TEMPER Enables Programmable One-Pot DNA Synthesis through Temperature Control

A research team led by Prof. Yeongjae Choi of the Graduate School of Engineering Biology at KAIST developed TEMPER (TEmperature Mediated Primer Exchange Reaction), a programmable one-pot DNA synthesis platform where temperature determines which DNA strand is generated. By treating temperature as an active programming input rather than a fixed reaction condition, TEMPER extends DNA molecular programming beyond systems that mainly process pre-synthesized strands through hybridization and strand displacement.

TEMPER combines multiple temperature-responsive hairpin DNA building blocks in one reaction mixture. Each hairpin is designed to operate within a distinct temperature window. Changing the incubation temperature thus selectively activates one building block while the others remain inactive. The extended product becomes the primer for the next step, allowing a programmed order of temperature changes to determine the order of DNA synthesis without repeated solution or reagent exchange. Because the active building block is selected by temperature rather than by the delivery of an external reagent, the same reaction mixture can be reprogrammed simply by changing the temperature profile.

The researchers established reaction windows from 20 to 60 °C in 10 °C increments and described the system with a thermodynamic model integrating primer hybridization, product dissociation, and polymerase activity. The model clarifies how these competing processes define the temperature range in which each hairpin can participate in synthesis. Using a standard thermal cycler, TEMPER achieved multi-step molecular information writing with a stepwise synthesis efficiency of 96.45%. The same principle was further demonstrated in DNA data storage and temperature-responsive molecular recording.

TEMPER differs from conventional DNA circuits by creating selected strands on demand as the program proceeds, rather than only rearranging strands already present in a reaction. Newly synthesized strands can become inputs for subsequent reactions, enabling molecular signals to propagate through synthesis itself. This design adds a temporal dimension to molecular programming: temperature controls not only reaction conditions, but also when new informational components are generated and made available to downstream processes. The study was published in Nature Communications on July 2, 2026, under the title “Programmable one-pot polymerase-mediated DNA synthesis via temperature control.” Jangho Choi and Jinho Kim served as co-first authors, and Prof. Yeongjae Choi as a corresponding author.

This work was supported by the Pioneer Research Center Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (RS-2022-NR067569), the research program through the NRF funded by the Korean government (MSIT) (RS-2022-NR072078, RS-2025-02218242), the Basic Science Research Program through the NRF funded by the Ministry of Education (RS-2024-00410005), the InnoCORE program of the Ministry of Science and ICT (N10260100), and the KAIST Institute’s own research project (N10260061) (Y.C.).