Team:UChile Biotec/Proyect

Our Motivation

Chile has a very extensive coastline. A significant portion of the national economy is related to marine resources, providing an important source of employment. Therefore, HAB causes significant social and environmental problems not only in Chile but also in other parts of the world. In 2016, Chile was affected by an unprecedented algal bloom that seriously damaged artisanal fishing in the south, particularly in Chiloe Island. This epidemic provoked many social protests by the local population, eager for answers and solutions. The standard method used to detect paralytic toxins in Chile is a mice bioassay, using injections of potentially-contaminated shellfish samples. Although this method is robust, it comes with a variety of problems such as 1) elevated costs, 2) the use of thousands of mice and 3) a requirement for highly-trained professionals in certified laboratories. Considering that HABs will continue to occur, yet current screening methods are laborious and time-consuming, we were motivated to develop a simple early warning device for the detection of marine toxins.

Overview

BiMaTox

BiMaTox is a DNA aptazyme biosensor for different marine toxins that are produced during harmful algal blooms (HAB), also known as red tides. The main paralytic toxins produced during such events are Saxitoxin (STX), Brevetoxin-2 and Okadaic Acid. Of these, STX is the most deadly, as it attacks the human nervous system by blocking sodium channels present in neurons, impeding synapse formation.

The biosensor consists of a cell-free cellulose matrix device that displays a color in the presence of the toxin.  The aptazymes will consist of a specific toxin aptamer connected to a horseradish peroxidase (HRP)-mimicking DNAzyme. When the toxin binds to its specific aptamer, the HRP activity of DNAzyme is triggered and produces the oxidation of a compound called ABTS, which generates a color that is readily visible to the human eye.

Our BiMaTox is a rapid, reliable and accessible biosensor that will facilitate the detection of these toxins. Our synthetic biology device will be easy to use for people without highly-specialized training. We believe in reducing the time and costs associated with the current methods, and we expect to protect the health of people, and provide tools for the authorities for better and faster decision-making.

Project Description

For the better understanding of our project we want you to know how it is composed, to fulfill this goal we want to introduce to you the villain of this story and of course.... the heroes of our system! The aptamer and the DNAzymes, that when conjugated they form an Aptazyme!

First... The Villain!!: Saxitoxin(STX)

The structure of Saxitoxin

STX is a trialkyl tetrahydropurine extremely stable in biological and physiologic solutions unless it is exposed for a long time to alkaline conditions. In watery solutions, STX has two pKas 8,22 and 11,28 which belongs to the guanidinium groups 7, 8, 9 y 1, 2, 3 respectively. This polar nature explains the solubility of STX in water and lower alcohols and its insolubility in organic solvents

To be such a small molecule, STX shows a surprising number of natural variations!! There are more than 20 isomeres described until 2005!

Biological activities of STX discovered to date

As it’s known STX and other types of paralytic toxins are capable to inhibit the flux of sodium ions trough Sodium voltage-dependent channels. This activity it‘s known for a lot of decades and until a few years, it was considerate the only pharmacologic activity that it possessed. Nowadays it‘s also known that it is capable to block potassium and calcium channels. In addition, it has been described as a weak inhibitor of the nitric oxide synthase at neural levels. This enzyme is responsible for the production of nitric oxide from the guanidine group of arginine. This could be the origin of the guanidine groups presents in the paralytic toxins.

Mammals also express several Na+ channel isoforms to which STX can bind with only micromolar affinity. They exist in mammalian sensory neurons, cardiac muscle, skeletal muscle in early stages of development and experimentally denervated skeletal muscle. Most mammalian Na+ channels are sensitive to STX and so human nerves and other non-cardiac excitable tissue are unable then to resist the action of these toxins. Differences in Na+ channel amino acid sequences underlie this diversity in toxin sensitivity, and mutation of a single amino acid can convert a toxin insensitive Na+ channel to a channel easily blocked by STX.

Now enters the Heroes!!!

Aptamers

The aptamers are like antibodies in the sense of their capability to bind itself to proteins and modulate the function of them. The aptamers are referred as chemical antibodies due to their synthetic production. Aptamers are short, single-stranded DNA or RNA oligonucleotides that can bind to their targets with high specificity and affinity through van der Waals forces, hydrogen bonding, salt bridges, and hydrophobic and other electrostatic interactions. Aptamers can fold into complex and stable three-dimensional shapes, which allows them to fold within or around their targets. The aptamers can fold around or between their targets by forming tridimensional stable and complex structures. DNA aptamers have a high stability, while RNA aptamers can produce more structures because of its high flexibility.

