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In this study, we describe the development of a new siRNA delivery method for enhanced gene silencing, which is based on HER2 RNA aptamer- and cell penetrating peptide (CPP)-mediated intracellular delivery of multimeric antisense strands of siRNAs. By making use of HER2 RNA aptamer or peptide nucleic acid-linked CPP as delivery moiety for multimeric antisense strands of siRNAs, the multimeric siRNAs were observed to show significant gene silencing effects even without any delivery vehicles, in comparison with the conventional duplexed monomeric siRNAs mixed with lipofectamine. Our observations suggest that the introduction of RNA aptamer or CPP to multimeric siRNA as delivery moiety of siRNA delivery system can allow efficient target gene suppression with slow and steady but persistent intracellular uptake capacity.

A single nucleotide polymorphism (SNP) is a single base-pair substitution that commonly constitutes the genetic variation in individuals. Because of its association with disease susceptibility and drug resistance, SNP detection is of great value in studying the variation in drug responses. Here we report four types of washing-free (i.e., separation-free or sequential addition of reagents) and polymerase chain reaction (PCR)-free fluorescence signal production platforms for quantitative detection of a single-base mismatch in RNA and compare their analytical efficiency. The RNA-templated SNP detection methods in this study are based on the allele-specific hybridization approach and/or on the allele-specific oligonucleotide ligation approach, and have been successfully applied to the quantitation of single-base mutation (C → U) in RNA of the BCR–ABL fusion gene, by endpoint measurements of fluorescence intensity. These methods have several advantages for practical applications, such as direct discrimination of single-base mismatch of the RNA extracted from cells, no requirement of PCR amplification, performance of homogeneous detection, easy design of detection probes, and suitability for large-scale genotyping.

Aptamers are oligonucleotides or peptide molecules that are able to bind to their specific target molecules with high affinity via molecular recognition. In this study, we present development of aptamer-based precipitation assays (or simply aptamoprecipitation) for His-tagged proteins and thrombin to compare their purification efficiency with other conventional affinity precipitation methods. A crosslinking method was employed to immobilize thiol-functionalized aptamers onto the surface of polystyrene resins, enabling them to specifically bind to His-tag and to thrombin, respectively. The resulting aptamer-functionalized resins were successfully applied via a one-step experiment to purification of His-tagged proteins from complex E. coli and to thrombin extraction, exhibiting superior or at least comparable purification results to the conventional immobilized metal affinity precipitation or immunoprecipitation.

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.

Department of Chemistry and Research Institute for Basic Sciences, Kyung Hee University, Seoul 131-701, Korea *E-mail: sshah@khu.ac.kr Department of Laboratory Medicine, Center for Diagnostic Oncology, Research Institute and Hospital, National Cancer Center, Goyang-si, Gyeinggi-do 410-769, Korea Hematologic Malignancy Branch, Research Institute and Hospital, National Cancer Center, Goyang-si, Gyeinggi-do 410-769, Korea. *E-mail: mindcatch@ncc.re.kr Received January 13, 2014, Accepted January 22, 2014

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.

This paper aims to address the cellular toxicity of ultra-pure titanium dioxide () and zinc oxide (ZnO) nanoparticles (NPs) frequently employed in sunscreens as inorganic physical sun blockers to provide protection against adverse effects of ultraviolet (UV) radiation including UVB (290-320 nm) and UVA (320-400 nm). In consideration that the production and the use of inorganic NPs have aroused many concerns and controversies regarding their safety and toxicity and that microsized and ZnO have been increasingly replaced by and ZnO NPs ( and ZnO NPs. NPs were prepared through a physical process of irradiating solid targets in liquid phase, enabling verification of the toxicity of ultra-pure NPs with nascent surfaces free from any contamination. Our results show that NPs are essentially non-poisonous and ZnO NPs are more toxic than NPs based on the cell viability assays.

Investigation of the intracellular fate of small interfering RNAs (siRNAs) following their delivery into cells is of great importance to elucidate their dynamics in cytoplasm. Here we describe the use of an advanced fluorescence-based method to probe the dissociation and/or degradation of double-labeled siRNAs in HeLa cells in comparison with that in human embryonic kidney 293T (HEK293T) cells. This work was performed with three siRNAs labeled with fluorescence resonance energy transfer (FRET) dyes, allowing a non-destructive and non-invasive assessment of the dissociation and degradation state of siRNAs in cultured cells. Our FRET analysis not only shows the asymmetric degradation as well as the time-dependent dissociation of each siRNA strand during the measured time period, underlining the high intrinsic nuclease resistance of duplex siRNAs, but also reveals the longer sustainability of siRNAs in HeLa cells compared with that in HEK293T cells, explaining the gene silencing in HeLa cells is more efficient than that in HEK293T cells. In addition, our single-molecule FRET assays demonstrate the potential of the delineated fluorescence-based technique for future research on biological behavior of siRNAs even at the single-molecule level. The fluorescence-based method is a straightforward technique to gain direct information on siRNA integrity inside living cells, which can provide a detection tool for dynamics of biological molecules.

