Biomod/2013/Aarhus/Introduction

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Apoptosis served on an origami plate

Introduction

Cancer is the leading cause of death worldwide, with metastasizing cancer types accounting for roughly 90 % of cancer deaths[1]. Traditional chemotherapeutics target all rapidly dividing cells, which often causes severe toxic effects on the organism, as well as drug resistance in many patients. The development of targeted therapeutics is therefore of great interest, as this allows higher local concentration of drugs at the desired tissue, thus lowering side effects.

The aim of our project is to design, synthesize and test a nano drug that is specifically activated by cancer cells and can induce apoptosis in these cells. The system is designed based on the DNA origami method, in which a long, single stranded plasmid DNA is folded into a large 3D structure.

The overall design consists of a two-layered origami plate to which the active components of the drug is attached (Figure 1A). To protect the active components during the transport, a dome-shaped origami is attached on top of the plate (Figure 1B). The dome is attached to the plate through a peptide sequence which is specifically recognized and cleaved by an extracellular matrix degrading enzyme, matrix metalloprotease 2 (MMP2). This enzyme is overexpressed in some types of metastasizing cancer cells, and this enables the drug to very specifically open and activate the system in close proximity to these cells.


Figure 1. Computer design of the overall design. (A) Double layered DNA origami plate with indicated attachment sites for the active components (shown in red) and peptide locks (shown in green). B: Origami dome locked in place with peptide locks.
Figure 1. Computer design of the overall design. (A) Double layered DNA origami plate with indicated attachment sites for the active components (shown in red) and peptide locks (shown in green). B: Origami dome locked in place with peptide locks.


The plate is made in two different variations that employ different stragedies to kill the cancer cell, one based on chemical modifications and one that utilizes a biological pathway in the cell. For the first approach (Figure 2A), we attached cholesterol molecules to the plate. These molecules will be incorporated into the lipid bilayer of the cell membrane and enable the plate to stick to the cell. Furthermore, the same plate was modified with photosensitizers which produce singlet oxygen when irradiated with light of specific wavelengths. Singlet oxygen is highly cytotoxic, and causes the cell to undergo apoptosis.

In the second approach (Figure 2B) we attach small internally segmented RNAs (sisiRNAs) to the plate. sisiRNA is a more stable and specific variant of small interfering RNAs which are able to induce silencing of a chosen gene through a pathway, known as RNA interference (RNAi).

As the sisiRNAs are unable to cross the cell membrane unaided, we conjugate two cell penetrating peptides (CPPs) to the 5’ ends of the two segments of the nicked passenger strand. CPPs are peptides, roughly 30 amino acids in length, which are able to cross cell membranes to the interior of the cell, where the sisiRNAs can exert their effect.


Figure 2. Strategies for for localization and killing of cancer cells. MMP2 enzymes cleave the peptide lock and release the plate from the dome. (A) The cholesterols bind the plate to the cell membrane and the photosensitizer produces singlet oxygen, or (B) MMP2 realeases the sisiRNAs from the plate, and these are transported across the cell membrane by the conjugated CPPs.
Figure 2. Strategies for for localization and killing of cancer cells. MMP2 enzymes cleave the peptide lock and release the plate from the dome. (A) The cholesterols bind the plate to the cell membrane and the photosensitizer produces singlet oxygen, or (B) MMP2 realeases the sisiRNAs from the plate, and these are transported across the cell membrane by the conjugated CPPs.

    Goals for the project

  • Design an origami plate and a dome structure
  • Fold the structures in self assembly reactions and characterize them
  • Connect the two origamis
  • Synthesize and incorporate a peptide sequence into DNA
  • Test the cleavability of the lock by MMP2
  • Synthesize a cholesterol derivative and a photosynthesizer for DNA conjugation
  • Conjugate the cholesterol and the photosensitizer to DNA strands
  • Conjugate different CPPs to the sisiRNA passenger strand and anneal the product to the guide strand
  • Test the constructs on cells, and compare the activities of the different constructs
  • Anneal a doubly CPP-modified sisiRNA and test it on cells with and without a transfection agent
  • Test the interdependency of the photosensitizer and cholesterols the induction of apoptosis
  • Attach the functional elements to the origami plate and test it on cells


SITEMAP | BIOMOD 2013 NANO CREATORS | Aarhus University

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