Allele Publishes mNeonGreen as the Brightest Monomeric Fluorescent Protein for Super-resolution Imaging

SAN DIEGO–(BUSINESS WIRE, Yahoo! Finance)–

This week scientists from Allele Biotechnology and its partner non-profit research institute, the Scintillon Institute, present their latest fluorescent protein, mNeonGreen, in the journal Nature Methods (Nature Publishing Group). In the paper, entitled “A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum,” the scientists describe the development of the brightest monomeric fluorescent protein to date.

The scientific efforts to develop this novel fluorescent protein were led by Dr. Nathan Shaner, a leader in the field of fluorescent protein engineering. Fluorescent proteins are highly valuable research tools that allow the labeling and imaging of individual proteins within a living cell, and tracking of their movements and localization in real time through a microscope. However, since the discovery of the original green fluorescent protein in 1993, imaging technology has advanced rapidly beyond the capability of most fluorescent proteins. The newly described fluorescent protein, mNeonGreen, allows researchers to take full advantage of modern super-resolution optical microscopy techniques that enable visualization of structures in living and fixed cells at much smaller scales than are possible using traditional optical microscopy. This improvement will lead to countless new insights into human health and a greater understanding of protein interactions at very small distance scales within living cells. According to Dr. Jiwu Wang, the CEO of Allele Biotechnology, “Super-resolution imaging will become the standard for publication in a short period of time, and mNeonGreen allows researchers to meet this standard while still being compatible with the equipment and methods they already use.”

Prominent researchers within the fluorescent protein field are touting mNeonGreen as a replacement for jellyfish-derived Aequorea GFP, one of the most commonly used fluorescent proteins today. According to lead researcher Dr. Nathan Shaner, “mNeonGreen can be directly substituted for other green fluorescent proteins such as EGFP without the need for any equipment changes,” making the upgrade an attractive prospect for many researchers.

Allele Biotechnology and Pharmaceuticals Inc. is a San Diego-based biotechnology company specializing in the fields of RNAi, stem cells, viral expression, camelid antibodies and fluorescent proteins. The company has co-developed a number of fluorescent proteins and other products for PALM or STORM super-resolution imaging 3D-SIM, and STED imaging. With the arrival of mNeonGreen, Allele plans to collaborate with leading imaging labs, microscope manufacturers, and journals such as Nature Methods to further promote the advantages and capabilities of the latest imaging methods. Additionally, this announcement will coincide with the launch of a new super-resolution imaging web portal and plasmid depository via collaboration with the Scintillon Institute. The Scintillon Institute is a non-profit research institute established in 2012 using seed funding from Allele Biotech. The institute’s researchers are focused on the development of biological tools to improve human health and quality of life, including applications to cancer imaging, regenerative medicine, and sustainable energy and food production.

For details about Allele’s new Superresolution FP distribution method, read our departmental and institutional usage page.

Tags: 3D, fluorescence, in vivo imaging, microscope, PALM, protein label, SIM, STED, STORM, super resolution imaging

3 Ways of Making DNA Libraries through Oligo Synthesis

Pools of DNA molecules of related but non-identical sequences are often used for selecting cDNAs that encode polypeptides with desired functions (such as in antibody screening), or DNA segments as protein binding sites (through SELEX), or DNA molecules that can catalyze reactions (DNA enzymes or deoxyribozymes), etc. The most direct way of creating DNA libraries is to introduce mixed bases during the synthesis of the oligos that will be used in creating the libraries.

1) The most commonly used method of generating degenerate oligos is to use mixed phosphoramidites (aka amidites, the building blocks of oligo synthesis) at desired positions in an oligo, e.g. using “N” to incorporate dA, dC, dG, and dT nucleotides, or “Y” for pyrimidines, “R” for purines. Mixed base oligos from most oligo suppliers are simple to order (and at no extra charge from Allele and a few other sources). During automated chemical synthesis of oligos, the synthesizer consecutively adds dT, dA, dC, or dG in the case of “N” at a pre-set ratio (e.g.25% each). This procedure does not always result in expected usage of each amidite because different amidites have different coupling efficiency, and the order of addition may also bias against amidites that are added later.

2) Using mixed bases like in method 1) leaves little control to achieve ratios of codons for specific amino acids. On the other hand, by using trimer amidites, which can be used for adding 3 nucleotides in each synthesis cycle, one can create oligos encoding selected amino acids at pre-determined percentages. However, this procedure is difficult to perform because trimer amidites are bulky and hard to couple to the elongating oligo; any moisture present during synthesis would have even more severe adverse effects than with regular amidites. Trimer oligo synthesis projects cost several thousand dollars per oligo on materials alone, and the risk is quite high that the oligos would not turn out of desired properties and qualities. For commercial users, this process has another problem—it is patented.

3) Another method for making library oligos is the so called “split-and-pool”, which is particularly suitable for having diversified amino acids embedded in otherwise common sequences like the CDRs within antibody variable regions. The latest oligo we made last month was a ~72 nt oligo with 8 locations that have pre-determined composition of amino acids, i.e. 20% Ala, 10% Gly, 12% His, etc. The procedure took us about 8 hours and we estimated the cost to be about $1,000. The subsequent sequencing results confirmed that ~70% of the clones using this oligo have desired degeneracy, compared to a similar oligo made by a bigger oligo company, at only 40%. In addition, we did not see any stop codon interruptions or major abnormalities.

DNA pools can also be generated by error-prone PCR, or more specifically with overlapping PCR using degenerate primers. The bottleneck for a library screening is how to handle big enough a number of colonies to accommodate the population, e.g. 10e10, or at least 10e8 clones are needed for finding high affinity antibodies. The second critical point is to have a robust and consistent selection readout such as fluorescence in cell sorting.