Aptamers are oligonucleotides or peptide molecules that are able to bind to their specific target molecules with high affinity via molecular recognition. In this study, we present development of aptamer-based affinity purification for His-tagged proteins for comparison of purification efficiency with the conventional Ni2+-based affinity chromatography. Thiol-functionalized aptamers able to specifically bind to His-tag were immobilized employing two crosslinking methods onto the surface of polystyrene resins. The resulting aptamer-anchored resins were successfully applied for purification of His-tagged proteins from complex E. coli and human cell lysates, respectively, and superior or at least comparable purification results to the conventional immobilized metal affinity chromatography were obtained via one-step purification.

Nanoparticles (NPs) are increasingly used in consumer products, which have aroused many concerns and debates regarding their fate in biological systems from a point of their safety/toxicity. Although a number of studies on the biological effects of NPs have been published, these are often complicated by the possible toxicity of conventional NPs, caused by contamination with chemical precursors or additives during their synthesis and/or purification procedures. To explicitly understand the toxicity basis of NPs, it is necessary to directly address a main problem related to their intrinsic/inherent toxicity and/or incompatibility with biological objects. The present study is designed to take advantage of a novel laser-assisted method called laser ablation to generate Ag, Au, Co, and Cu NPs in biocompatible aqueous solution, and to evaluate the toxicity of the resulting ultra-pure NPs. Our results show that the ultra-pure NPs with nascent surfaces possess moderate cytotoxicity to human cells in a cell-dependent manner.

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.

A highly sensitive accelerator mass spectrometry (AMS)based assay for determination of the 14 C content in the scraped silica gel after thin layer chromatography (TLC) has been established. In the present study, 14 C-labeled inositol or its cellular derivatives was selected as a model analyte to evaluate the designed assay, since a series of phosphatidyl inositols (PIs) are known to be derived from inositol and to play pivotal roles as precursors or second messengers for several cell signaling processes. After incubating HeLa cells with 14 C-labeled inositol, allowing the dosed 14 C-labeled inositol to be converted to a series of 14 C-labeled PIs and phosphoinositides through biochemical processes, the phospholipids were extracted and separated by the conventional TLC. The resulting TLC plates were scraped and graphitized for AMS measurement. The obtained data demonstrates the intracellular distribution of the phosphatidyl inositols, implying that the developed assay is better and more conv

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.

)1,2-Diaminocyclohexaneoxalatoplatinum(II) (oxaliplatin, also known as Eloxatine) is a novel platinum co-ordination compound, recently approved for the treatment of colorectal cancer, a major cause of cancer deaths worldwide, and has been licensed in the European Union since 1999, and in the United States since 2002 (structure shown in Figure 1).

The mechanisms through which estrogens induce breast carcinogenesis have not been definitely unraveled, although corresponding catechol and quinine metabolite formation is known to produce ROS. There are two main hypotheses about how estrogen initiates cancer: one through estrogen receptor- mediated processes and the other by DNA damage caused from the interaction of specific estrogen metabolites with DNA (see Figure 1 for the major pathway of estrogen activation in the for-mation of depurinating estrogen-DNA adducts).

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.

(trans-R, R)-1,2-diaminocyclohexaneoxalatoplatinum(II) (oxaliplatin) is a recently approved platinum analogue for use in the chemotherapy of metastatic colorectal cancer. Like many cytotoxic drugs, oxaliplatin exerts its antitumor effects by covalent modification of DNA. We report an accelerator mass spectrometry (AMS) assay to measure the kinetics of oxaliplatin-induced DNA damage and repair. We determined the apparent rate of oxaliplatin adduction to salmon sperm DNA. The oxaliplatin-DNA adduct distribution was further investigated at the nucleoside level by HPLC-AMS. Cultured platinum-sensitive testicular (833K) and platinum-resistant breast and bladder (MDA-MB-231 and T24, respectively) cancer cells were incubated with a subpharmacological concentration of oxaliplatin (0.2 microM). Both cellular and DNA radiocarbon contents in the drug-sensitive testicular cells had approximately twice the area under the curve as compared to the more platinum-resistant cell lines, implying that differential accumulation of the drug may be responsible for the sensitivity of cancer cells to platinum treatment. The lowest concentration of radiocarbon measured was approximately 1+/-0.1 amol/microg of DNA, when assaying 1 microg of DNA. This sensitivity for measuring oxaliplatin-DNA adducts is the highest reported to date. The sensitivity offered by this method may be applicable to other DNA-damaging drugs, metabolisms studies, and diagnostics development.

