Temperature-Controlled Programable One-Pot Polymerase-Mediated DNA Synthesis

Abstract

Polymerase-mediated DNA synthesis is central to many biotechnology applications. However, existing programmable synthesis methods require repeated exchange of DNA building blocks, which increases reagent consumption and complicates multistep workflows. Here, we introduce the TEmperature Mediated Primer Exchange Reaction, TEMPER, a programmable platform for arbitrary DNA synthesis that operates solely through temperature control in a one-pot reaction. TEMPER uses hairpin DNA as temperature-responsive DNA building blocks whose synthesis activity is confined to defined temperature ranges. These temperature ranges are determined by the lengths of the primer hybridization site and product dissociation site within each hairpin, which regulate DNA hybridization, DNA dissociation, and polymerase-mediated extension. By assigning different hairpin DNA building blocks to distinct temperature ranges, TEMPER enables selective and sequential DNA synthesis in a one-pot reaction. We demonstrate its versatility by developing a DNA data storage writer, a colorimetric temperature indicator, and a temperature data logger, highlighting its broad potential in nanotechnology and biotechnology applications.