A DNA aptamer for binding and inhibition of DNA methyltransferase 1.

DNA methyltransferases (DNMTs) are enzymes responsible for establishing and maintaining DNA methylation in cells. DNMT inhibition is actively pursued in cancer treatment, dominantly through the formation of irreversible covalent complexes between small molecular compounds and DNMTs that suffers from low efficacy and high cytotoxicity, as well as no selectivity towards different DNMTs. Herein, we discover aptamers against the maintenance DNA methyltransferase, DNMT1, by coupling Asymmetrical Flow Field-Flow Fractionation (AF4) with Systematic Evolution of Ligands by EXponential enrichment (SELEX). One of the identified aptamers, Apt. #9, contains a stem-loop structure, and can displace the hemi-methylated DNA duplex, the native substrate of DNMT1, off the protein on sub-micromolar scale, leading for effective enzymatic inhibition. Apt. #9 shows no inhibition nor binding activity towards two de novo DNMTs, DNMT3A and DNMT3B. Intriguingly, it can enter cancer cells with over-expression of DNMT1, colocalize with DNMT1 inside the nuclei, and inhibit the activity of DNMT1 in cells. This study opens the possibility of exploring the aptameric DNMT inhibitors being a new cancer therapeutic approach, by modulating DNMT activity selectively through reversible interaction. The aptamers could also be valuable tools for study of the functions of DNMTs and the related epigenetic mechanisms

2 X 20: Works by 20 of Kentucky\u27s Finest Working Folk Artists

2012 Kentucky Folk Art Center exhibition catalog of the twenty finest working folk artists.https://scholarworks.moreheadstate.edu/kfac_exhibition_catalogs/1009/thumbnail.jp

Asymmetrical Flow Field Flow Fractionation Coupled to Nanoparticle Tracking Analysis for Rapid Enrichment and Online Characterization of Nanoparticles

AF4 is a size separation technique that is routinely used for nanoparticle analysis due to the open channel design, the gentle forces used, and separation being done in native and stable buffers. NTA is an excellent detector for AF4 due to the flow cell fluidics and the individual counting and sizing NTA performs, making it an excellent candidate to use as an online detector to allow simultaneous fractionation and characterization of samples. NTA is label free, making general detection of all NPs above a certain size possible, making this a very versatile detector that can be used for many types of nanoparticles and materials that may be separated by AF4. Chapter 2 focuses on the coupling connection and validation of the AF4-NTA system. The initial design required a split flow design. COMSOL simulations were done to determine the best strategy for splitting the eluent into multiple lines of equivalent sample. A splitter manifold was used to control pressure and flow while a syringe pump was used to control the flow rate going through the NTA. The AF4-NTA system showed great particle counting ability and consistent sizing of the different size populations of standards tested. Some drawbacks were identified with the split flow design, which was mainly band broadening from the larger dead volume of the NTA channel. The main counting ability showed very strong linearity when evaluating sequential increases in injection amounts and produces simple and attractive three-dimensional fractograms. Chapter 3 uses AF4-NTA for the analysis of non-spherical particles and soft-shelled and flexible materials like exosomes. AF4-NTA was used to separate and analyze two different aspect ratios of gold nanorods. The NTA hydrodynamic size and TEM data were used to determine the conversion factor between the NTA hydrodynamic size and the rod length. Additionally, it was shown that offline NTA had difficulty identifying the two populations but were easily identified when utilizing AF4 separations before NTA analysis. AF4-NTA was also used to analyze 2D nanomaterials and the protein corona formation around it once it was incubated in cell culture medium. AF4-NTA showed very rapid analysis of nanomaterials, allowing quick determination of aggregation and protein corona thickness. Finally, the system was tested against softer and more flexible materials, which AF4 has advantages over SEC. Good recovery, quantification, and sizing indicate the AF4-NTA is quite versatile and applicable to a wide array of sample types, matrices, sizes, shapes, and composition. Chapter 4 focuses on the development of AF4-NTA for exosome analysis. AF4-NTA showed applicability in measuring extracellular vesicles, but additional downstream analysis is needed to make use of the BioNP information. AF4 was used as a preparatory step for the downstream analysis while the offline NTA data characterizes the BioNPs collected. The AF4 recovery was compared to other common isolation techniques as well as the purity and native size investigated and showed comparable purification abilities. In-channel labeling was performed as an efficient way to wash exosomes of unbound probes, allowing simple plating and quantification of the probes. This technique removes every step of blocking, incubating, and washing because AF4 can complete this on the analyte directly during the size separation. While AF4-NTA was never ran with the final probe designs due to the COVID-19 shutdowns, it was shown that in-channel labeling and protein label quantification within the ranges of NTA is possible. AF4-NTA is versatile with broad applicability. The online NTA data saves time, sample, and allows direct relation of particle counts to downstream results. The throughput is very quick compared to individual steps of purification, characterization, and protein quantification. Some drawbacks still exist, such as band broadening from slow flow rates in NTA, AF4 dilution factor, and limited working ranges by NTA. Some of these can be further improved while some are inherent drawbacks of the individual instruments used, but AF4-NTA was shown to be an ideal candidate for general nanoparticle analysis, especially when dealing with complex matrices or mixtures of multiple size populations

