Structural and biochemical basis for the inhibition of cell death by APIP, a methionine salvage enzyme.

APIP, Apaf-1 interacting protein, has been known to inhibit two main types of programmed cell death, apoptosis and pyroptosis, and was recently found to be associated with cancers and inflammatory diseases. Distinct from its inhibitory role in cell death, APIP was also shown to act as a 5-methylthioribulose-1-phosphate dehydratase, or MtnB, in the methionine salvage pathway. Here we report the structural and enzymatic characterization of human APIP as an MtnB enzyme with a Km of 9.32 µM and a Vmax of 1.39 µmol min(-1) mg(-1). The crystal structure was determined at 2.0-Å resolution, revealing an overall fold similar to members of the zinc-dependent class II aldolase family. APIP/MtnB exists as a tetramer in solution and exhibits an assembly with C4 symmetry in the crystal lattice. The pocket-shaped active site is located at the end of a long cleft between two adjacent subunits. We propose an enzymatic reaction mechanism involving Glu139* as a catalytic acid/base, as supported by enzymatic assay, substrate-docking study, and sequence conservation analysis. We explored the relationship between two distinct functions of APIP/MtnB, cell death inhibition, and methionine salvage, by measuring the ability of enzymatic mutants to inhibit cell death, and determined that APIP/MtnB functions as a cell death inhibitor independently of its MtnB enzyme activity for apoptosis induced by either hypoxia or etoposide, but dependently for caspase-1-induced pyroptosis. Our results establish the structural and biochemical groundwork for future mechanistic studies of the role of APIP/MtnB in modulating cell death and inflammation and in the development of related diseases.

Fluorometric Detection of MicroRNA Using Isothermal Gene Amplification and Graphene Oxide.

We have developed a facile fluorometric system for the detection of microRNA (miRNA), using rolling circle amplification (RCA), graphene oxide (GO), and fluorescently labeled peptide nucleic acid (F-PNA). The padlock probe DNA complementary to a target miRNA was selectively ligated to form circular DNA that was then used as the template for RCA. F-PNAs complementary to the target miRNA were annealed to multiple sites of the isothermally amplified single-stranded RCA product (RCAP) containing multiple target miRNA sequences. This F-PNA/RCAP duplex is less adsorbed onto the GO monolayer, thus attenuating the quenching of F-PNA fluorescence by GO. In the absence of target miRNA (and hence the absence of RCA and duplex formation), the free F-PNA is completely adsorbed onto the GO monolayer and fluorescence quenching ensues. Thus, GO-based fluorescence detection coupled with isothermal gene amplification would be a simple and convenient method for the quantitative detection of miRNA.

Aptamer selection for fishing of palladium ion using graphene oxide-adsorbed nanoparticles.

A new aptamer selection method using graphene oxide (GO)-adsorbed nanoparticles (GO-adsorbed NPs) was employed for specific fishing of palladium ion. High affinity ssDNA aptamers were isolated through 13 rounds of selection and the capacity of the selected DNA aptamers for palladium ion uptake was measured, clarifying that DNA01 exhibits the highest affinity to palladium ion with a dissociation constant (Kd) of 4.60±1.17 µM. In addition, binding ability of DNA01 to palladium ion was verified against other metal ions, such as Li(+), Cs(+), Mg(2+), and Pt(2+). Results of the present study suggest that future modification of DNA01 may improve palladium ion-binding ability, leading to economic recovery of palladium from water solution.

Improved ligand binding by antibody-aptamer pincers.

