Team:Thessaloniki/Description
From the Code of Life, to Logical Programming
DNA Computing has vigorously emerged in science, taking nucleic acids outside their biological context and constructing molecular motifs of autonomous self-assembly, capable of information processing. DNA circuitry builds on the nucleic acids’ intrinsic ability to convert molecular networks into computational layouts. Moreover, the cornerstones of DNA, nucleobases, are capable of constructing proficient programming languages that can integrate limitless mathematically defined operations into biological systems. Hence, DNA Computing proves to be a promising tool for the construction of programmable networks with computational proficiency, feasible in an abundance of research fields.
POSEIDON: A toolkit for the examination of DNA-Protein interactions
Introducing POSEIDON; our team deploys the first DNA Computer able of quantifying DNA-Protein interactions. Using toehold-mediated DNA Strand Displacement, we design multilayer molecular circuits that examine a protein’s properties and quantify its binding affinity towards a particular sequence. After running through the molecular processing algorithm, an output signal in the form of fluorescence is produced making the signal acquisition obtainable by a variety of laboratory equipment. To that extend, our team developed an alternative method of reporting measurements based on an electrochemical assay where a gold sensing pad is able to distinguish between the active and inactive states of our molecular circuit. Our toolkit focuses on the needs of basic research;the use of low-cost materials and the ever-growing demand in the clarification of molecular interplay. Through our assay, we are able to identify the equilibrium state and sequence-specific binding site of DNA-binding proteins, as well as possible mutations or epigenetic modifications that are responsible for the dissociation of these interactions. Characterization of the conditions needed for completion of the binding reaction is achievable, while our toolkit can also process multiple signals at a time.
POSEIDON runs on DNA Strand Displacement (DSD) reactions;chemical cascades that operate based on the hybridization ability of DNA. According to their mechanism, each computational unit carries a distinct toehold sequence that is used universally amongst the circuit to introduce DSD reactions. During this process, a single-stranded DNA fragment invades a partially double-stranded DNA molecule through the binding of their toehold sequences. This occurrence induces the formation of a Holliday junction and further accelerates a strand displacement reaction, delineating it as thermodynamically favorable. As a result, a double-stranded molecule incapable of further reaction is formed, while a second single-stranded fragment is also released. This fragment is considered an output of this level of computation, while it can also act as an input for a downstream subsequent reactions downward similar reaction.
Our Assay's Importance
Molecular networks are the fundamental units through which a cell can process information and mediate primary operations regarding its integrity. Fundamental processes that keep the cell alive are consistently mediated by a myriad of molecules that connect and form perplexing networks of interaction. Such chemical grids prove to be especially sensitive and their malfunction is the prevailing cause of cell defect and the process of malignancy. In particular, DNA-Protein interactions have major effects upon many phenotypic features, such as gene regulation and protein maturation that rely on the identification of target sites in the genome containing distinct sequences. Elucidation of these interactions and the conditions under which they are dissociated are of major importance for a plethora of future applications that concern disease diagnosis, therapeutics, drug discovery and many more. To this day, most assays directed towards the characterization of such intracellular interactions are limited to qualitative identification, while those performing quantitative measurements tend to be rather costly. Most importantly, the majority of information concerning a wide variety of proteins come from in silico predictions that meet no immediate validation from in vitro experiments and, therefore, there is an immediate need for novel methodologies of DNA-Protein interaction examination.
Such methodologies could strengthen biological research in its whole spectrum, especially endeavors oriented around gene regulatory mechanisms, their disengagement, and cell dysregulation. Regulatory proteins are held accountable for a variety of primary cellular functions like metabolic and homeostatic procedures, signaling, cellular division, and differentiation. Amongst others, Transcription Factors ensure the cell’s canonical operation while also delineating the cell’s properties through gene regulation. That being said, methods able to outline a TF’s interaction with deoxyribonucleic acids as well as their behavior around other cofactors hold great promise in the elucidation of complex biochemical pathways and the identification of novel therapeutic targets.
Proof Of Concept
On the basis thereof, our team decided to investigate DNA-Protein interactions related to gene regulatory devices and Transcription Factor binding. As a proof of concept, we examined the of Transcription Factors related to the metastatic stages of melanoma. During this process, we conducted experiments using two transcription proteins, NFκ-B p65 and ELK-1. Experiments occurred with both consensus sequences and sequences containing SNPs in the protein’s binding site, unveiling the percentage at which the mutation reflects on the binding reaction.
Team’s Notice
Our iGEM journey this year introduced us to the captivating world of synthetic biology and allowed us to design, test and validate POSEIDON, bringing our idea to life! We had the opportunity to construct our very own DNA Computer, fulfil its design and deploy it towards research, providing novel solutions to basic science puzzles. We developed an alternative, electrochemical assay of circuit output measurement, delivered a complete molecular toolkit and proved that our solution works! We stayed long days and even longer nights in the lab working towards our goal… And we cherished every single moment of it!