Activity modulation and allosteric control of a scaffolded DNAzyme using a dynamic DNA nanostructure.

Recognition of the fundamental importance of allosteric regulation in biology dates back to not long after its discovery in the 1960s. Our ability to rationally engineer this potentially useful property into normally non-allosteric catalysts, however, remains limited. In response we report a DNA nanotechnology-enabled approach for introducing allostery into catalytic nucleic acids. Specifically, we have grafted one or two copies of a peroxidase-like DNAzyme, hemin-bound G-quadruplex (hemin-G), onto a DNA tetrahedral nanostructure in such a manner as to cause them to interact, modulating their catalytic activity. We achieve allosteric regulation of these catalysts by incorporating dynamically responsive oligonucleotides that respond to specific "effector" molecules (complementary oligonucleotides or small molecules), altering the spacing between the catalytic sites and thus regulating their activity. This designable approach thus enables subtle allosteric modulation in DNAzymes that is potentially of use for nanomedicine and nanomachines

Programming cell entry of molecules via reversible synthetic DNA circuits on cell membrane

Cellular uptake of biomolecules is crucial for regulating cell function. However, powerful and biocompatible tools for dynamically manipulating the cell entry of single-stranded DNAs (ssDNAs) remain elusive. Herein, we constructed synthetic DNA circuits on the cell membrane to program the cell entry of ssDNAs, using toehold-mediated DNA strand displacement reactions. We found that the dimerization and trimerization of cholesterol-ssDNAs enhanced membrane-anchoring and cellular uptake of ssDNAs. Moreover, we demonstrated that de-dimerization and de-trimerization of cholesterol-ssDNAs could be accomplished by inputting recovery ssDNAs into the synthetic DNA circuits, which could simultaneously decrease the cellular uptake of ssDNAs. We speculate that operating the synthetic DNA circuits on the cell membrane will be a powerful strategy for regulating the cellular uptake of exogenous materials, which has important implications for bioimaging, drug delivery, and gene therapy

Comparison of therapeutic effects between big-bubble deep anterior lamellar keratoplasty and penetrating keratoplasty for medically unresponsive Acanthamoeba keratitis

Abstract Purpose To compare the outcomes of big-bubble deep anterior lamellar keratoplasty (BB-DALK) and penetrating keratoplasty (PKP) in the management of medically unresponsive Acanthamoeba keratitis (AK). Methods This retrospective study included 27 eyes of BB-DALK and 24 eyes of PKP from a tertiary ophthalmology care centre. Glucocorticoid eye drops were subsequently added to the treatment plan 2 months postoperatively based on the evaluation using confocal laser scanning microscopy. The clinical presentations, best-corrected visual acuity (BCVA), postoperative refractive outcomes, graft survival, and Acanthamoeba recurrence were analyzed. Results The AK patients included in the study were in stage 2 or stage 3, and the percentage of patients in stage 3 was higher in the PKP group (P = 0.003). Clinical presentations were mainly corneal ulcers and ring infiltrates, and endothelial plaques, hypopyon, uveitis and glaucoma were more common in the PKP group (P = 0.007). The BCVA and the graft survival rate showed no statistically significant differences between the two groups at 1 year after surgery. However, 3 years postoperatively, the BCVA of 0.71 ± 0.64 logMAR, the graft survival rate of 89.5%, and the endothelial cell density of 1899 ± 125 cells per square millimeter in the BB-DALK group were significantly better than those of the PKP group (P = 0.010, 0.046, and 0.032, respectively). 3 eyes (11.1%) in the BB-DALK group and 2 eyes (8.3%) in the PKP group experienced Acanthamoeba recurrence, but the rates showed no statistically significant difference between the two groups (P = 1.000). In the PKP group, immune rejection and elevated intraocular pressure were observed in 5 and 6 eyes, respectively. Conclusion Corneal transplantation is recommended for AK patients unresponsive to antiamoebic agents. The visual acuity and graft survival can be maintained after BB-DALK surgery. Acanthamoeba recurrence is not related to the surgical approach performed, whereas complete dissection of the infected corneal stroma and delayed prescribing of glucocorticoid eye drops were important to prevent recurrence

Programming Intracellular Clustering of Spiky Nanoparticles via Liposome Encapsulation