Rapid Enrichment and Detection of Extracellular Vesicles Enabled by CuS-Enclosed Microgels.

Extracellular vesicles (EVs) are cell-derived membranous vesicles that exist in nearly all biological fluids, including blood and urine; and carry a great number of cargo molecules such as protein, nucleic acids, and lipid. They may play important roles in cell-cell communication and modulation of pathological processes, which, however, are not yet well understood, calling for highly sensitive, specific, and rapid methods for EV detection and quantification in biological samples. Here, we report the CuS-enclosed microgels that not only help enrich EVs carrying specific protein markers from complex biomatrices, but also produce strong chemiluminescence (CL) to realize sensitive detection of the target EVs. A detection limit of 104 EV particles/mL was achieved with these microgels by targeting EV proteins like CD63 and HER2, with a dynamic range up to 108 particles/mL. Direct detection of EVs in human serum and cell culture medium without tedious sample preparation was demonstrated, consuming much less sample compared to ELISA and Western Blot. We envision that our method will be valuable for quick quantification of EVs in biological samples, benefiting disease monitoring and functional study

Rapid Enrichment and Detection of Extracellular Vesicles Enabled by CuS-Enclosed Microgels.

Extracellular vesicles (EVs) are cell-derived membranous vesicles that exist in nearly all biological fluids, including blood and urine; and carry a great number of cargo molecules such as protein, nucleic acids, and lipid. They may play important roles in cell-cell communication and modulation of pathological processes, which, however, are not yet well understood, calling for highly sensitive, specific, and rapid methods for EV detection and quantification in biological samples. Here, we report the CuS-enclosed microgels that not only help enrich EVs carrying specific protein markers from complex biomatrices, but also produce strong chemiluminescence (CL) to realize sensitive detection of the target EVs. A detection limit of 104 EV particles/mL was achieved with these microgels by targeting EV proteins like CD63 and HER2, with a dynamic range up to 108 particles/mL. Direct detection of EVs in human serum and cell culture medium without tedious sample preparation was demonstrated, consuming much less sample compared to ELISA and Western Blot. We envision that our method will be valuable for quick quantification of EVs in biological samples, benefiting disease monitoring and functional study

Highly Efficient Exosome Isolation and Protein Analysis by an Integrated Nanomaterial-Based Platform

Exosomes play important roles in mediating intercellular communication and regulating a variety of biological processes, but clear understanding of their functions and biogenesis has not been achieved, due to the high technical difficulties involved in analysis of small vesicular structures that contain a high proportion of membrane structures. Herein, we designed a novel approach to integrate two nanomaterials carrying varied surface properties, the hydrophilic, macroporous graphene foam (GF) and the amphiphilic periodic mesoporous organosilica (PMO), for efficient exosome isolation from human serum and effective protein profiling. The high specific surface area of GF, after modification with the antibody against the exosomal protein marker, CD63, allowed highly specific isolation of exosomes from complex biological samples with high recovery. Since the organic solvent, methanol, turned out to be the most effective lysis solution for releasing the exosomal proteins, the amphiphilic PMO was employed to rapidly recover the exosomal proteins, including the highly hydrophobic membrane proteins. The fine pores of PMO also acted as the nanoreactors to accelerate protein digestion that produced peptides subject to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. A total of 334 proteins with 111 membrane proteins [31% of these contained >2 transmembrane domains (TMD)] were identified using the integrated GF/PMO platform. In contrast, with the commercial exosome isolation kit and the in-solution protein digestion method, only 151 proteins were found, with 28 being membrane proteins (only one contained three TMDs). Our results support that the integrated GF/PMO platform is of great value to facilitate the comprehensive characterization of exosomal proteins for better understanding of their functions and for identification of more exosome-based disease markers

Calibration-free analysis of surface proteins on single extracellular vesicles enabled by DNA nanostructure.

