Digital Imprinting of RNA Recognition and Processing on a Self-Assembled Nucleic Acid Matrix

Abstract

The accelerating progress of research in nanomedicine and nanobiotechnology has included initiatives to develop highly-sensitive, high-throughput methods to detect biomarkers at the single-cell level. Current sensing approaches, however, typically involve integrative instrumentation that necessarily must balance sensitivity with rapidity in optimizing biomarker detection quality. We show here that laterally-confined, self-assembled monolayers of a short, double-stranded(ds)[RNA-DNA] chimera enable permanent digital detection of dsRNA-specific inputs. The action of ribonuclease III and the binding of an inactive, dsRNA-binding mutant can be permanently recorded by the input-responsive action of a restriction endonuclease that cleaves an ancillary reporter site within the dsDNA segment. The resulting irreversible height change of the arrayed ds[RNA-DNA], as measured by atomic force microscopy, provides a distinct digital output for each dsRNA-specific input. These findings provide the basis for developing imprinting-based bio-nanosensors, and reveal the versatility of AFM as a tool for characterizing the behaviour of highly-crowded biomolecules at the nanoscale.

Ribonuclease III mechanisms of double‐stranded RNA cleavage

“… 117 RNase III can function in dense nanopatches of dsRNA, and can provide a permanent topographic ‘imprint’ of dsRNA recognition by either a protein or inhibition by an intercalating agent. 118 Such approaches can provide the basis for detecting dsRNA and related molecules as disease biomarkers at the single-cell level.…”

Computational Evolution of Beta-2-Microglobulin Binding Peptides for Nanopatterned Surface Sensors

“…Ultra-flat gold substrates were prepared as described in past publications from our group [ 31 , 37 , 51 ]. Briefly, a sequential deposition of gold was employed using electron beam evaporation.…”

Noncanonical DNA Cleavage by BamHI Endonuclease in Laterally Confined DNA Monolayers Is a Step Function of DNA Density and Sequence

“…The inherent capacity of nucleic acids to self-assemble via canonical Watson-Crick base pairing allows the generation of programmed DNA architectures either on surfaces or in solution [9]. As one example, in chemical nanoreactors, biomolecular components are positioned with nanoscale precision and controlled orientation to locally permit or enhance reaction progression [10][11][12]. This technology is broadly inspired by reactions occurring inside biological cells and may provide novel routes for the multiplexed chemical manipulation of nucleic acids and other biopolymers by directing reactions that normally occur in bulk solution.…”

“…Combining the immobilisation of DNA on solid surfaces using micro and nanolithographic techniques allows the generation of spatially confined DNA brushes (monolayers) with surface densities that closely approximate that occurring in intracellular microenvironments [11][12][13][14]. By varying the height, composition, orientation, and density of the DNA molecules in such brushes (also termed patches or laterally confined DNA monolayers (LCDMs)), it is possible to investigate biomolecular reactions and interactions to an extent that cannot be achieved in dilute solutions [11,12,15,16]. While other studies have investigated the physical properties of DNA brushes [17][18][19], key applications essentially fall in the area of synthetic biology, including the generation of cell-free gene expression systems [14].…”

“…Our group used the atomic force microscopy (AFM)-based nanolithography technique termed nanografting [20] to generate LCDMs on ultra-flat gold surfaces [12,13,16,21]. We used such DNA patches to investigate endonuclease action on the immobilised DNA by AFM measurement of the height change in the DNA patch relative to the surrounding inert surface [13,16].…”