To increase the affinities of antibodies or aptamers for their targets, we designed antibody-aptamer pincers (AAPs) or heterodimers for thrombin or human epidermal growth factor 2 (HER2) as a model system. For this purpose, we first conjugated a 15-mer or 29-mer anti-thrombin aptamer, which are well-known to bind to thrombin in two specific epitopes, with an anti-thrombin antibody to enable each binding part of the AAP to simultaneously recognize a different part of the thrombin molecule. The AAP comprising a 15-mer aptamer and an anti-thrombin antibody has an apparent dissociation constant (Kd(app)) value of 567 pM, and this value is approximately 1/100 of that of the antibody alone or 1/35 of that of the aptamer monomer alone. The AAP comprising a 29-mer aptamer and an anti-thrombin antibody has a much lower Kd(app) value than that of 15-mer aptamer-conjugated antibody. Furthermore, this concept of the AAP system was employed to HER2-targeted drug delivery system (DDS) based on both antibody and drug-loaded aptamer. Anti-HER2 aptamer was conjugated with anti-HER2 antibody and loaded with doxorubicin, and the resulting AAP-HER2-Dox was found to have approximately 3- and 6-fold higher cytotoxicity than drug alone and antibody alone, respectively. Therefore, this novel AAP system constructed by conjugation of the antibody with the aptamer can effectively improve the affinities of the resulting AAPs for their target molecules and the drug-loaded AAP system can possibly serve as a platform for targeted DDS against many malignancies.

TAMRA- and Cy5-labeled probe for efficient kinetic characterization of caspase-3.

Our objective was to create a novel fluorogenic substrate for efficient in vitro kinetic assays on caspase-3. We designed a TAMRA (5'-tetramethylrhodamine-5(6)-carboxamide)- and Cy5 (cyanine 5)-labeled probe that allowed us to evaluate the caspase-3 activity via the changes in fluorescence intensity and wavelength. The prepared probe was found to be an efficient and selective substrate of caspase-3, with V(max) of 41.4±3.3 nM/min and K(M) of 1.60±0.23 µM. The strategy used in the design of this fluorogenic substrate can be applied in future endeavors to development of substrates for caspase-3 inhibitor screening assays or for real-time detection of apoptosis in living cells.

A colorimetric sandwich-type assay for sensitive thrombin detection based on enzyme-linked aptamer assay.

A colorimetric sandwich-type assay based on enzyme-linked aptamer assay has been developed for the fast and sensitive detection of as low as 25 fM of thrombin with high linearity. Aptamer-immobilized glass was used to capture the target analyte, whereas a second aptamer, functionalized with horseradish peroxidase (HRP), was employed for the conventional 3,5,3',5'-tetramethylbenzidine (TMB)-based colorimetric detection. Without the troublesome antibody requirement of the conventional enzyme-linked immunosorbent assay (ELISA), as low as 25 fM of thrombin could be rapidly and reproducibly detected. This assay has superior, or at least equal, recovery and accuracy to that of conventional antibody-based ELISA.

Aptamer-based competitive binding assay for one-step quantitation of hepatitis B surface antigen.

An aptamer-based competitive binding assay for one-step (i.e. no requirement of pre-treatment) quantitation of target molecules of interest has been developed. This method has been successfully employed for the fast and sensitive detection of the surface antigen of the hepatitis B virus (HBsAg). The key features of our method include its low intrinsic background noise, low costs, high resolution, and high sensitivity, enabling the detection of as low as 1.25 mIU mL(-1), approximately 40-fold better than that of the most widely used Abbott Architect assay for HBsAg detection, without the tedious extraction and/or washing procedures. Moreover, this assay has better recovery and accuracy than that of conventional competitive binding assay or others for HBsAg quantitation.

Poly(A)-targeting molecular beacons: fluorescence resonance energy transfer-based in vitro quantitation and time-dependent imaging in live cells.

Quantitation of poly(A)-RNA, time-dependent visualization of intracellular poly(A)(+)-RNA localization in living mammalian cells, and time-resolved intracellular binding dynamics of molecular beacons at the single-molecule level using a fluorescence resonance energy transfer (FRET)-based molecular beacon are described. FRET-based molecular beacons were designed as poly(A)-targeting probes to be oligonucleotides that contained Cy5 and Cy3 fluorescent dyes at the strand ends and a poly(A)-targeting sequence inside the strand. Our ratiometric analysis using poly(A)-targeting probes allowed for highly specific and wide-ranging detection (from 1.25nM to 0.5µM) of poly(A)-RNA, as well as for determination of K(d) values, and revealed a distribution of the probe itself and localization of the target RNA sequence in cells. Furthermore, time-dependent FRET-mediated fluorescence changes at the single-molecule level caused by the folding-induced gradual conformation changes in live cells were observed.

