User:Fangdi Sun/Notebook/WF Purple: Applying XNA

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Project Description/Abstract

Idea/Topic

Engineer a novel genetic polymer that is both nuclease resistant and optimized for intracellular delivery in order to target vascular endothelial growth factor (VEGF).


Background/Overview

A number of synthetic genetic polymers have been shown to be capable of both heredity and evolution through the use of polymerase evolution and design. These polymers are typically resistant to nucleases, making them attractive for intracellular delivery. Short aptamers of these synthetic genetic polymers can theoretically be used to target a variety of molecular structures. These aptamers could be developed via a SELEX-type procedure.

For our project we seek to develop a synthetic genetic aptamer that targets VEGF and prevents VEGF binding to VEGF receptor.


Details

Activity Assay: The VEGF gene will be encoded next to a fluorophore to express a chimeric protein containing VEGF linked to the fluorophore. The same will be done with VEGF receptor, with a different fluorophore. (Perhaps this isn't necessary if there are already fluorescent tags for VEGF and VEGF receptor?) FACS will be able to distinguish between VEGF that can bind its receptor and VEGF that does not.

SELEX plan: Analogous to Module 1, except instead of heme, we will use VEGF. There will also be another selection within one cycle. VEGF proteins bound with the putative XNA aptamer will be put through a column with VEGF receptors. The XNA sequences of interest will disable the VEGF from binding. Evolution of the XNA will be made possible by polymerase and RT identified by site-directed mutagenesis.


References

[1] Pinheiro VB, Taylor AI, Cozens C, Abramov M, Renders M, Zhang S, Chaput JC, Wengel J, Peak-Chew SY, McLaughlin SH, Herdewijn P, and Holliger P. Synthetic genetic polymers capable of heredity and evolution. Science 2012; 336(6079): 341-344.

DNA and RNA have been viewed as unique polymers that propogate and store information. This study showed that the ribofuranose ring can be replaced with synthetic moieties termed “xeno-nucleic acids” (XNAs). They developed polymerases and reverse transcriptases to demonstrate heredity for XNAs. They also showed that XNAs can fold and possess binding properties. Through this SELEX-like protocol, they showed that XNAs have the capacity to evolve. XNAs therefore increase the sequence space and introduce greater chemical diversity for aptamers.

[2] Nielsen PE. DNA analogues with nonphosphodiester backbones. "Annu Rev Biophys Biomol Struct" 1995; 24: 167-83.

This review article looks at synthetic DNA analogs that incorporate nonphosphodiester backbones. Due to their modifications, these synthetic polymers have the attractive property that they are generally biologically stable (nuclease-resistant). Nonetheless, these analogs are still capable of forming stable hybrids with complementary DNA or RNA molecules, suggesting their feasibility as "anti-sense" and "anti-gene" drugs. Nielsen claims that the utility of these synthetic genetic polymers is in their potential for use in RNA interference (RNAi) rather than for their structural novelty.

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