Aptamers have been identified against targets that include small organic and inorganic molecules, such as dyes, nucleotides, amino acids, and drugs; biopolymers, such as peptides, proteins, and polysaccharides; ions; phospholipids; nucleic acids; viruses; bacteria; cell fragments and whole cells The aptamers can be used for detecting and characterizing their targets and modify the activity of them. First reported in 1990, aptamers were conceptualized independently by three groups of researchers, Ellington and Szostak (1990), Robertson and Joyce (1990), and Tuerk and Gold (1990). Tuerk and Gold named the production process the Systematic Evolution of Ligands by Exponential Enrichment.

SELEX

The process of screening large nucleic acid pools to develop aptamers, SELEX, has been optimized since its development to meet a variety of needs. Mimicking natural selection, SELEX involves screening pools of random-sequence nucleic acid libraries for oligonucleotides that bind a particular target.

Automated DNA synthesizers prepare the libraries of up to approximately 1017 random sequence oligonucleotides using equimolar mixtures of nucleotide bases during generation of the random regions.

Bigger libraries allow more diverse sequences and a bigger capacity to develop more specific and more affined aptamers.

Library oligonucleotides have a central randomized sequence of approximately 30 to 80 bases with defined terminal binding sites on each end for capturing and enzymatic manipulation. An immobilized target is incubated with the library, and the oligonucleotides that bind to the target are isolated, eluted, and amplified.

Applications of Aptamers

The aptamers can be used for the same applications that monoclonal antibodies produced in animals do, including basic science investigations, regulatory testing and clinical applications.

Basic research

In basic research the aptamers can be conjugated with peptide tags, proteins and nanoparticles to give them fluorescent properties to identify and detect concentrations of molecules, biological compounds, viruses, diseased cells and residues in food.

Aptamers can be used in common essays like immunofluorescence microscopy, microarrays, flow cytometry and blotting essays like Western Blots.

Regulatory testing

Aptamer’s properties are ideal for helping in efficiency testing in regulatory processes. For example, the aptamers can be used in quality controls of proteins of therapeutic use by the detection of little differences between the protein products that were not detected using monoclonal antibodies from animal origin.

For example the U.K.'s Department of Environment, Food and Rural Affairs (DEFRA) conducted a project to develop aptamers for rabies batch potency testing with the aim of replacing mice used to develop antibodies for the detection of contaminants in vaccine preparations.

Therapeutics

Recombinant antibodies and aptamers have been used in therapeutic applications to alter target activity, for example, by binding to cell surface receptors, or by delivery of therapeutic agents to target cells via conjugation to antibiotics, RNA interference, toxins, enzymes, or drugs.

Aptamers are used to combat autoimmune conditions, toxins, chronical diseases, cancer and diseases caused by pathogens.

DNAzymes

Deoxyribozyme also known as DNAzyme or catalytic DNA, are oligonucleotides of DNA that are capable to do specific chemical reactions and were described for the first time in 1997 by Ronald Breaker. Its function is similar to other biological enzymes like proteins or ribozymes (enzymes composed of RNA). None the less in contrast with the abundance of enzymatic proteins that are present in biological systems and the discovery of ribozymes in 1980, there is no record of the existence of DNAzymes in nature.

DNAzymes should not be confused with DNA aptamers, the main difference between both is the ability of DNAzymes to catalyze a chemical reaction. Except for ribozymes, nucleic acid molecules inside cells main function is the storage of genetic material due to their complementary between their bases, this gives the chance to generate copies of high fidelity and transfer the genetic information

DNAzima have proven to be useful in a variety of areas such as:

• Clinical essays

• Use as biosensors in different systems.

This last function is the one we found the most interesting and among the variety of DNAzymes available to use, we chose to work with an HRP like-DNAzyme that possess the same peroxidase properties of the Horseradish peroxidase.

References

1. Llewellyn L. (2005),"Saxitoxin, a toxic marine natural product that targets a multitude of receptors", Natural Product Report.

2. Willner I. et al (2008),"DNAzymes for sensing, nanobiotechnology and logic gate applications", ChemRevSoc (1153-1165).

3. Groff K. et al (2015),"Modern affinity reagents: Recombinant antibodies and aptamers", Biotechnolgy Advances 33(1787-1798).