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Tags: antibody, CDR, degenerate oligos, DNA library, DNA primer, fluorescence, mixed bases, oligo, oligos, split-and-pool, trimer oligo

Do You Know How Well Your Sunscreen Works?

Skin diseases caused by sun exposure include melanoma, basal cell carcinoma, squamous cell carcinoma, photoaging, as well as sunburn and many other conditions. According to the Skin Cancer Foundation, skin cancer is the most common type of cancer in the US. The vast majority of mutations found in melanoma, according to a 2009 study published in Nature [1], are caused by UV radiation.

Currently, commercial sunscreens are composed of physical sunblocks including zinc oxide and titanium dioxide, and chemical UV (ultraviolet lights) absorbers/filters such as octinoxate for UVB and benzophenone for UVA. The compositions of commercial sunscreen products are disclosed by the manufacturer and regulated by the health product regulatory authorities such the FDA in the US. The UV absorbers/filters are organic chemicals that absorb UV lights within a very limited range of wavelength. Consequently, a combination of different chemicals is needed to achieve “broad-spectrum” protection.

Currently the FDA required test of effectiveness of UV protection measures only UVB, which means there is no way of knowing how effective a sunscreen product is against cancer-causing UVA and damaging visible lights [2]. Even though the life style changes in recent time result in more damaging light exposure such as extended sun bathing on beach or tanning in beauty saloons, etc., only 3 new sunscreen active components (and none of new chemical class) have been introduced to the US market in more than 3 decades. There seems to be a gap between the need and the effort for developing substantially improved skin protection products.

1. Pleasance, E.D., R.K. Cheetham, P.J. Stephens, D.J. McBride, S.J. Humphray, C.D. Greenman, I. Varela, M.L. Lin, G.R. Ordonez, G.R. Bignell, K. Ye, J. Alipaz, M.J. Bauer, D. Beare, A. Butler, R.J. Carter, L. Chen, A.J. Cox, S. Edkins, P.I. Kokko-Gonzales, N.A. Gormley, R.J. Grocock, C.D. Haudenschild, M.M. Hims, T. James, M. Jia, Z. Kingsbury, C. Leroy, J. Marshall, A. Menzies, L.J. Mudie, Z. Ning, T. Royce, O.B. Schulz-Trieglaff, A. Spiridou, L.A. Stebbings, L. Szajkowski, J. Teague, D. Williamson, L. Chin, M.T. Ross, P.J. Campbell, D.R. Bentley, P.A. Futreal, and M.R. Stratton, A comprehensive catalogue of somatic mutations from a human cancer genome. Nature. 463(7278): p. 191-6.
2. Botta, C., C. Di Giorgio, A.S. Sabatier, and M. De Meo, Genotoxicity of visible light (400-800 nm) and photoprotection assessment of ectoin, L-ergothioneine and mannitol and four sunscreens. J Photochem Photobiol B, 2008. 91(1): p. 24-34.

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Tags: fluorescence, light exposure, melanoma, skin cancer, skin care, spectra, spectrum, sunburn, sunscreen, UVA, UVB, visible light

Getting the most from fluorescent proteins, Part 2

On Feb 3^rd in our previous blog entry on fluorescent proteins, we discussed some basic tips on setting yourself up for success with fluorescent protein based experiments. Here are some more ideas to help boost your imaging success:

1.    Know your background.

All cells contain endogenous fluorescent materials which can confound image interpretation, especially when your fluorescent protein signal is weak.  Make sure you’re familiar with the autofluorescence of your cell type before starting your FP experiments: take some images of non- expressing control cells using the same filters and excitation wavelength as you plan to use for your FP imaging.

Keep in mind that for mammalian cells, autofluorescence is confined mainly to the blue and green regions of the visual spectrum, while in other organisms (e.g. plants, yeast, and bacteria), some cell types may contain fluorescent compounds in other regions of the spectrum. For any given species and cell type, there is likely to be a wavelength “window” with the least autofluorescence; try to choose a fluorescent protein in this wavelength range for maximum signal above background.

2.    Sometimes two (or more) FPs are better than one.

If you are having trouble obtaining sufficient fluorescent signal from a fluorescent protein fusion construct, consider adding an extra copy of the fluorescent protein to boost your brightness.  While this is not recommended unless all else has failed, for low-abundance proteins it can substantially increase the likelihood of detection.  It is possible to create a functional fusion of two or more copies of fluorescent protein in many cases, although the larger size of such a tag increases the chances of mislocalization, so proper controls and validation are essential if you use this technique.  Also, remember that it is generally difficult to use PCR to amplify tandem copies of any gene, including FPs, so restriction-based subcloning is the most reliable way to create multi-copy FP tags.

3.    The best fluorescent proteins don’t stick together!

Truly monomeric fluorescent proteins make the best fusion tags, since they don’t produce localization artifacts due to multimerization. Even weak dimers, such as EGFP and its derivatives, can cause trouble if your fusion protein is at high concentration or in a confined space like a membrane or vesicle.

Are you still using your old EGFP fusion constructs?  If so, make sure to validate your localization results by other methods, or switch to a truly monomeric FP such as mTFP1 or mWasabi.  If you prefer to keep your original constructs, note that any Aequorea GFP-derived FP can be made completely monomeric by adding the A206K mutation.

One final warning — many commercially available FPs that were initially advertised as being monomeric later turned out to be dimers!  With any new FP you try, validate your results before making your conclusions.

Tags: cell marker, cellular, cellular localization, fluorescence, fluorescent protein, FRET, GFP, imaging, mTFP1, mWasabi