Growing evidence suggests that oxidative damage to cells generates mutagenic 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG), which may initiate diseases related to aging and carcinogenesis. Kinetic measurement of 8-oxodG metabolism and repair in cells has been hampered by poor assay sensitivity and by difficulty characterizing the flux of oxidized nucleotides through the relevant metabolic pathways. We report here the development of a sensitive and quantitative approach to characterizing the kinetics and metabolic sources of 8-oxodG in MCF-7 human breast cancer cells by accelerator mass spectrometry. We observed that [ 14 C]8-oxodG at medium concentrations of up to 2 pmol/ml was taken up by MCF-7 cells, phosphorylated to mono-, di-, and triphosphate derivatives, and incorporated into DNA. Oxidative stress caused by exposure of the cells to 17β-estradiol resulted in a reduction in the rate of [ 14 C]8-oxodG incorporation into DNA and an increase in the ratio of 8-oxodG monophosphate (8-oxodGMP) to 8-oxodG triphosphate (8-oxodGTP) in the nucleotide pool. 17β-Estradiol-induced oxidative stress up-regulated the nucleotide pool cleansing enzyme MTH1 and possibly other Nudix-related pyrophosphohydrolases. These data support the conclusion that 8-oxodGTP is formed in the nucleotide pool by both 8-oxodG metabolism and endogenous reactive oxygen species. The metabolism of 8-oxodG to 8-oxodGTP, followed by incorporation into DNA is a mechanism by which the cellular presence of this oxidized nucleoside can lead to mutations.

A general protocol is described for measuring the incorporation of radiocarbon-labeled 2′-deoxynucleosides into DNA using accelerator mass spectrometry (AMS). This technology provides attomole (10 −18 mol) sensitivity, with detection limits for DNA analysis in the range of one 14 C atom per 10 11 –10 12 total carbons. In practice this corresponds to approximately 1 labeled nucleoside per 10 11 normal bases. A key aspect of the method is the use of precautions aimed at prevention of artifactual DNA oxidation during the sample preparation by the use of antioxidants and chaotropic salts during the DNA isolation. In principle, any type of appropriately labeled nucleoside derivative can be studied using the described protocol, provided that there is incorporation of the deoxynucleoside into DNA. We demonstrated this protocol using MCF-7 human breast cancer cells and a mouse model for mammary carcinoma, which we dosed with 14 C-labeled 2′-deoxyguanosine (dG) and 14 C-labeled 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG). The nucleoside 8-oxodG is a ubiquitous compound that forms in cells by the reaction of dG with reactive oxygen species which has been associated with numerous disease, carcinogenesis and aging. DNA from cells treated with 14 C-labeled nucleosides was isolated and prepared for analysis by AMS in order to measure the DNA-bound radioactivity. The method allows the generation of reliable and sufficient yields of pure DNA from human cells and animal tissues for analysis of radiocarbon levels. Ultimately, this protocol will be applied to understanding the role of modified nucleoside incorporation into DNA in cancer initiation and progression, but could also be used to study any DNA metabolism process where 14 C-labeled nucleosides are used.

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.

Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra- and interstrand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [14C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. Only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, which allowed for calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 h. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was approximately 100-fold slower than that reported for the more potent cisplatin analogue, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [14C]carboplatin, and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 amol/10 microg of DNA. This sensitivity may allow the method to be used for clinical applications.

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.

