Team:Vilnius-Lithuania-OG/Nucleotide Design


Substrate Nucleotide Design Prospects


2'-Deoxycytidine-5'-triphosphate (dCTP) - one of the four building blocks of DNA - is as a versatile scaffold for a specific enzyme substrate immobilization. Cytosine 4th amine (-NH2) and deoxyribose 3’ hydroxy (-OH) chemical groups can be easily manipulated to add modifications of interest.

Such substrate modified dCTP molecules (sub-dCTP) establish two separate states of cytidine nucleotides: Substrate dCTP and catalytically converted Product dCTP.

The switching of substrate dCTP to product dCTP is achieved when an active enzyme catalyzes the removal of the unique modification. Take this into consideration, there could be 3 ways such stable equilibrium of different cytidines could be utilized for activity recording:

1. Substrate nucleotides can be too bulky and thus are structurally hindered to be incorporated by phi29, meaning only catalytically converted product nucleotides are incorporated.

2. Substrate nucleotides can be incorporated into the DNA sequence, however modification did on substrate as it its converted to product restricts the incorporation of product nucleotides

3. Both, substrate and product nucleotides are accessible to phi29 DNA polymerase and can be incorporated into the DNA sequence.

This creates a number of new variable – substrate deoxycytidine concentration and product deoxycytidine concentration – which are proportional to the activity of the enzyme that catalyzed the reaction. These variable can be imbedded in to the very sequence of DNA as incorporated nucleotides and read during next generation sequencing as a ratio to a reference nucleotide. The ratio of incorporated reference and substrate/product nucleotide is proportional to the catalytic activity of the biomolecule, which catalyses the catalytic conversion.

The spectrum of attachable substrates defined defines the capabilities of such catalysis recording system. Taking advantage of the chemical synthesis, virtually any substrate can be linked to the 2'-Deoxycytidine-5'-triphosphate 2’-OH and N4- functional groups. The combination of modifications added to the nucleotide, chemical and biochemical reactions allows to create a dynamic system which can account for almost all of the enzyme catalytic reactions.

1. Oxidoreductases

As the name implies, oxidoreductases catalyze the oxidation and reduction reactions. A range of substrates containing alcohol, carbonyl or carboxylic acid, amine or ether groups with different carbon backbone can be used to measure the catalytic activity of oxidoreductases - alcohol dehydrogenases, monooxygenases, reductases, cytochromes. The main approach, shown below., utilizes the instability or chemical properties of formed reaction products. Covalently linked product molecules spontaneously and easily (beta elimination reaction) are removed from the modified cytosine, switching the state of the nucleotide to accessible.

2. Transferases

Transferases catalyze the transfer of functional group from one compound to the other. The most popular transferases are phosphotransferases and aminotransferases. Cytosine linked substrates bearing carbonyl/amino, hydroxyl groups with a specific carbon backbone are easily synthesized and can be used for specific catalytic reaction activity recording. Image below. depicts main example of such catalytic “activity” recording. Reaction product, bearing carbonyl group takes part in beta elimination reaction to producing accessible 2'-Deoxycytidine-5'-triphosphate.

3. Hydrolases

Hydrolases catalyze the transfer of a chemical group from substrate to water molecule. Functional groups of interest present in the substrate can be easily linked to 2'-Deoxycytidine-5'-triphosphate 2’-OH and N4- positions and can act, not necessary, as the connecting bond. Utilizing the vast majority of connected substrates, different amidases, esterases, phosphoesterases, glucosidases and sulphotases can be screened. Image below. shows an example reaction catalyzed by amidase. The enzyme hydrolases the functional group and disconnects the linked product molecule in one step forming a native dCTP nucleotide.

4. Lyases

Lyases catalyze the elimination reaction of various chemical groups by breaking C-C, C-O, C-N and other bonds. As a consequence of bond breakage, lyases allow the substrate nucleotide to lose a part of linked substrate, therefore newly formed product nucleotide becomes accessible to Phi29 DNA polymerase and in turn is incorporated into a DNA sequence. Additionally, lyase catalyzed reactions can be linked with simple chemical conversions, leading to removal of whole substrate as depicted below. Image below visualizes the cleave of C-C bond leading to a product nucleotide, which spontaneously undergoes beta elimination reaction, leading to native dexocytinine nucleotide.

5. Isomerases

Isomerases catalyze the conversion of one isomer to another. Combination of isomerases with other stereospecific enzymes, such as hydrolases, can be employed to change the configuration of nucleotide linked substrate molecules which are in turn removed by secondary enzyme, thus recording the activity of rate limiting enzyme. Image below shows a substrate bound nucleotide. The stereospecific amidase only catalyzes the removal of S isomer substrate. Introduction of isomerase enzyme allows to record the catalytic activity of this enzyme in a form of amidase hydrolyzed nucleotides.