Extracellular vesicles (EVs) are essential intercellular communicators that are of increasing interest as diagnostic biomarkers. Exploring their biological functions and clinical values, however, remains challenging due to their small sizes and high heterogeneity. Herein, we report an ultrasensitive method that employs target-initiated construction of DNA nanostructure to detect single EVs with an input as low as 100 vesicles/μL. Taking advantage of both DNA nanostructure labeling and EV membrane staining, the method can also permit calibration-free analysis of the protein profiles among different EV samples, leading to clear EV differentiation by their cell of origin. Moreover, this method allows co-localization of dual protein markers on the same EV, and the increased number of EVs carrying dual tumor proteins present in human serum could differentiate cancer patients at the early developmental stage from healthy controls. Our results demonstrate the great potential of this single-EV visualization method in non-invasive detection of the EV-based protein biomarkers for cancer diagnosis and treatment monitoring

Rapid Enrichment and Sensitive Detection of Multiple Metal Ions Enabled by Macroporous Graphene Foam

Nanomaterials have shown great promise in advancing biomedical and environmental analysis because of the unique properties originated from their ultrafine dimensions. In general, nanomaterials are separately applied to either enhance detection by producing strong signals upon target recognition or to specifically extract analytes taking advantage of their high specific surface area. Herein, we report a dual-functional nanomaterial-based platform that can simultaneously enrich and enable sensitive detection of multiple metal ions. The macroporous graphene foam (GF) we prepared displays abundant phosphate groups on the surface and can extract divalent metal ions via metal-phosphate coordination. The enriched metal ions then activate the metal-responsive DNAzymes and produce the fluorescently labeled single-stranded DNAs that are adsorbed and quenched by the GF. The resultant fluorescence reduction can be used for metal quantitation. The present work demonstrated duplexed detection of Pb²⁺ and Cu²⁺ using the Pb- and Cu-responsive DNAzymes, achieving a low detection limit of 50 pM and 0.6 nM, respectively. Successful quantification of Pb²⁺ and Cu²⁺ in human serum and river water were achieved with high metal recovery. Since the phosphate-decorated GF can enrich diverse types of divalent metal cations, this dual-functional GF-DNAzyme platform can serve as a simple and cost-effective tool for rapid and accurate metal quantification in determination of human metal exposure and inspection of environmental contamination

A Single Extracellular Vesicle (EV) Flow Cytometry Approach to Reveal EV Heterogeneity.

Extracellular vesicles (EVs) actively participate in intercellular communication and pathological processes. Studying the molecular signatures of EVs is key to reveal their biological functions and clinical values, which, however, is greatly hindered by their sub-100 nm dimensions, the low quantities of biomolecules each EV carries, and the large population heterogeneity. Now, single-EV flow cytometry analysis is introduced to realize single EV counting and phenotyping in a conventional flow cytometer for the first time, enabled by target-initiated engineering (TIE) of DNA nanostructures on each EV. By illuminating multiple markers on single EVs, statistically significant differences are revealed among the molecular signatures of EVs originating from several breast cancer cell lines, and the cancer cell-derived EVs among the heterogeneous EV populations are successfully recognized. Thus, our approach holds great potential for various biological and biomedical applications

A DNA aptamer for binding and inhibition of DNA methyltransferase 1.

DNA methyltransferases (DNMTs) are enzymes responsible for establishing and maintaining DNA methylation in cells. DNMT inhibition is actively pursued in cancer treatment, dominantly through the formation of irreversible covalent complexes between small molecular compounds and DNMTs that suffers from low efficacy and high cytotoxicity, as well as no selectivity towards different DNMTs. Herein, we discover aptamers against the maintenance DNA methyltransferase, DNMT1, by coupling Asymmetrical Flow Field-Flow Fractionation (AF4) with Systematic Evolution of Ligands by EXponential enrichment (SELEX). One of the identified aptamers, Apt. #9, contains a stem-loop structure, and can displace the hemi-methylated DNA duplex, the native substrate of DNMT1, off the protein on sub-micromolar scale, leading for effective enzymatic inhibition. Apt. #9 shows no inhibition nor binding activity towards two de novo DNMTs, DNMT3A and DNMT3B. Intriguingly, it can enter cancer cells with over-expression of DNMT1, colocalize with DNMT1 inside the nuclei, and inhibit the activity of DNMT1 in cells. This study opens the possibility of exploring the aptameric DNMT inhibitors being a new cancer therapeutic approach, by modulating DNMT activity selectively through reversible interaction. The aptamers could also be valuable tools for study of the functions of DNMTs and the related epigenetic mechanisms