Coomassie blue is sufficient for specific protein detection of aptamer-conjugated chips.

An aptamer-based biochip for protein detection and quantitation which combines the recent biochip technology and the conventional staining methods, is described. Using a model system comprising His-tagged proteins as the analyte and single-stranded RNA aptamers specific for His-tagged proteins as immobilized ligands on chips, we could demonstrate that aptamers were equivalent or superior to antibodies in terms of specificity and sensitivity, respectively. The sensor has the characteristics of good stability, reproducibility and reusability, with detection limit as low as 85 ng/mL His-tagged protein. It has been demonstrated that the sensor can be stored for at least 4 weeks and reused with reasonable reduction rate of staining intensity. In conclusion, we could show the suitability of nucleic acid aptamers as low molecular weight receptors on biochips for sensitive and specific protein detection and quantitation.

Molecular beacon-based quantitiation of epithelial tumor marker mucin 1.

Mucin 1 (Muc1) is a glycoprotein expressed on most epithelial cell surfaces, which has been confirmed as a useful biomarker for the diagnosis of early cancers. In this study, we demonstrate that a quantum dot (QD)-aptamer beacon acts by folding-induced dissociation of a DNA intercalating dye, BOBO-3, in the presence of the target molecules, Muc1. Release of intercalated BOBO-3s from the QD-conjugated aptamers results in a decrease in QD fluorescence resonance energy transfer (FRET)-mediated BOBO-3 emission, allowing for label-free Muc1 detection and quantitation. We attain highly specific and wide-range detection (from 50nM to 20µM) of Muc1, suggesting that our QD-aptamer beacon can be a potential alternative to immuno-based assays for Muc1 detection. The detection methodology is expected to be improved for the early diagnosis of different types of epithelial cancers of large populations.

Detection of single-base mutation in RNA using T4 RNA ligase-based nick-joining or DNAzyme-based nick-generation.

A single nucleotide polymorphism (SNP) is a common genetic variation when a single nucleotide differs between members of a species or paired chromosome. Due to its association with disease susceptibility and drug resistance, SNP detection is of great value in studying the variation in drug responses. Here we present two quantitative SNP detection methods for a single-base mismatch in RNA, based on nick-joining and nick-generating activities of T4 RNA ligase and DNAzyme, respectively. T4 RNA ligase successfully discriminated a one-base mismatch in the ligation junction, and the designed DNAzyme cleaved RNA by discerning a single-base mismatch in the cleaving site.

Binding of uranyl ion by a DNA aptamer attached to a solid support.

A UO(2)(2+)-specific DNA aptamer was attached to aminopolystyrene (aminoPS) using sulfo-SMCC as a crosslinking agent in view of high affinity of DNA for uranyl ion. Capacity of the aptamer-conjugated aminoPS resins for uranyl uptake was measured, revealing that about 0.63 µg of uranium can be complexed to 1g of the resins, which clearly demonstrates that most of DNA aptamers introduced to the resins can strongly bind to uranyl ion. In the presence of 21 mM bicarbonate ion at pH 8.01, apparent dissociation constant (K(d)(app)) of about 84.6 pM and log formation constant (K(f)) of about 22.9 were obtained. Results of the present study strongly suggest that modification of the aptamer-containing resins can improve uranyl-binding ability, probably leading to economical recovery of uranium from seawater.

Functionalized quantum dots to quantify NADPH and their use for NADP+-dependent biocatalyzed transformations.

Quantum dots are semiconducting nanoparticles that can be prepared with interesting optical properties. The fluorescent properties of quantum dots are one of the key advantages for their use as optical labels for biorecognition events and biocatalytic processes. We have prepared semiconductor quantum dots conjugated with Nile Blue (NB), and demonstrate that NB-functionalized quantum dots can act as versatile probes to analyze different biocatalyzed transformations, and can be used for the quantitative detection of NADPH as well as NADH. This approach provides a new path for the optical detection of NAD(P)H and for the quantitative analysis of NAD(P)(+)-dependent biotransformations.

Towards biomarker-dependent individualized chemotherapy: exploring cell-specific differences in oxaliplatin-DNA adduct distribution using accelerator mass spectrometry.

