-switching Onco-Morpholinos will favor the expression of a particular splice isoform (this applies to splice isoforms that are due to switching ‘cassette exons’ in or out).

Whiteboard2_2018_1.gif

Our Onco-Morpholinos are pre-designed sequences in collaboration with our manufacturer, Gene Tools.  To maximize efficacy & specificity, RNA targeting was assessed using Gene Tools proprietary design program.  We evaluate Morpholino sequence parameters including sequence length, predicted Tm, self-complementarity, %G and %GC content to select optimized sequences.  miRNA target selection includes an additional step using miRBase.  In some instances, we may wish to offer an Onco-Morpholino with properties outside the normal range of our desired parameters because of sequence-dependent limitations in the RNA code.  In this case, you will find Product Notes on the individual product or gene page that describe the product’s non-optimal parameters, and we will continue to design and offer new Morpholino sequences to improve the properties of available products that target cancer-associated genes.

We want to emphasize the importance of knowing your target RNA through experimental identification of the RNA sequence.  On our gene and product pages, we provide a portion of the RNA target sequence that we used for product design and the NCBI Accession ID from which the target sequence originates.  Comparison of our target sequence with your experimentally-determined RNA sequences will help ensure our product is a good fit for your application.  We will provide the exact Onco-Morpholino sequence, molecular weight and molar absorptivity on an Oligo Spec sheet delivered with the product.

Onco-Morpholino product will be shipped to you dry and sterile in a small glass vial.  We recommend re-suspension in sterile, untreated water to a concentration of 0.5 mMolar (200 µL to 100 nmol).  More recommendations for product handling can be downloaded here PDF LINK or accessed at our manufacturer's website.

 


Endo-Porter:  Cell delivery reagent

Endo-Porter is a novel peptide that utilizes the endo-lysosomal system to facilitate cellular entry of a variety of substances – including peptides, small proteins, and Onco-Morpholinos.  Endo-Porter requires no interaction with the cargo and can deliver high concentrations of cargos exceeding 70kD into both adherent and non-adherent cells.  Over 100 papers have been published using Endo-Porter, and you can read about the delivery mechanism, properties, protocols, and citations at our manufacturer’s website.

We offer Endo-Porter as 1.0 mL of 1 mMolar in aqueous solution with 10% polyethylene glycol 1500 MW.  This amount is enough to provide high delivery efficiency in 50 mL of cell culture medium.

>Click here to order Endo-Porter<

The protocol is simple: squirt and swirl. Our recommended delivery procedure:

  1. Replace spent culture medium with fresh medium (preferably 2-10% serum).
  2. Add the desired cargo and swirl to mix.
  3. Add 2-20 µL of 1.0 mM Endo-Porter for every 1 mL of culture medium & cargo and immediately swirl to mix.

In many cases, long-term (72 hour) and short-term (24 hour) delivery is effective and non-toxic up to 20 µM (20 µL of 1mMolar Endo-Porter / mL of culture medium); however, cell lines vary in their response to Endo-Porter and it is prudent to start experiments with a new cell line by doing a ranging study for efficacy and toxicity at 2, 5, 10 and 20 µL/mL Endo-Porter.  Check the cells at 72 hours for signs of toxicity, then use the highest concentration that the cells tolerate for subsequent delivery procedures. Unlike most delivery solutions, 10% serum concentrations yield effective results. However, reducing the serum concentration down to 2% or 1% will markedly improve Endo-Porter activity (Image 2). 

Additional cargo and Endo-Porter can be added with each media replacement if desired.  For delivery of Onco-Morpholino oligos, we suggest that you start with 1 µM oligo final concentration in the culture medium and then adjust concentrations as needed to achieve effective and specific gene knockdown.  For adherent cells in a 96-well plate format, we found that pre-mixing Endo-Porter by pipetting into the Morpholino/media mixture before adding the mixture to the cell well produces more consistent results; however, delivery effectiveness may be significantly reduced.