Photo-Morpholinos incorporate a photo-sensitive subunit cleaved by 365 nm light. Photo-cleavable Morpholinos (Photo-Morpholinos) retain the specificity and efficacy of an unmodified Morpholino oligo but have a photo-sensitive subunit near the middle of the oligo that is cleaved by 365 nm light. The shorter fragments generated from the cleavage have little binding affinity to their target . Gene Tools will design an optimal Photo-Morpholino for you whether working on translation knockdowns, splice targets, or RNA targets like miRNAs. Photo-Morpholinos give you temporal control of expression, a virtual ON or OFF switch. As shown in the figure above , the photo-sensitive subunit takes up roughly the same space in the molecule as an unmodified Morpholino subunit and has little impact on Morpholino oligo binding. Photo-Morpholinos are suitable for use in transparent systems as 365 nm light is required to break the photo-subunit.
Photo-Morpholinos can be used to target and bind strongly to the same RNA targets as an unmodified Morpholino oligo but in addition Photo-Morpholinos can be cleaved into fragments with light to restore the normal activity of the RNA target whenever you wish.
An unmodified antisense Morpholino oligo can be bound to a sense Photo-Morpholino and the RNA target will maintain normal expression until the unmodified Morpholino oligo is released by cleaving the bound Photo-Morpholino with light. Then the unmodified antisense Morpholino will be released to bind to its RNA target and alter gene expression.
The following sections describe in more detail Photo-Morpholino design, pricing and modifications, uses, experimental examples, control oligos, stock and storage solutions, and frequently asked questions.
Depending on the strategy chosen, the optimal Photo-Morpholino properties will be based either on binding to mRNA (antisense Photo-Morpholino strategy) or binding to another Morpholino oligo (sense Photo-Morpholino strategy). Gene Tools typically designs a Photo-Morpholino to bind strongly with complementary target sequence and positions the photo-linker at or near the middle of the oligo sequence so both of the cleavage fragments bind so weakly with complementary sequence that they dissociate. The photo-sensitive subunit closely matches the backbone spacing of an unmodified Morpholino subunit so that the Morpholino strongly binds its target until cleaved. In many cases the length of a Photo-Morpholino may be shorter that the typical 25 base antisense Morpholino to optimize the binding energies of full length oligo and fragments for both efficient binding and post-cleavage release.
Pricing and modifications
Photo-Morpholinos cost $700 for 300 nanomoles and $1500 for 1000 nanomoles with lengths up to 25 bases (including the photo-cleavable subunit as one base). Photo-cleavable Vivo-Morpholinos are not available.
Photo-Morpholinos can be used to assess all of the same processes targeted with unmodified Morpholino oligos but with additional temporal control. To block translation we have two strategies:
- an antisense Photo-Morpholino strategy in which your antisense Photo-Morpholino oligo is actively knocking down your gene until cleaved with 365 nm light to allow expression of your gene;
- a sense Photo-Morpholino strategy in which your antisense Morpholino is paired with a sense Photo-Morpholino and gene expression continues until the sense photo-Morpholino is cleaved with 365 nm light and releases the antisense Morpholino to knockdown your gene.
When modifying splicing, on illumination the antisense Photo-Morpholino strategy restores normal splicing and the sense Photo-Morpholino strategy frees an antisense Morpholino to alter pre-mRNA splicing. If micro-RNA activity is targeted, the antisense Photo-Morpholino strategy suppresses the miRNA activity until the oligo is cleaved, while the sense Photo-Morpholino strategy allows normal miRNA activity until the Photo-Morpholino is cleaved and the antisense oligo binds the miRNA or its mRNA target.
It is important to use a light source with 365 nm light to cleave Photo-Morpholinos and a narrow-bandwidth light source is optimal to avoid damage by shorter UV wavelengths. For example, unfiltered broad-band trans-illuminators will likely kill embryos at an exposure time that is insufficient to cleave the Photo-Morpholinos. Instead, you should use a narrow-band 365 nm light source such as an appropriately equipped microscope, a laser, or our Gene Tools lightbox. Photo-Morpholinos are cleaved in up to 50 injected embryos at a time with a 5 minute exposure in our light box. Choosing a laser or microscope-mounted source may provide very different results. The light system you choose should provide reproducible exposure time, distance from the light source, spectrum and radiant power. Start by assessing the toxicity to embryos of a light source in a time-course study without Photo-Morpholinos. Find an exposure time that is toxic and choose an exposure time that is not toxic for initial experiments with Photo-Morpholinos. For many microscope mounted light sources, one minute may be sufficient to cleave the Photo-Morpholino delivered into a single embryo.
Method of use in embryos
Do you have an embryonic lethal phenotype with your current antisense Morpholino oligo? Now you can use a complementary sense Photo-Morpholino paired with your antisense Morpholino to block the antisense oligo’s activity until the embryonic lethal period is past, then release your antisense Morpholino later in development with 365 nm light.
Would you like to knock down gene expression in only a small region of the embryo? Now you can use a complementary sense Photo-Morpholino paired with your antisense Morpholino to block the antisense oligo’s activity except where you expose the embryo to focused 365 nm light.
Would you like to have a Morpholino oligo work for a specific amount of time post-fertilization? Inject embryos with an antisense Photo-Morpholino and cleave it at a particular point in development with 365 nm light to ”turn off” the Morpholino oligo activity.
