- Molecular biology assays (TUNEL assay)
- Enzymatic de novo DNA synthesis with free dTNPs and polymerase-dNTP conjugates at elevated temperatures
- Enhanced thermostability
- Increases the elongation rate for de novo DNA synthesis
Terminal deoxynucleotidyl transferase (TdT) is used in several molecular biology methods. A major shortcoming of TdT is inhibition by secondary structures of a single stranded DNA substrate reducing the efficiency of nucleotide incorporation by the enzyme. Reduced efficiency can negatively impact enzymatic DNA synthesis reactions based on polymerase-dNTP conjugates or using reversible terminator dNTPS (RTdNTPs)). Commercially available TdT enzyme and the TdT variant both encounter this difficulty.
Currently there are not many techniques for de novo DNA synthesis. However, the TUNEL assay was recently used to develop an enzymatic de novo DNA synthesis method. TdT inhibition by secondary structures of a single stranded DNA substrate creates problems for the technique making it harder to advance de novo DNA synthesis. Secondary structures can be dissolved by increasing the reaction temperature. However, murine TdT is not stable above 40°C.
Researchers at Berkeley Lab’s Joint BioEnergy Institute (JBEI) engineered two murine TdT variants with enhanced thermostability properties using the web server, Fireprot.
In order to enhance the activity of TdT on hairpins, the scientists optimized the concentrations of the divalent cation cofactors and innovated a TdT for enhanced thermostability, allowing reactions at elevated temperatures. By combining these improvements, a ~10-fold increase in the elongation rate of a guanine-cytosine hairpin was observed.
Increasing the reaction temperature can reduce inhibitory effects arising from the formation of secondary structures in single stranded DNA, the substrate that TdT uses. The stability of the mutants is increased by about 10°C therefore elevating the reaction temperature enables faster turnover of single stranded DNA with secondary structures. Elevating the reaction temperature from the current 37°C can help to avoid inhibiting effects of secondary structures on TdT activity.
LBL PRINCIPAL INVESTIGATORS: Jay D. Keasling
DEVELOPMENT STAGE: Proven principle
IP STATUS: Patent pending
OPPORTUNITY: Available for licensing or collaborative research.
“Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3′ Terminal Structures for Enzymatic De Novo DNA Synthesis,” MDPI, Volume 11, Issue 1