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RESEARCH PROPOSAL:
Cellular targeting through gold nanoparticles functionalized with bivalent RNA aptamers
WAYS TO ASSAY GNPs:
“Simple and Rapid Colorimetric Biosensors Based on DNA Aptamer and Noncrosslinking Gold Nanoparticle Aggregation” by Zhao et al. (2007) ChemBioChem 8: 727-731.
Advantages of GNPs:

  • (1) simplicity (2) no need for complicated or expensive analytical equipment needed to assay (3) extremely high extinction coefficients (≥ 1000 x greater than organic dyes).
  • Oligonucleotide-modified GNPs crosslinked by a DNA aptamer sequence form aggregates. Addition of desired target causes dissociation of AuNP aggregates.
  • Hybridization can be used to attach GNP to aptamer and radiolabeled aptamer can be used to determine GNP-aptamer attachment.
  • Challenge: [MgCl2] impacts DNA aptamer stability.
  • Transmission electron microscopy can be used as a second diagnostic (TEM).
  • Question: dsDNA-GNP has a lower affinity than ssDNA-GNP

APPLICATIONS OF GNPs:
“Gold nanoparticle-assisted delivery of small, highly structured RNA into the nuclei of human cells” by Ryou et al. (2011) Biochemical and Biophysical Research Communication 416: 178-183.

  • Advantages of aptamers: small in size, nontoxic, minimally or not immunogenic, and highly specific to target molecules.
  • Delivery to living systems is one of the greatest challenges of aptamers (i.e. viral vector-based systems elicit a strong immune response).
  • Advantages to GNPs: easy to modify and not toxic to mammalian cells.
  • Obstacle of GNP gene delivery system: construction is both time-consuming and inconvenient.
  • MTT assay for cell viability: test whether GNPs are toxic to cells. Additionally, if aptamer elicits apoptotic response, can test for efficacy of aptamer and GNP delivery system.
  • Fluorescence microscopy can be used to detect RNA aptamer delivery into cells by labeling RNA aptamer with fluorophore (i.e. cy3).
  • Chose RNA aptamer that had previously shown efficiency and specificity to β-catenin.
  • PAGE used as a diagnostic tool to estimate number of aptamers bound to each GNP.
  • GNPs are functionalized with thiolated oligonucleotide of anti-RNA aptamer.
  • Used ssDNA functionalized GNP. Question: Can we design a specific GNP that is better suited for RNA?

ENGINEERING GNPs:
“Competitive Protection of Aptamer-Functionalized Gold Nanoparticles by Controlling the DNA Assembly” by Li et al. (2011) Analytical Chemistry 83: 6464-6467.

  • DNA assembly method to protect aptamer-functionalized GNP from non-desirable binding to interfering molecules using a designed DNA that reduced cross reaction by competitive hybridization to aptamer and overcoming nonspecific adsorption by restricted access of GNP surface.
  • Applied to enhance signal for visual detection of minute amounts of protein (thrombin).
  • One key problem with GNPs: Trying to reduce non-specific interactions. Used oligo-DNA to reduce non-specific binding.