One-electron oxidation of 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) yielded a guanidinohydantoin derivative (dGh) and a spiroiminodihydantoin derivative (dSp), both putatively mutagenic products that may be formed in vivo. The nucleoside dGh was the major product at room temperature, regardless of pH. The results are contrary to previously published model studies using 2′,3′,5′-triacetoxy-8-oxo-7,8-dihydroguanosine (Luo, W.; Miller, J. G.; Rachlin, E. M.; Burrows, C. J. Org. Lett. 2000, 2, 613; Luo, W.; Miller, J.G.; Rachlin, E.M.; Burrows, C.J. Chem. Res. Toxicol. 2001, 14, 927), who observed a spiroiminodihydantoin derivative as the major product at neutral pH. Clearly, the functional groups attached to the ribose moiety of 8-oxodG influence the oxidation chemistry of the nucleobase derivative. To explore this chemistry in vivo, 14C-labeled 8-oxodG was synthesized and incubated with growing MCF-7 human breast cancer cells, resulting in the incorporation of the compound into cellular DNA as measured by a novel accelerator mass spectrometry assay.

The DNA oxidation product 7,8-dihydro-8-oxoguanine (8-oxoG) forms several mutagenic oxidation products, including a metastable oxaluric acid (Oa) derivative. We report here that a synthetic oligonucleotide containing Oa hydrolyzes under simulated "in vivo" conditions to form a mutagenic urea (Ua) lesion. Using the Oa 2'-deoxyribonucleoside as a model, the hydrolysis rate depended strongly upon the concentrations of bicarbonate and divalent magnesium. In buffered solutions containing physiologically relevant levels of these species, the half-life of Oa nucleoside was approximately 40 h at 37 degrees C. The mutagenic properties of Ua in DNA were investigated using a M13mp7L2 bacteriophage genome containing Ua at a specific site. Transfection of the lesion-containing genome into wild-type AB1157 Escherichia coli allowed determination of the mutation frequency and DNA polymerase bypass efficiency from the resulting progeny phage. Ua was bypassed with an efficiency of 11% as compared to a guanine control and caused a 99% G-->T mutation frequency, assuming the lesion originated from G, which is at least an order of magnitude higher than the mutation frequency of 8-oxoG under the same conditions. SOS induction of bypass DNA polymerase(s) in the bacteria prior to transfection caused the mutation frequency and type to shift to 43% G-->T, 46% G-->C, and 10% G-->A mutations. We suggest that Ua is instructional, meaning that the shape of the lesion and its interactions with DNA polymerases influence which nucleotide is inserted opposite the lesion during replication and that the instructional nature of the lesion is modulated by the size of the binding pocket of the DNA polymerase. Replication past Ua, when formed by hydrolysis of the 8-oxoG oxidation product Oa, denotes a pathway that nearly quantitatively generates point mutations in vivo.

Department of Emergency Medicine, Kyung Hee University Hospital at Gangdong, Seoul 134-727, KoreaReceived November 29, 2012, Accepted December 26, 2012Key Words : Aptamer, Quantum dot, His-tagged proteinOne of the fundamental formats in biological research is tomonitor and understand the spatio-temporal activity ofbiomolecules from the cellular to the integrative level. Tostudy these interactions, researchers commonly use selectivefluorescent labeling both in vivo cellular imaging and invitro assay detection.

Although the modern biological systems are by large based on DNA genomes and protein enzymes, RNA plays important roles in many fundamental processes in cells, including regulation of protein biosynthesis, RNA splicing, and retroviral replication, with remarkable features. From this point of view, site-specific substitution and derivatization of RNA can provide powerful tools for elucidating RNA structures and functions. The modification of either the 3'and 5'-termini or an internal position of the oligonucleotides with a primary alkylamine group is a widely used method for introducing additional functional groups to the RNA. In particular, several 5'-modifications of RNA molecules such as sulfhydryl modification for functionalizing the 5'-terminus of RNA by a transcription or kinase reaction have been shown to have broad applications in studying RNA structures, mapping RNA-protein interactions, and in vitro selection of catalytic RNAs, since a unique functional group incorporated into the RNA can be subsequently conjugated to the desired molecule by a selective chemical reaction. However, there is still a need to develop coupling chemistry with high stability and yield to modify RNA and other biomolecules. In addition, the coupling functional groups are ideally required to be stable under aqueous reaction conditions, and the coupling reaction should be highly chemoselective. In this regard, Cu-catalyzed azide-alkyne cycloaddition (CuAAC or click chemistry) to form the triazole version of Huisgen’s [2+3] cycloaddition family may be the best choice, because this reaction only occurs between alkynyl and azido functional groups with high yield, and because the resulting 1,2,3-triazoles are stable at aqueous conditions and high temperature. Indeed, the azide group is one of the most utilized bioorthogonal chemical tags for biomolecule-conjugate experiments because of its small size and inertness to most components in a biological environment. We recently reported a two-step synthetic method for 5'azido-5'-deoxyguanosine (azido-G) by adapting literature procedures (Scheme 1) and its efficiency for click chemistry using 6-heptynoyl p-nitroaniline, in consideration that the click chemistry with rapid reaction between the azide and alkyne groups to form a covalent triazole linkage without cross reacting with other functional groups could be used in bioconjugation chemistry. In the present study, we sought enzymatic methods to incorporate azido groups in RNA and measured incorporation efficiency of azido-G into the 5'terminus of RNA by in vitro transcription using T7 RNA polymerase that requires guanosine to efficiently initiate transcription. This is important in consideration that a terminal azido group is able to be introduced to the 5'termini of RNA molecules, and that azides are unstable under solid-phase synthesis conditions. A 97-mer single-stranded DNA containing a T7 promoter at the 3'-end (5'CAG GAC TGC TCT CAC TCT CAC GCA CCA AGA AGC TGC CAT TGA TCC CGC TGC

