Nucleic acid aptamer selection against supercharged GFP variant to enhance cancer targeted drug delivery

Alice Zhang

September 4, 2012

Abstract

The intracellular delivery of macromolecule cargo into mammalian cells as therapeutic agents is often restricted by the inability of certain macromolecules to penetrate cell membranes unaided [1].  However, green fluorescent protein (GFP) variants with extremely high positive net charges up to +48 [2] can enter mammalian cells successfully through endocytosis by interacting with sulfated peptidoglycans of the surface of the cells. These “supercharged” GFP variants has been shown to deliver nucleic acids effectively across numerous mammalian cell lines, including those that are unreceptive to standard transfection methods [3].

Targeted cancer cell therapies focus on delivering drugs to specific tumor sites without harming normal cells during treatment. Aptamers are nucleic acid ligands that bind to protein targets with high specificity and high affinity. To improve the efficacy of drug delivery into cancer cells, an aptamer that binds to the GFP variant can function as an anchor to a guidance molecule and cytotoxic payload, creating a complex targeted drug delivery system.

Specific Aim 1: Selection of RNA aptamers against +22 GFP variant

An aptamer for the GFP variant will act as a fundamental component for a targeted drug delivery system. The aptamer will aid in anchoring the GFP variant to a cytotoxic drug and a guidance module for selective cell recognition. Supercharged GFP provides a shuttle for the assembly to enter the cell and deliver its payload due to the protein’s potent capability to penetrate mammalian cells.

Figure 1. An example of an assembly of the supercharged GFP, aptamer, and cytotoxic payload to create a drug delivery system that will deliver the payload into the cell. A guidance module can also possibly be attached to the assembly for selective cell recognition.

Ideally, the entire complex will result in cell death of selective cancer cells targeted by the guidance module. An aptamer against supercharged GFP will create a novel drug delivery system that can help deliver medications that cannot effectively enter cells or might be sensitive to external interference. Because the protein potently delivers cargo internally, these obstacles can be avoided and potentially require lower drug doses for effectiveness.

+22 GFP can be obtained from Christien Kluwe in the Ellington Lab. 

References:

1.      Cronican, J.J., Beier, K.T., Davis, T.N., Tseng, J., Li, W., Thompson, D.B., Shih, A.F., May, E.M., Cepko,  C.L., Kung, A.L., Zhou, Q., Liu, D.R. (2011). “A class of human proteins that deliver functional proteins into mammalian cells in vitro and in vivo.” Cell. (18)7: 833-38.

2.      Cronican, J.J., Thompson, D.B., Beier, K.T., McNaughton, B.R., Cepko, C.L., Liu, D.R. (2010). “Potent delivery of functional proteins into mammalian cells in vitro and in vivo using supercharged protein.” ACS Chemical Biology. (5)8: 747-52.

3.      McNaughton, B.R., Cronican, J.J., Thompson, D.B., Liu, D.R. (2009). “Mammalian cell penetration, siRNA transfection, and DNA transfection by supercharged proteins.” PNAS. (106)15: 6111-16. 

Full Proposal 
Progress Report 1
Progress Report 2
Final Manuscript