Thresholding DNA Nanomachine as a Highly Cooperative and Efficient Enzyme‐Like System for Controlled RNA Cleavage

Abstract

Therapeutic nucleic acid agents (TNA) can be activated by a marker RNA sequence followed by initiation of targeted RNA cleavage. This property can be used in conditional cell suppression, e. g., cancer marker‐dependent cell death. However, healthy cells often express lower levels of cancer markers, thus jeopardizing TNA activation exclusively in cancer cells. Earlier, we developed a conditionally activated split deoxyribozyme construct (DNA thresholder or DTh) that can be activated by high but not by low concentrations of cancer markers. It's activity, however, was suppressed by very high marker concentrations. Herein, we combine the DTh functional units in a single DNA association (Thresholding DNA nanomachine or Th‐DNM). Th‐DNM maintains a high level of RNA cleavage activity in the presence of marker concentrations above the threshold level. Th‐DNM differentiated fully complementary miR17 markers sequence from double base mismatched miR‐20. Th‐DNM can become a building block of DNA nanorobots for cancer treatment.

Distinguishing High from Low Concentrations of RNA Cancer Marker: Five‐Input DNAzyme Thresholding Gate

“…[16] The threshold miR-17 concentration could be changed from 42, 630 and 1100 pM depending on the number of input-recognition module. [17] Therefore, we were able to change the threshold concentration in the range of more than 1 order of magnitude. In this study, we aimed at pushing the idea of DTh to limit by widening the range of threshold concentrations by adding up to 5-input recognition modules.…”

“…This agent showed efficient RNA cleavage activity in the presence of high concentrations of mi‐R17 and lower activity at lower concentrations of mi‐R‐17 or in the presence of double base‐pair mismatched miR‐20 [16] . The threshold miR‐17 concentration could be changed from 42, 630 and 1100 pM depending on the number of input‐recognition module [17] . Therefore, we were able to change the threshold concentration in the range of more than 1 order of magnitude.…”

Towards the development of a DNA automaton: modular RNA-cleaving deoxyribozyme logic gates regulated by miRNAs

“…It has been proven earlier that the DNA binding strand can help avoid the decomposition of biDz in the presence of increased input amounts in the sample. 28 Kahan-Hanum M. et al 26 previously used the 1 : 20 substrate to DNA logic gate concentration ratio. We, on the other hand, have used a substrate to DNA logic gate concentration ratio of 2 : 1 which yielded highly efficient substrate cleavage.…”

“…However, all these developments do not exhibit complex logical operations and are capable of recognising a single marker. 28,30,41,42 In this study we would like to propose our approach on strict input-dependent regulation of Dz activity as a possible advancement on the cancer marker-dependent activation of therapeutic agents. Subsequently, we have adapted all our DNA logic gates to target a 60 nt long sequence naturally found in the mRNA of GFP, as a model system to prove that the principle and rules of our designs could be used for long natural mRNAs in the future.…”

“…29 As we have suggested before, this leads to the inhibition of the formation of the entire enzymatic unit and to a detrimental decrease in its activity. 28,30 The DNA logic gates associated with the DNA strand act as standalone systems with interchangeable modules, allowing for diverse logic operations. However, with increasing inputs, design optimization is required due to system complexity and challenges with signal leakage.…”