Attack Site Density of a Highly-efficient PET Hydrolases.

INTRODUCTION: Poly (ethylene terephthalate) (PET) is one of the most abundant polyester materials used in daily life and it is also one of the main culprits of environmental pollution. ICCG (F243I/D238C/S283C/Y127G) is an enzyme that performs four modifications on the leaf branch compost keratase (LCC). It shows excellent performance in the hydrolysis of PET and has a great potential in further applications. METHOD: Here, we used ICCG to degrade PET particles of various sizes and use the density of attack sites (Γattack) and kinetic parameters to evaluate the effect of particle size on enzyme degradation efficiency. We are surprised to observe that there is a certain relationship between Km and Γattack. In order to further confirm the relationship, we obtained three different enzymes (Y95K, M166S and H218S) by site-directed mutagenesis on the basis of ICCG. RESULT: The results confirmed that there was a negative correlation between Km and Γattack. In addition, we also found that increasing the affinity between the enzyme and the substrate does not necessarily lead to the increase of degradation rate. CONCLUSION: These findings show that the granulation of PET and the selection of appropriate particle size are helpful to improve its industrial application value. At the same time, additional protein engineering to increase ICCG performance is realistic, but it can't be limited to enhance the affinity between enzyme and substrate.

Photoregulation of Gene Expression with Ligand-Modified Caged siRNAs through Host/Guest Interaction.

Small interfering RNA (siRNA) can effectively silence target genes through Argonate 2 (Ago2)-induced RNA interference (RNAi). It is very important to control siRNA activity in both spatial and temporal modes. Among different masking strategies, photocaging can be used to regulate gene expression through light irradiation with spatiotemporal and dose-dependent resolution. Many different caging strategies and caging groups have been reported for light-activated siRNA gene silencing. Herein, we describe a novel caging strategy that increases the blocking effect of RISC complex formation/process through host/guest (including ligand/receptor) interactions, thereby enhancing the inhibition of caged siRNA activity until light activation. This strategy can be used as a general approach to design caged siRNAs for the photomodulation of gene silencing of exogenous and endogenous genes.

Photoregulation of Gene Expression with Amantadine-Modified Caged siRNAs through Host-Guest Interactions.

RNA interference is an essential and powerful tool for targeting and verifying specific gene functions. Conditional control of small interfering RNA (siRNA) activity, especially using light activation, is a potential method for regulating target gene expression and functions. In this study, a series of photolabile siRNAs with amantadine modification have been rationally designed and developed through host-guest interactions between amantadine and ß-cyclodextrin derivatives to enhance the blocking effect of siRNA binding and/or RNA-induced silencing complex processing. These caged siRNAs with amantadine modification at the 5' end of antisense-strand RNA were efficiently inactivated through the host-guest interactions between amantadine and ß-cyclodextrin. Photomodulation of the gene silencing activity of these amantadine-modified caged siRNAs targeting both exogenous and endogenous genes was successfully achieved, which indicates that host-guest interactions could be a new strategy for developing new caged siRNAs for gene photoregulation with low leaking activity.

Synthesis and Evaluation of Caged siRNAs with Single cRGD Modification for Photoregulating RNA Interference.

We designed and synthesized caged siRNAs with photolabile linker and single cRGD peptide modifications for the photoregulation of gene expression. Photolabile linker and cRGD were inserted at 5' terminus of siRNAs to obtain cRGD-modified caged siRNAs. All these caged siRNAs could be activated through light activation to release the native siRNAs and further achieve the photoregulation of gene silencing of two exogenous reporter genes (firefly luciferase and green fluorescent protein, GFP) and one endogenous gene (the mitosis motor protein, Eg5). The intracellular distribution and cellular uptake pathways of these caged siRNAs were also investigated. Tumor-bearing mice were further used to demonstrate the photoregulation of gene silencing with cRGD-modified caged siRNAs in vivo. Overall, the data support the use of this new generation of caged siRNAs in cancer therapy.

Multimerized self-assembled caged two-in-one siRNA nanoparticles for photomodulation of RNAi-induced gene silencing.

We rationally designed and developed caged siRNA nanoparticles (Multi-Chol-siRNA) self-assembled with cholesterol-modified multimerized caged siRNAs for photomodulation of siRNA gene silencing activity. Strong resistance to serum nuclease and RNase A was observed for these cholesterol-modified caged siRNA nanoparticles due to the formation of nanostructures with high intensity of siRNA. These caged Multi-Chol-siRNA self-assembled nanoparticles were successfully used to achieve photochemical regulation of both exogenous GFP and endogenous Eg5 gene expressions with a GFP/RFP transient transfection system and Eg5-associated assays, respectively. Further, Two-in-One caged Multi-Chol-siGFP/siEg5 self-assembled nanoparticles simultaneously targeting GFP and Eg5 genes were also developed. The caged Multi-Chol-siRNA self-assembled nanoparticles have demonstrated the effectiveness of enhancing photomodulation of multiple RNAi-induced gene silencing activities in cells.

Caged siRNAs with single folic acid modification of antisense RNA for photomodulation of exogenous and endogenous gene expression in cells.

Manually controlling siRNA activity is an essentially important way to spatiotemporally investigate gene expression and function. Owing to ease of operation and precise manipulation, light can be used for controlled regulation of siRNA-induced gene silencing. Here, we developed a series of caged siRNAs with folic acid modification at the 5' terminus of the antisense strand of the siRNA through a photolabile linker. The attachment of the folic acid moiety temporarily masked the corresponding siRNA activity. Upon illumination, these caged siRNAs were activated, and their gene silencing activities were restored. Based on this strategy, we successfully photomodulated gene expression of both an exogenous gene (for green fluorescent protein, GFP) and an endogenous gene (for mototic kinesin-5, Eg5) in cells.

Anti-photobleaching flower-like microgels as optical nanobiosensors with high selectivity at physiological conditions for continuous glucose monitoring.

Optical glucose detection holds considerable promise for continuous in vivo glucose monitoring with wireless transdermal transmission and long-lasting activity. To construct a new class of optical glucose nanobiosensors with high sensitivity and selectivity at physiological conditions, the first generation of fluorescent poly(amido amine) (G1.0 PAMAM), serving as the optical code, was introduced into glucose-sensitive poly(N-isopropylacrylamide-(2-dimethylamino)ethyl methacrylate-3-acrylamidephenylboronic acid) copolymer microgels via a facile method. The fabricated microgels display the ability of adapting to the surrounding medium of different glucose concentrations over a clinically relevant range (0-20 mM) and convert biochemical signals into optical signals. As nanobiosensors, the G1.0 PAMAM functionalized microgels exhibit high selectivity for glucose over various kinds of potential primary interferents, such as lactate, human serum albumin and metal ions, in the physiologically important glucose concentration range. Compared to traditional fluorescent dyes and quantum dots, which are limited by photobleaching and toxicity, this microgel with remarkable anti-photobleaching property and low toxicity makes it possible to be used for long-term continuous glucose monitoring. Through in vivo investigations, it can be observed that G1.0 PAMAM functionalized microgels can achieve wireless transdermal detection, indicating that the fabricated microgels have potential applications as a new generation of nanobiosensors for the highly sensitive and minimally invasive continuous glucose monitoring.