The intracellular clustering of anisotropic nanoparticles is crucial to the improvement of the localized surface plasmon resonance (LSPR) for phototherapy applications. Herein, we programmed the intracellular clustering process of spiky nanoparticles (SNPs) by encapsulating them into an anionic liposome via a frame-guided self-assembly approach. The liposome-encapsulated SNPs (lipo-SNPs) exhibited distinct and enhanced lysosome-triggered aggregation behavior while maintaining excellent monodispersity, even in acidic or protein-rich environments. We explored the enhancement of the photothermal therapy performance for SNPs as a proof of concept. The photothermal conversion efficiency of lipo-SNPs clusters significantly increased 15 times compared to that of single lipo-SNPs. Upon accumulation in lysosomes with a 2.4-fold increase in clustering, lipo-SNPs resulted in an increase in cell-killing efficiency to 45% from 12% at 24 μg/mL. These findings indicated that liposome encapsulation provides a promising approach to programing nanoparticle clustering at the target site, which facilitates advances in the development of smart nanomedicine with programmable enhancement in LSPR

Additional file 1 of Comparison of therapeutic effects between big-bubble deep anterior lamellar keratoplasty and penetrating keratoplasty for medically unresponsive Acanthamoeba keratitis

Supplementary Material

Programming Intracellular Clustering of Spiky Nanoparticles via Liposome Encapsulation

The intracellular clustering of anisotropic nanoparticles is crucial to the improvement of the localized surface plasmon resonance (LSPR) for phototherapy applications. Herein, we programmed the intracellular clustering process of spiky nanoparticles (SNPs) by encapsulating them into an anionic liposome via a frame-guided self-assembly approach. The liposome-encapsulated SNPs (lipo-SNPs) exhibited distinct and enhanced lysosome-triggered aggregation behavior while maintaining excellent monodispersity, even in acidic or protein-rich environments. We explored the enhancement of the photothermal therapy performance for SNPs as a proof of concept. The photothermal conversion efficiency of lipo-SNPs clusters significantly increased 15 times compared to that of single lipo-SNPs. Upon accumulation in lysosomes with a 2.4-fold increase in clustering, lipo-SNPs resulted in an increase in cell-killing efficiency to 45% from 12% at 24 μg/mL. These findings indicated that liposome encapsulation provides a promising approach to programing nanoparticle clustering at the target site, which facilitates advances in the development of smart nanomedicine with programmable enhancement in LSPR

Directing curli polymerization with DNA origami nucleators

The physiological or pathological formation of fibrils often relies on molecular-scale nucleators that finely control the kinetics and structural features. However, mechanistic understanding of how protein nucleators mediate fibril formation in cells remains elusive. Here, we develop a CsgB-decorated DNA origami (CB-origami) to mimic protein nucleators in Escherichia coli biofilm that direct curli polymerization. We show that CB-origami directs curli subunit CsgA monomers to form oligomers and then accelerates fibril formation by increasing the proliferation rate of primary pathways. Fibrils grow either out from (departure mode) or towards the nucleators (arrival mode), implying two distinct roles of CsgB: as nucleation sites and as trap sites to capture growing nanofibrils in vicinity. Curli polymerization follows typical stop-and-go dynamics but exhibits a higher instantaneous elongation rate compared with independent fibril growth. This origami nucleator thus provides an in vitro platform for mechanistically probing molecular nucleation and controlling directional fibril polymerization for bionanotechnology

DNAzyme-Based Rolling-Circle Amplification DNA Machine for Ultrasensitive Analysis of MicroRNA in <i>Drosophila</i> Larva

We present a highly sensitive colorimetric method for microRNA (miRNA) detection. This method is based on a rolling-circle amplification (RCA) DNA machine, which integrates RCA, nicking enzyme signal amplification and DNAzyme signal amplification. The DNA machine is triggered by the hybridization of target miRNA with a rational designed padlock DNA template and activated by RCA. The resulting RCA product then autonomously replicates a multiple machinery cutter cycle and generates accumulated amount of products. Specifically, the DNA product in the present work is designed as a horseradish peroxidase (HRP)-mimicking DNAzyme, which could that catalyze a colorimetric reaction and generate colored product. Through these cascade amplifications, microRNA (miRNA) as low as 2 aM could be detected. As an example of in vivo application, miRNA from single <i>Drosophila</i> larva was successfully analyzed. <i>Drosophila</i> is a model organism that provides a powerful genetic tool to study gene functions. Study of <i>Drosophila</i> miRNAs has brought us knowledge of its biogenesis and biological functions. The analysis of miRNA typically requires a pretreatment process of extracting total RNAs from target cells, followed by quantitative analysis of target miRNA in total RNA samples, which nevertheless suffers from laborious total RNA extraction and time-consuming processes and poor limit of detection. Meanwhile, the tiny size of <i>Drosophila</i> makes it difficult to accurately measure trivial changes of its cellular miRNA levels. The ability to detect ultralow concentration of miRNA of the proposed method enables the analysis the expression of mir-1 in single <i>Drosophila</i> larva. We thus expect that the strategy may open new avenues for in situ miRNA analysis in single cell or living animals