Department of Emergency Medicine, Kyung Hee University Hospital at Gangdong, Seoul 134-727, KoreaReceived January 6, 2012, Accepted January 20, 2012Key Words : Aging, Lipofuscin, Retinal pigment epithelium Retinal age pigments, or lipofuscin granula, contain anumber of fluorophores that accumulate as a consequence oflight-related vitamin A recycling in association with agingand with some retinal disorders in retinal pigment epithelial(RPE) cells.

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.

E-mail: sshah@khu.ac.krReceived August 4, 2011, Accepted August 8, 2011Key Words : 5'-Modification, RNA, Click chemistry RNA plays important roles in many fundamental pro-cesses in cells, including regulation of protein biosynthesis,RNA splicing, and retroviral replication, with remarkablefeatures.

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.

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.

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.

Emerging as alternatives to antibodies, a wide range of aptamers have been found to bind specifically to proteins. Aptamers, however, offer several advantages over antibodies, due to their relative ease of isola-tion and modification, tailored binding affinity, and resistance against denaturizing. Moreover, their target molecules include metal ions, small organic compounds, metabolites, proteins and even cells. Thus, aptamers have been used in many bioanalytical applications, such as for specific detection of proteins

Several 5′-modifications of RNA molecules have been shown to have broad applications in studying RNA structures, mapping RNA–protein interactions, and in vitro selection of catalytic RNAs. While phosphorothioate modification is one of the most popular methods for functionalizing the 5′-terminus of RNA by a transcription or kinase reaction, conjugation of terminal phosphorothioates with fluorophores has been achieved only with a low efficiency. To overcome this limitation, we have developed a two-step synthetic method for 5′-deoxy-5′-thioguanosine-5′-monophosphorothioate by combining two known reactions and measured its incorporation efficiency into the 5′-terminus of RNA by in vitro transcription using T7 RNA polymerase that requires guanosine to efficiently initiate transcription, followed by treatment of alkaline phosphatase, yielding a terminal sulfhydryl group at the 5′-termini of RNA molecules. Since the sulfhydryl group can be used as an alternative to phosphorothioates, our method may provide a useful route to efficiently introduce reporters, such as fluorophores, into the 5′-terminus of RNA via a stable thio-linker, or to tether the oligomer to a solid support.

A highly sensitive detection method for the determination of protein-ligand interactions has been developed. Radiocarbon-labeled 17beta-estradiol was incubated with estrogen receptor-alpha; as a selective binding partner, and covalently attached using crosslinking agents, to form covalently linked protein-ligand complexes. After separation using a denaturing gel, the (14)C content in the sliced gels was identified by accelerator mass spectrometry. The obtained data demonstrated specific binding of the small molecule to its binding partner. In theory, this method can be applied to most protein-ligand interaction studies.

The first organic artificial proteinase based on synthetic materials is obtained by building active sites on the backbone of poly(ethylenimine) (PEI). The active site comprising three salicylate residues is prepared by preassemblage of three molecules of a salicylate derivative with Fe(III) ion followed by cross-linkage of the salicylates with PEI and demetalation of the resulting polymer. Proteinase activity of the artificial enzyme is demonstrated with hydrolytic cleavage of γ-globulin (Gbn). Both the heavy (50 kDa) and the light (25 kDa) chains of Gbn are effectively cleaved into peptides smaller than 5 kDa. The optimum activity of the artificial proteinase is manifested at pH 5−7. The half-life for cleavage of the two chains of Gbn by the artificial proteinase is ∼1 h at pH 7 and 50 °C when the concentration of the artificial active site is 0.4−1 mM. The activity (at pH 7) of the artificial proteinase prepared with PEI is comparable to that (at pH 9) of a catalytic antibody elicited by a joint hybrido...

