Methods and compositions for controlling gene expression and treating cancer
View Patent ↗Disclosed are peptides which specifically bind to preselected pre-miRNA or pri-miRNA targets, methods of down-regulating EMT in a cell using such peptides, and methods of preparing such peptides. Also provided are therapeutic compositions comprising peptides that specifically bind a preselected cancer marker which is a peptide or which is a pre-miRNA or a pri-miRNA and methods of treatment using the same.
1. A method of screening for a peptide which binds a preselected pre-miRNA and/or pri-miRNA, with at least nanomolar affinity, and which is at least partially protease-resistant, comprising the steps of:
(a) preparing an mRNA library by transcribing a plurality of mRNAs from a plurality of synthetic DNAs with random base incorporation;
(b) preparing a plurality of peptides by translation from the mRNA library in a translational system;
(c) linking the resulting peptides to the mRNA encoding the peptides to form a plurality of peptide fusion products;
(d) contacting the plurality of the peptide fusion products with a preselected pre-miRNA target and/or a preselected pre-miRNA target, the fusion products which are specific for the pre-miRNA target or pre-miRNA target binding to the target; and
(e) isolating the peptide fusion products bound to the pre-miRNA and/or pre-miRNA targets.
2. The method of claim 1 , wherein the contacting step is performed in the presence of a competitor RNA.
3. The method of claim 2 , wherein the competitor RNA is tRNA.
4. The method of claim 1 , wherein the isolating step comprises immunoprecipitating the peptide fusion products bound to the pre-miRNA target or to the pri-miRNA target.
5. The method of claim 1 , further comprising selecting for the peptide fusion products which are membrane-permeable.
6. The method of claim 5 , wherein the peptide fusion products are selected by Immobilized Artificial Membrane (IAM) High Performance Liquid Chromatography (HPLC).
7. The method of claim 1 , further comprising subjecting the peptide fusion products to proteolysis with P450 enzymes, and selecting those peptide fusion products which are at least partially resistant to degradation.
8. The method of claim 1 , wherein the preselected pre-miRNA and/or pre-miRNA is pre-miRNA-221 or pri-miRNA-221.