Unit 2: Aptamers and RNAi / Chimeras

The main goal of NanoCAN's UNIT 2 is to develop LNA-containing aptamers with the ability to bind cancer stem cells with high affinity and specificity. Once developed, aptamers will be attached to the outside of the liposomal devices (Unit 1) or the non-liposomal nanospheres (Unit 3) where they will act as targeting devices for the particles. A third strategy includes the construction of aptamer-siRNA chimeras, i.e. of nucleic acid molecules, in which the siRNA is directly coupled to the aptamer via a linker region.

Aptamers are single-stranded DNA or RNA molecules which fold up into unique three-dimensional structures in order to strongly bind a target. Aptamers have been produced against diverse targets including small molecules, proteins, viruses, and cells, and typically dissociation constants are in the picomolar to nanomolar range.

An aptamer for a certain target is selected from a large pool of oligonucleotides by a process known as Systematic Evolution of Ligands by  Exponential Enrichment (SELEX). In SELEX an oligonucleotide library containing billions of sequences is generated by chemical synthesis. The library is then exposed to target and the species that do not bind are washed away. The binding species are amplified by PCR and the single-stranded aptamer strands are recovered from the PCR mixture. This aptamer pool enriched in target binders is then used for another round of selection, amplification and single strand isolation. Several rounds with increasing selection pressure are performed until strong target binders are obtained.

Locked Nucleic Acid (LNA) is a nucleotide modification in which the furanose ring of the nucleotide is locked. This provides several favorable features for oligonucleotides containing LNA. One advantage is increased stability against nucleases which is important for aptamers since unmodified DNA and RNA have short serum half-lives. Several existing aptamers have been modified with LNA to improve pharmacokinetics; however, this is a trial and error approach since it is not possible to predict whether modification at a particular location will result in improved aptamer performance. One of the goals of this module is to introduce the LNA modification in the selection of aptamers i.e. during SELEX.

selection round

To develop aptamers against entire cells a procedure known as cell-SELEX is employed. In cell-SELEX live cells are used as targets during the selection. Cell-SELEX is ideal to achieve aptamers against cell surface markers which are otherwise hard to use as targets. For this purpose, a cell line overexpressing the protein of interest is used for positive selection. After positive selection a negative selection is performed where the aptamer pool is exposed to the same cell line, in which the protein of interest is not expressed. These cell lines, only differing in the presence / absence of the respective target protein are engineered in Unit 4. By finally collecting the species that bind in positive selction and does not in the negative selection, aptamers for the cell surface marker are developed. 

In addition to functioning as a targeting device for particles containing siRNA, aptamers can also be directly linked to siRNA. These aptamer-siRNA chimeras are simpler than the particle counterparts and smaller in size, which may offer some advantages for addressing metastases without extensive vasularization. However, because the chimeras are not protected by lipid layers or other structures, in vivo stability needs to be ascertained. To modulate the properties of the nucleic acid chimeras UNIT 2 employs different other strategies, such as incorporation of UNAs (Unlocked Nucleic Acids), in addition to LNA technology.

 

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