Photo-Morpholinos are handled differently depending on whether you have chosen the light-on or light-off strategy. You might use the oligo alone or in combination with a complementary oligo.
For the antisense Photo-Morpholino strategy, a target should first be validated with a standard antisense Morpholino oligo. The Photo-Morpholino shares the same target as the original antisense oligo. This Photo-Morpholino can be delivered much like an unmodified Morpholino though a larger dose might be needed because the replacement of a base with a photo-cleavable linker may reduce the oligo efficacy (activity) slightly. For example, a Photo-Morpholino may require a 6 ng injection to get the same knockdown as 5 ng of an unmodified oligo. Treatment of the embryo(s) with the appropriate narrow-band 365 nm light will inactivate the Photo-Morpholino.
For the sense Photo-Morpholino strategy, your antisense Morpholino is paired with a complementary sense Photo-Morpholino. The concentration of your stock solutions should be checked prior to pairing and the molar ratio of sense Photo-Morpholino to antisense Morpholino should be tested in a range from 1.3 : 1.0 to 1.0 : 1.0 The sense Photo-Morpholino should not be paired at less than 1:1 with the antisense Morpholino and the optimum ratio of sense Photo-Morpholino to antisense Morpholino will likely be near 1.1 : 1.0. Your oligo stock concentrations should be re-checked by spectroscopy prior to each pairing, particularly if the oligo stocks have been stored for a while. Once paired, the duplex oligos can be delivered much like an antisense oligo. In the paired form, the total amount of Morpholino delivered to yield the same effect after light-cleavage of the paired Photo-Morpholino will be roughly twice that of a bare oligo. For example, a bare Morpholino may yield good results from a 5 ng injection. To yield the same results after photo-cleavage, the duplex will include 5 ng of each strand for a total of roughly 10 ng per injection. After injection, treatment of the embryo(s) with the appropriate narrow-band 365 nm light will inactivate the Photo-Morpholino and release the antisense oligo. Analyzing fluorescence of an average of more than 100 embryos per treatment, the gal4-UAS target was assessed for both sense Photo-Morpholino and antisense Photo-Morpholino (Fig 3 Sec A, columns 5 and 6) strategies with controls (Fig 3 Sec A, columns 1 and 2). GFP expression was directly assessed with the antisense Photo-Morpholino strategy ). The oligos used in this study are available as ready-made controls for assessment of your equipment with the appropriate transgenic line (See Tallafuss et al.* for details).
Control oligo sequences (all have been tested in an appropriate zebrafish strain)
- GFP MO ACAGCTCCTCGCCCTTGCTCACCAT
- GFP AS photo ACAGCTCCTCaaPaaTGCTCACCAT
Photo-MO shares GFP MO target for antisense Photo-Morpholino strategy
- gal4 MO GTTCGATAGAAGACAGTAGCTTCAT
- gal4 AS photo GTTCGATAGAtaPatGTAGCTTCAT
Photo-MO shares gal4 MO target for antisense Photo-Morpholino strategy
- gal4 S photo ATGAAGCTACaaPaaTCTATCGAAC
Photo-MO pairs with gal4 MO for sense Photo-Morpholino strategy
Storage and stock solutions
Photo-Morpholinos will arrive freeze-dried in an amber vial. When ready for first use, dissolve the freeze-dried Photo-Morpholino with sterile water to a concentration no greater than 1 mM. For 300 nanomoles oligo, dissolve with 300 microliters of water to produce a 1mM stock. Vortex well and determine the concentration of your oligo using our acidic UV spectroscopy protocol, available online. Store the unused Photo-Morpholino tightly sealed in the provided amber vial at room temperature in a dark room or drawer. Prior to each use, we recommend heating the Photo-Morpholino stock for 30 min in a 65°C water bath or heat block and vortexing.
Avoid using dyes that have absorbance at 365 nm such as phenol red in stocks or injection solutions. These dyes will tend to shade the photo-labile subunit in the Photo-Morpholino and inhibit cleavage. Note that expression of fluorescent proteins such as Kaede protein in your embryos may have a similar shading effect.
Notes on Morpholino storage are here.
A lightbox is offered by Gene Tools for cleaving Photo-Morpholinos. The lightbox includes a narrow-band 365 nm LED shining though a lens and diffuser system, a digital timer, an opaque cap and safety interlock and a cooling system comprised of a heatsink and blower, all housed in an anodized aluminum enclosure. The lightbox will cleave Photo-Morpholino oligos within transparent biological samples, such as zebrafish embryos, by illuminating the bottom of a flat-bottomed glass sample tube containing the biological samples. The LED and sample tube are enclosed within an opaque chamber during the light exposure, with an optical interlock turning off the LED if the opaque cap is removed during a light exposure. The narrow-band LED minimizes the sample’s exposure to higher-energy photons, which can damage biological samples. The light intensity is calibrated against a standard lightbox at the factory so the light output intensities of different lightboxes are similar.
Several transgenic zebrafish and Xenopus lines have been developed using gal4-UAS (Upstream Activation Sequence) regulation of genes along with a gal4-UAS regulated reporter gene, typically green fluorescent protein (GFP). Such lines are readily available from zebrafish and Xenopus stock centers. Using a transgenic zebrafish line expressing GFP under the control of gal4-UAS, Tallafuss et al. successfully demonstrated both the antisense Photo-Morpholino and sense Photo-Morpholino strategies (see figure 3 below).
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