Oxaliplatin is a third-generation platinum-based anticancer drug that is currently used in the treatment of metastatic colorectal cancer. Oxaliplatin, like other platinum-based anticancer drugs such as cisplatin and carboplatin, is known to induce apoptosis in tumor cells by binding to nuclear DNA, forming monoadducts, and intra- and interstrand diadducts. Previously, we reported an accelerator mass spectrometry (AMS) assay to measure the kinetics of oxaliplatin-induced DNA damage and repair [Hah, S. S.; Sumbad, R. A.; de Vere White, R. W.; Turteltaub, K. W.; Henderson, P. T. Chem. Res. Toxicol.2007, 20, 1745]. Here, we describe another application of AMS to the measurement of oxaliplatin-DNA adduct distribution in cultured platinum-sensitive testicular (833K) and platinum-resistant breast (MDA-MB-231) cancer cells, which resulted in elucidation of cell-dependent differentiation of oxaliplatin-DNA adduct formation, implying that differential adduction and/or accumulation of the drug in cellular DNA may be responsible for the sensitivity of cancer cells to platinum treatment. Ultimately, we hope to use this method to measure the intrinsic platinated DNA adduct repair capacity in cancer patients for use as a biomarker for diagnostics or a predictor of patient outcome.

An alternative to Western blot analysis using RNA aptamer-functionalized quantum dots.

To make full use both of optical properties of quantum dots (QDs) and of specific interactions between aptamers and their ligands of interest, we employed QD-conjugated RNA aptamer interactions with histidine tag. QDs offer revolutionary fluorescence performance due to their long-term photostability, brilliant colors, fixability, and narrow, symmetrical emission spectra, and aptamers are known to specifically bind to their target molecules, including metal ions, small molecules, and macromolecules. In this study, we have synthesized RNA aptamer-functionalized QDs, and demonstrated their application to specific protein detection, as an alternative to the conventional Western blot analysis. We observed that our RNA aptamer-functionalized QD system dramatically reduced the time and effort required for conventional Western blot analysis, whereas the selectivity was comparable to that of the conventionally available anti-histidine tag antibody and the sensitivity was comparable to that of the Coomassie blue staining method. In principle, owing to the remarkable optical properties of QDs and a wide versatility of aptamers for selection, our system can harness the high brightness, stability and reusability to quantitatively detect aptamer-recognizable proteins. Furthermore, multiplex detection for several proteins on a single blot can be achieved by our new method, which thus may be able to facilitate and simplify the routinely used protein detection procedure, and make a variety of proteomics analysis possible.

Incorporation of extracellular 8-oxodG into DNA and RNA requires purine nucleoside phosphorylase in MCF-7 cells.

7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) is a well-known marker of oxidative stress. We report a mechanistic analysis of several pathways by which 8-oxodG is converted to nucleotide triphosphates and incorporated into both DNA and RNA. Exposure of MCF-7 cells to [(14)C]8-oxodG combined with specific inhibitors of several nucleotide salvage enzymes followed with accelerator mass spectrometry provided precise quantitation of the resulting radiocarbon-labeled species. Concentrations of exogenously dosed nucleobase in RNA reached one per 10(6) nucleotides, 5-6-fold higher than the maximum observed in DNA. Radiocarbon incorporation into DNA and RNA was abrogated by Immucillin H, an inhibitor of human purine nucleoside phosphorylase (PNP). Inhibition of ribonucleotide reductase (RR) decreased the radiocarbon content of the DNA, but not in RNA, indicating an important role for RR in the formation of 8-oxodG-derived deoxyribonucleotides. Inhibition of deoxycytidine kinase had little effect on radiocarbon incorporation in DNA, which is in contrast to the known ability of mammalian cells to phosphorylate dG. Our data indicate that PNP and RR enable nucleotide salvage of 8-oxodG in MCF-7 cells, a previously unrecognized mechanism that may contribute to mutagenesis and carcinogenesis.

Recent advances in biomedical applications of accelerator mass spectrometry.

