Hybrid Nucleic Acid-Quantum Dot Assemblies as Multiplexed Reporter Platforms for Cell-Free Transcription Translation-Based Biosensors

Abstract

Cell-free synthetic biology has emerged as a valuable tool for the development of rapid, portable biosensors that can be readily transported in the freeze-dried form to the point of need eliminating cold chain requirements. One of the challenges associated with cell-free sensors is the ability to simultaneously detect multiple analytes within a single reaction due to the availability of a limited set of fluorescent and colorimetric reporters. To potentially provide multiplexing capabilities to cell-free biosensors, we designed a modular semiconductor quantum dot (QD)-based reporter platform that is plugged in downstream of the transcription-translation functionality in the cell-free reaction and which converts enzymatic activity in the reaction into distinct optical signals. We demonstrate proof of concept by converting restriction enzyme activity, utilized as our prototypical sensing output, into optical changes across several distinct spectral output channels that all use a common excitation wavelength. These hybrid Förster resonance energy transfer (FRET)-based QD peptide PNA-DNA-Dye reporters (QD-PDDs) are completely self-assembled and consist of differentially emissive QD donors paired to a dye-acceptor displayed on a unique DNA encoding a given enzyme’s cleavage site. Three QD-based PDDs, independently activated by the enzymes BamHI, EcoRI, and NcoI, were prototyped in mixed enzyme assays where all three demonstrated the ability to convert enzymatic activity into fluorescent output. Simultaneous monitoring of each of the three paired QD-donor dye-acceptor spectral channels in cell-free biosensing reactions supplemented with added linear genes encoding each enzyme confirmed robust multiplexing capabilities for at least two enzymes when co-expressed. The modular QD-PDDs are easily adapted to respond to other restriction enzymes or even proteases if desired.

Design and Characterization of a Gene-Encoding DNA Nanoparticle in a Cell-Free Transcription–Translation System

“…Thereafter, the DNA NPs were purified via PEG precipitation 47 and resuspended in Tris buffer (as suggested by the TXTL kit directions) supplemented with an optimal amount of MgCl 2 for DNA NP stability in tandem to TXTL reaction efficiency. 48 The supernatant from the PEG-based purification of the DNA NPs was reserved for characterization of assembly and function (termed the PEG supernatant in Figure 2). We prepared a few other control samples; namely, an M13-scaffolded DNA wireframe snub cube NP served as a negative control for protein expression in TXTL.…”

Passivating quantum dots against histag-displaying enzymes using blocking peptides: salient considerations for self-assembling quantum dot biosensors

“…[38][39][40][41] Within our group alone it has been used as a biosensor scaffold for endopeptidases, exopeptidases, lipases, nucleases, restriction enzymes, and kinases. 12,13,15,22,40,[42][43][44][45][46][47][48] The general design was the selfassembly of a dye-labeled peptide substrate onto the QD surface through a histag, allowing the system to function as a FRET-based biosensor. In all cases it was important that the enzyme approach the QD, interact with the substrate, but not bind irreversibly.…”

“…9,11 Such QDs are used in a broad array of biosensing applications to detect peptides, proteins, antibodies, nucleic acids, viruses, small molecules, and a plethora of other targets, as well as having numerous applications in fluorescence imaging (cellular and in vivo), cell-free biosynthesis, enzyme kinetics enhancement, bioluminescent imaging, and energy harvesting. [12][13][14][15][16][17][18][19][20][21][22][23][24][25][26] In some of these applications, histag-modified enzymes are employed advantageously to selectively bind to QDs. However, it is important to recognize that most histag-enzymes are modified solely for IMAC purification and are not intended for conjugation to nanomaterials.…”

Pursuing excitonic energy transfer with programmable DNA-based optical breadboards

“…The unique combination of access to any designer nanoscale architecture in combination with all the processes and properties inherent to FRET is making these hybrid materials useful across a wide swath of research applications including especially as sensors and probes. Indeed, many of the examples described in the preceding section epitomize such applications where, for example, dye-labeled DNA structures either on their own or as interfaced with NPs such as QDs are providing for a variety of targeted sensors for monitoring force on the nanoscale, 432 pH, 429 voltage changes, 435 multiplexed enzyme activity, 431,445,448 along with providing for multiplexed diagnostics, and biomarker detection. 419,436,460 The always growing library of available fluorescent materials, ranging from new dye families to noble metal clusters and NPs manifesting unique quantum confined or other complex photophysical properties such as upconversion will also contribute to new FRET-based applications and probes that utilize DNA scaffolds.…”