Ribonuclease-Responsive DNA Nanoswitches

Abstract

DNA has been used in the construction of dynamic DNA devices that can reconfigure in the presence of external stimuli. These nanodevices have found uses in fields ranging from biomedical to materials science applications. Here, we report a DNA nanoswitch that can be reconfigured using ribonucleases (RNases) and explore two applications: biosensing and molecular computing. For biosensing, we show the detection of RNase H and other RNases in relevant biological fluids and temperatures, as well as inhibition by the known enzyme inhibitor kanamycin. For molecular computing, we show that RNases can be used to enable erasing, write protection, and erase-rewrite functionality for information-encoding DNA nanoswitches. The simplistic mix-and-read nature of the ribonuclease-activated DNA nanoswitches could facilitate their use in assays for identifying RNase contamination in biological samples or for the screening and characterization of RNase inhibitors.

Figure 1.. DNA Nanoswitch Design and Operation

(A) The nanoswitch is locked in a looped conformation with a pre-hybridized RNA lock strand. On the addition of RNase H, the lock strand is digested, resulting in unlooping the nanoswitch to the open state. (B) Mechanism of cleavage of the RNA lock strand by RNase H and release of the DNA latches. (C) The DNA nanoswitch (1) is locked by an RNA lock strand into a looped configuration (2). RNase H causes cleavage of the RNA lock, causing the nanoswitch to unlock and reconfigure into the linear state (3). This conformational change can be read out on an agarose gel to detect the presence of RNase H (inset).

Figure 2.. RNase H Assay

(A) Sensitivity plot showing nanoswitch unlocking with different enzyme concentrations (gel image shown as inset). Error bars represent standard deviation obtained from triplicate experiments. (B and C) Detection of RNase H in 10% FBS (B) and human (HeLa) and murine (C2C12) cell lysates (C). (D) The nanoswitch-based RNase assay works at a range of temperatures. (E) Activity of different RNases (H, T, I_f, and A) in the nanoswitch assay. (F) Inhibition efficiency of kanamycin on RNase H activity. (G) Rapid readout can be obtained by mixing nanoswitches with the sample containing RNase H and a quick gel run.

Figure 3.. Multi-input DNA Nanoswitches and Information Processing

(A) DNA latches can be placed at specific locations on the scaffold, resulting in different loop sizes. (B) A nanoswitch mixture with 5 different nanoswitches can recognize specific RNA lock strands to reconfigure and yield specific bands on a gel. The presence or absence of these 5 bands can be used as a 5-bit code to encode information in DNA nanoswitches. (C) Combination of different locked states of the 5 nanoswitches is used as a 5-bit code to encode information (e.g., “R,” “N,” “A”), and the information can be erased by the addition of RNase H. (D) By using DNA locks, specific nanoswitches can be protected from unlocking, providing a write-protection feature for information encoding. (E) Information (locked bit) erased using RNase H can be rewritten using a DNA lock of the same sequence as the previously used RNA lock.