β-Cyclodextrin was attached to two dendrimer poly(ethylenimine)s. The resulting cyclodextrin-containing dendrimers, CD-I and CD-II, can be considered either as dendrimers equipped with specific binding sites or as cyclodextrins containing amino groups around the cavities. Amines of CD-I and CD-II remarkably resisted protonation compared with those of the parent dendrimers. A compact conformation of CD-I or CD-II in which the dendrimer wraps itself around the cyclodextrin is proposed as a conformation consistent with the suppressed protonation. Esters containing t -butylphenyl groups were complexed by CD-I and CD-II and underwent fast deacylation. Kinetic data were obtained with several ester substrates, which revealed that two amino groups located in the vicinity of each cyclodextrin cavity of CD-I or CD-II participated as nucleophiles. In addition, optimum reactivity was attained when the spacer connecting the t -butylphenyl and the ester groups was –O–CH 2 – or –CH=CH–. Structures of the active sites for the accelerated deacylation of esters were elucidated on the basis of the kinetic data.

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.

Nanoparticles (NPs) are attractive materials owing to their physical and electrochemical properties, which make them extremely useful in diagnostic applications. Photon upconversion is the phenomenon where high-energy photons are emitted upon excitation of low-energy photons. Nucleic acids detection based on upconversion nanoparticles (UCNPs), which display a high signal-to-noise ratio and no photobleaching, has been widely applied. We evaluated whether UCNPs can improve polymerase chain reaction (PCR) specificity and affect PCR amplification. The effects of UCNPs with a diameter size of 40, 70, and 250 nm were evaluated using 3 PCR kits (AccuPower PCR PreMix, AmpliTaq Gold 360 Master Mix, and HotStarTaq Plus Master Mix) and 3 real-time PCR kits (AccuPower GreenStar qPCR PreMix, SYBR Green PCR Master Mix, and QuantiTect SYBR Green PCR Kit). Quantum dots were used for comparison with the UCNPs. In the presence of an appropriate concentration of UCNPs, PCR specificity was optimized. UCNPs of 40-nm size improved PCR specificity more effectively than did UCNPs sized 70 or 250 nm. As the size and concentrations of the UCNPs were increased, PCR amplification was more severely inhibited. At lower annealing temperatures (25 degrees C-45 degrees C), addition of the 40 nm UCNP (1 mu g/mu L) to the PCR reagent produced specific PCR products without nonspecific sequence amplification. Therefore, UCNPs of different sizes, with different DNA polymerases used in the commercial kits, showed different inhibitory effects on PCR amplification. These results demonstrate that optimization of UCNPs, added to reaction mixtures at appropriate concentrations, can improve PCR specificity. However, the mechanism underlining UCNPs effect on PCR remains unclear and will require further investigation.

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 103 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.

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.

For clinical epidemiologic and proteomic studies, the control of preanalytic variation, including sample processing and storage, is important. We evaluated the stability of progastrin-releasing peptide (ProGRP) as a marker for the quality control of stored serum and plasma samples. The ProGRP from 23 healthy volunteers was measured serially for 8 hours at room temperature, and the results were validated with clinical samples from the biobank. A significant difference in ΔProGRP was also noted between good-quality (time delay

Investigation of the intracellular fate of small interference RNA (siRNA) following their delivery into cells is of great interest to elucidate dynamics of siRNA in cytoplasm. However, its cellular delivery and sustainability should be understood at the molecular level and improved for the successful in vivo application of siRNA. Here we present a fluorescence resonance energy transfer (FRET) based method using oligonucleotide probes to study intracellular dissociation (or melting) and sustainability of siRNAs in live cells. The FRET probes were specifically designed to observe intracellular dissociation (or melting) and degradation of short synthetic RNAs in real-time, thus providing the desired kinetic information in cells. Intracellular FRET analysis shows that siRNA duplex is gradually diffused into cytosol, dissociated, and degraded for a duration of 3.5 h, which is confirmed by confocal microscopy colocalization measurements. In addition, our FRET assays reveal the asymmetric degradation as well as the time-dependent dissociation of each siRNA strand. The application of this FRET technique can allow for direct information on siRNA integrity inside living cells, providing a detection tool for dynamics of biological molecules.

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.