Efficient delivery of a DNA aptamer-based biosensor into plant cells for glucose sensing through thiol-mediated uptake

Abstract

DNA aptamers have been widely used as biosensors for detecting a variety of targets. Despite decades of success, they have not been applied to monitor any targets in plants, even though plants are a major platform for providing oxygen, food, and sustainable products ranging from energy fuels to chemicals, and high-value products such as pharmaceuticals. A major barrier to progress is a lack of efficient methods to deliver DNA into plant cells. We herein report a thiol-mediated uptake method that more efficiently delivers DNA into Arabidopsis and tobacco leaf cells than another state-of-the-art method, DNA nanostructures. Such a method allowed efficient delivery of a glucose DNA aptamer sensor into Arabidopsis for sensing glucose. This demonstration opens a new avenue to apply DNA aptamer sensors for functional studies of various targets, including metabolites, plant hormones, metal ions, and proteins in plants for a better understanding of the biodistribution and regulation of these species and their functions.

Phosphorothioated DNA Engineered Liposomes as a General Platform for Stimuli‐Responsive Cell‐Specific Intracellular Delivery and Genome Editing

“…CM vesicles were pretreated with Ellman's reagent [5,5′‐dithio‐bis‐(2‐nitrobenzoic acid]) (DTNB, a thiol oxidizer), or dithiothreitol (DTT, a thiol reductant), [25] before incubating with the PS DNA‐Lipo. As shown in Figure 2f, the content mixing was inhibited by DTNB, indicating that the PS DNA facilitated membrane fusion through the formation of a disulfide bonding [26] . While another inhibitor DTT didn't impede fusion, we speculated that DTT reduces the disulfide bond of membrane proteins to thiol that further improves the formation of a dynamic dithiol network, therefore unable to inhibit the fusion with PS DNA [27] …”

“…As shown in Figure 2f, the content mixing was inhibited by DTNB, indicating that the PS DNA facilitated membrane fusion through the formation of a disulfide bonding. [26] While another inhibitor DTT didn't impede fusion, we speculated that DTT reduces the disulfide bond of membrane proteins to thiol that further improves the formation of a dynamic dithiol network, therefore unable to inhibit the fusion with PS DNA. [27]…”

Modern analytical and bioanalytical technologies and concepts for smart and precision farming

“…Another alternative to delivering DNA-aptamer-based biosensors into plants is thiol-mediated uptake through the roots and leaves of plants. 97 Due to its small size, the disulfide unit efficiently delivers nucleic acid through the cell wall. This approach could be an excellent pathway to introduce DNA-based bioristors for imaging.…”

Toward a Traceless Tag for the Thiol‐Mediated Uptake of Proteins

“…Since the introduction of cell-penetrating polydisulfides, [12] followed by AspA for TMU with small molecules, [13] a variety of CAXs has been shown to deliver diverse substrates into various targets. [7] This includes the delivery of genome editing machinery, [14] various forms of genes, [15][16][17][18][19] phosphorothioate oligonucleotides, [20] antibodies [8] and other proteins, [21][22][23][24] and nanoparticles, [8,[25][26][27][28][29] to living animals, [14] plant cells, [18] bacteria, [30] and into deep tissue. [24] Inhibition of TMU has been linked to drug discovery with regard to antiviral, antithrombotic and antitumor potential.…”

“…[7] The proven power of TMU for cell penetration calls for general TMU tools to solve practical delivery problems in a simple and reliable manner. While various TMU methods for oligonucleotide delivery are rapidly evolving, [14][15][16][17][18][19]38] traceless small-molecule tags accessible for the broader community to easily and reliably deliver proteins of interest (POIs) do not exist. Such ideal small-molecule TMU tags could be attached to the POI in situ under bioorthogonal conditions prior to delivery and detached after uptake in the cytosol to release the POI in its native form (Figure 1c).…”