Dynamic Bonds for Membrane Transport
The lipid bilayer membrane is the dynamic barrier that Nature has selected to keep ions and biomolecules confined in the appropriate location. This cellular compartmentalization is of critical importance for all the processes that allow life in complex organisms. However, the membrane barrier represents a problem when trying to introduce probes and/or deliver therapeutics. Therefore it is imperative to find new conceptual ways to enter cells and cross the lipid bilayer. As organic chemists we design and synthesize new molecules that can cross cell membranes and help in the delivery of other molecules. We focus our efforts in the use of dynamic covalent chemistry for the incorporation of the hydrophobic part of amphiphilic molecules that translocate the membrane and help in the delivery of polymers of biological interest. We use simple and facile synthetic methods to make molecules that we can evaluate in green fluorescent HeLa cells for the delivery of interference RNA (siRNA).
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Cas9 direct delivery
This methodology has been applied to polymers and peptides and has been successful for the delivery in several cell lines of interference RNA (siRNA), plasmids, or Cas9 ribonucleoprotein of the CRISPR system.
Conceptual scheme for the formation of dynamic amphiphiles to transport polymers of biological interest. Cas9 ribonucleoprotein delivery using peptides modified with hydrophobic tails via a hydrazone bond. The disruption of the eGFP gene in HeLa cells constitutively expressing the fluorescent protein is illustrated.
(1) “In situ” Functionalized Polymers for siRNA Delivery”, Priegue, J. M.; Crisan, D. N.; Martínez-Costas, J; Granja, J. R.; Fernandez-Trillo, F.; Montenegro, J. .
(2) “Hydrazone-modulated peptides for efficient gene transfection” Louzao, I.; García-Fandiño, R.; Montenegro, J.
(3) “Peptide/Cas9 Nano-structures for Ribonucleoprotein Cell Membrane Transport and Gene Edition” Lostalé-Seijo, I.; Louzao, I.; Juanes, M.; Montenegro, .
(4) “Supramolecular Recognition and Selective Protein Uptake by Peptide Hybrids” Juanes, M., Lostalé-Seijo, I., Granja, J., Montenegro, J. .
(5) “Different Length Hydrazone Activated Polymers for Plasmid DNA Condensation and Cellular Transfection” Priegue, J., Lostalé-Seijo, I., Crisan, D., Granja, J., Fernández-Trillo, F., Montenegro, J. .
(6) “Synthetic materials at the forefront of gene delivery” Lostalé-Seijo, I., Montenegro, J.
The origin of life and the transition from chemistry to biology remains one of the great unknown questions in science. Top-down approaches have revealed the necessity of various components in modern organisms, they failed to provide fundamental understanding on the minimum requirements necessary for life. Bottom-up approaches toward developing minimal “protocells” could shed light on the interface of biology and chemistry and reveal the principles required for the formation and maintenance of self-sustaining autonomous cells. We are engaged in a research program aimed to better understand the synergies required between proteins and lipids by proposing the development of synthetic cells that combine the complexity and evolved sophistication of biological processes with the control and robustness of modern synthetic and materials chemistry.
(1) “Self-Assembled Micro-Fibres by Oxime Connection of Linear Peptide Amphiphiles” Booth, R. Insua, I., Bhak, G. Montenegro,
(2) “pH-Triggered Self-assembly and Hydrogelation of Cyclic Peptide Nanotubes Confined in Water Micro-droplets” Méndez-Ardoy, A., Granja, J. R., Montenegro, .
Supramolecular Nanotechnology Control
Controlling and manipulating matter at the nanometric scale represents one of the mayor goals of nowadays science. Along these lines, the self-assembly of small monomers offers a great opportunity to generate nanometric structures with controlled topology. We know how to design and prepare peptide nanotubes and we can control the diameter and properties of these nanotubes by simply varying the sequence of the peptide used. We have recently used rationally designed hydrophilic cyclopeptides with a pyrene artificial amino acid to prepare hybrid tubular structures with carbon nanotubes.
Proposed model of coupling between SCPNs and SWCNTs.
Differential Sensing in Lipid Bilayers
The olfactory reception system allow us, the human beings, to distinguish around 10.000 different odours with only 350 olfactory receptors. Incompatibility with a 1 to 1 recognition this sensorial system requires the assistance of cross-responsive interactions to create spatial maps that are recognized as unique fingerprints in the brain. We have applied these lesson from nature to construct differential sensors for the discrimination of many different analytes. For instance volatile hydrophobic molecules such as single atom carbon atoms (octanal, nonanal, decanal) cis/trans isomers or even enantiomers. Click to read more about about it! We have recently developed a differential sensing system, operating in lipid bilayer membranes, for the challenging discrimination of complex polymers with biological relevance. We have been able to differentiate single, double stranded and short nucleotides sequences with single nucleobase resolution. Click the graph to read the tale of the first DNA artificial nose!
A) Set of different cationic reactive counteirons Arg1Ox3, Arg2Ox3, Arg2Ox4. B) Formation of the oxime-amphiphiles Arg1(OxT10)3/DNA activated neutral complex that translocate the membrane. C) Fluorescence fingerprints of different anionic biopolymers allow discrimination trough pattern recognition.