The use of radioisotopes has a long history in biomedical science, and the technique of accelerator mass spectrometry (AMS), an extremely sensitive nuclear physics technique for detection of very low-abundant, stable and long-lived isotopes, has now revolutionized high-sensitivity isotope detection in biomedical research, because it allows the direct determination of the amount of isotope in a sample rather than measuring its decay, and thus the quantitative analysis of the fate of the radiolabeled probes under the given conditions. Since AMS was first used in the early 90's for the analysis of biological samples containing enriched 14C for toxicology and cancer research, the biomedical applications of AMS to date range from in vitro to in vivo studies, including the studies of 1) toxicant and drug metabolism, 2) neuroscience, 3) pharmacokinetics, and 4) nutrition and metabolism of endogenous molecules such as vitamins. In addition, a new drug development concept that relies on the ultrasensitivity of AMS, known as human microdosing, is being used to obtain early human metabolism information of candidate drugs. These various aspects of AMS are reviewed and a perspective on future applications of AMS to biomedical research is provided.

Scanning probe-based fabrication of 3D nanostructures via affinity templates, functional RNA, and meniscus-mediated surface remodeling.

Developing generic platforms to organize discrete molecular elements and nanostructures into deterministic patterns on surfaces is one of the central challenges in the field of nanotechnology. Here we review three applications of the atomic force microscope (AFM) that address this challenge. In the first, we use two-step nanografting to create patterns of self-assembled monolayers (SAMs) to drive the organization of virus particles that have been either genetically or chemically modified to bind to the SAMs. Virus-SAM chemistries are described that provide irreversible and reversible binding, respectively. In the second, we use similar SAM patterns as affinity templates that have been designed to covalently bind oligonucleotides engineered to bind to the SAMs and selected for their ability to mediate the subsequent growth of metallic nanocrystals. In the final application, the liquid meniscus that condenses at the AFM tip-substrate contact is used as a physical tool to both modulate the surface topography of a water soluble substrate and guide the hierarchical assembly of Au nanoparticles into nanowires. All three approaches can be generalized to meet the requirements of a wide variety of materials systems and thereby provide a potential route toward development of a generic platform for molecular and materials organization.

Upconversion nanoparticle-based Förster resonance energy transfer for detecting the IS6110 sequence of Mycobacterium tuberculosis complex in sputum.

Upconversion nanoparticles (UCNPs), which are excited at near-infrared wavelength (980 nm), emit high-energy photons. Since UCNPs display a high signal-to-noise ratio and no photobleaching, they are extremely useful for diagnostic application. In this study, we applied UCNPs for detecting the IS6110 sequence of the Mycobacterium tuberculosis complex (MTBC) and evaluated the feasibility of the system for use in molecular diagnostics. Using biotinylated primers, IS6110 DNA PCR was performed and the PCR amplicon was then mixed with streptavidin-conjugated UCNPs, followed by intercalation with SYTOX Orange dye. Fluorescence detection for the Förster resonance energy transfer (FRET) of the UCNPs (UCNP-FRET) was then performed. The estimated lowest detection by UCNP-FRET was 10(2) copies/µL of IS6110 DNA (157 bp). The kappa agreement of the UCNP-FRET assay with conventional PCR was 0.8464 (95% confidence interval, 0.7442-0.9486) and false-negative results were reduced. Our results demonstrated the successful implementation of the UCNP-FRET system in detecting the IS6110 sequence of the MTBC and its potential application for molecular diagnostics.

Selective and quantitative cell detection based both on aptamers and the conventional cell-staining methods.

Aptamer-based biochips for selective cell detection and quantitation in combination of the recent biochip technology and the conventional cell staining methods are described. Using a model system comprising HER2- or PSMA-positive cells as the analytes and single-stranded RNA aptamers specific for HER2 or PSMA as immobilized ligands on chips, we could demonstrate that aptamers were equivalent or superior to antibodies in terms of specificity and sensitivity, respectively. In particular, our PSMA-specific sensor was found to have the characteristics of good stability, reproducibility and reusability, with detection limit as low as 10(3) LNCaP cells. In conclusion, we could show the suitability of nucleic acid aptamers as low molecular weight receptors on biochips for sensitive and specific cell detection and quantitation for future diagnostics development.