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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.

Rosenecker's lab synthesized a tetramer of the PKKKRKV NLSV404 peptide sequnece spaced by glycine residues. (may want to consider finding a lab that has a Applied Biosystems Automatic Synthesizer) [4]. A strong paper, with luciferase transfection assay, in situ flourescent hybridization to detect plasmid dna, and a nuclear import assay with labelled BSA-BODIPY to be transported by the peptide. The SV40 NLS sequence they used is the minimal sequence observed on the T-antigen of SV40 necessary. HeLa cells showed 58.9% transfection within 2 hours, much faster than with the dendrimer alone or with a mutant peptide control. Analysis shows quicker localization of the peptide-plasmid in the nucleus, indicating that the peptides probably helped transport the plasmid across the pore if not through the cytosol as well. Early cell transfection also indicates a lessened need for mitosis to occur. Also did a nuclear transport inhibition protocol by Sebestyen [5], which had also showed linking of peptides to plasmids by the cyclo-propayrroloindole cross-linker. Remember: Karyopherins are the proteins found to accumulate in the nucleus.

Dr. Davis at Case Western also showed that DNA was binded the nucleolin receptor on the cell membrane, and may indicate a new cellular uptake mechanism with increased transfection efficiency. The DNA nanoparticles were formed by PEGlyated polylysine. We probably don't want to pursue this mechanism, but the effectiveness of the internalization and transport of the nanoparticles is impressive [6].

Dr. Dean is one of the first to have described the nuclear targeting sequence of the SV40 DNA itself. In his review [7], Dean describes several methods to nucleari import of nonviral vectors. The classic SV40 NLS PKKKRKV. He also describes the 72 bp sequence that is shown to have binding sites for a number of mammalian transcription factors that are usually brought into the nucleus for RNA transcription. Reich has also idenifitied NFkappaB binding sites that show enhanced expression as well. Using the DNA sequences have an obvious advantage to us in that it eliminates the need for the peptides, but I'm worried that the origami will not allow for the normal binding of the factors even if the binding sequence is spaced and free. Also, we also need to take pore size into account, peptides keep the origami size efficiently small. If we use the peptides, it is important we use a mutant peptide as a negative control.

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 |
4. Ritter W, Plank C, Lausier J, Rudolph C, Zink D, Reinhardt D, and Rosenecker J. A novel transfecting peptide comprising a tetrameric nuclear localization sequence. J Mol Med (Berl). 2003 Nov;81(11):708-17. DOI:10.1007/s00109-003-0483-2 |
5. Sebestyén MG, Ludtke JJ, Bassik MC, Zhang G, Budker V, Lukhtanov EA, Hagstrom JE, and Wolff JA. DNA vector chemistry: the covalent attachment of signal peptides to plasmid DNA. Nat Biotechnol. 1998 Jan;16(1):80-5. DOI:10.1038/nbt0198-80 |
6. Chen X, Kube DM, Cooper MJ, and Davis PB. Cell surface nucleolin serves as receptor for DNA nanoparticles composed of pegylated polylysine and DNA. Mol Ther. 2008 Feb;16(2):333-42. DOI:10.1038/sj.mt.6300365 |