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Paul Gruenbacher

Notebook

Will be using links to google drive for files.

Friday May 10

Submitted cadnano design of 66 helices, non-hollow: 66helicev1. Will note that midline there are scaffold crossovers that do not have staples nearby, had incorrectly assumed that staple crossovers must avoid scaffold crossovers. Design approximately 40 nm long and less than 22 nm in diameter.

Sunday May 12

New version edited by Dr. Castro.66 helices, 2-layered, hollow: 66helicev3. Will note that diameter remains the same, is approximately 44 nm long now. Should not be an issue

'Literature review': It seems well accepted that crossing the nucleolus membrane is the greatest challenge facing gene therapy. The calcium phosphate protocol as it stands only takes us to the cytosol, so the origami must be designed to be able to enter the nucleus. A paper in 1988 used a range of gold particles to measure the size able to enter through the nuclear pore complex [1] and found that the limiting size was 26 nm. This suprised some people as some viral proteins are larger than this, including Hepatitis nucleocapsids. A later paper in 2002 used gold particles with protein complexes to measure a larger size of 32-36nm [2]. We would need to add targeting sequences or nuclear localization signals to the origami. For NLS peptide conjugation to the DNA staples, one would need DNA staples with a free alkylamino group of a thymine. see A_single_nuclear_localization_signal_peptide_is_sufficient_to_carry_DNA_to_the_cell_nucleus

However, peptide conjugation and staple modification is making things more complicated than what is desired or allowable. Perhaps nuclear targeting sequences would be better, though a recent paper in 2011 downplays their importance [3].

Monday May 13th

Ordered staples for version 3 origami design edited by Castro. The templated had now been decreased to the smaller 7202 template strand. Will be leaving for Puerto Rico while waiting for staples to come in, will be back on the 23rd.

Corresponded with Dr. James Adair at PennU and Dr. Robert 'Bob' Lee of OSU pharma.

Literature Review: In general, some better indications that the origami can be functionalized with cationic peptides to facilitate transport across the nuclear pore complex.

References

1. Dworetzky SI, Lanford RE, and Feldherr CM. The effects of variations in the number and sequence of targeting signals on nuclear uptake. J Cell Biol. 1988 Oct;107(4):1279-87. DOI:10.1083/jcb.107.4.1279 |

2. pmid=PMC65638

3. van Gaal EV, Oosting RS, van Eijk R, Bakowska M, Feyen D, Kok RJ, Hennink WE, Crommelin DJ, and Mastrobattista E. DNA nuclear targeting sequences for non-viral gene delivery. Pharm Res. 2011 Jul;28(7):1707-22. DOI:10.1007/s11095-011-0407-8 |