Seonmi Shin

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.

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.

Aptamer-Based Alternatives to the Conventional Immobilized Metal Affinity Chromatography for Purification of His-Tagged Proteins

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.

Sniffing for Gene-Silencing Efficiency of siRNAs in HeLa Cells in Comparison with That in HEK293T Cells: Correlation Between Knockdown Efficiency and Sustainability of siRNAs Revealed by FRET-Based Probing

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.