Biomedical subjects

Suhyung Cho

Publications and source records attributed to Suhyung Cho.

3 recordsLinked to original sources

PubMed2005

Bis-aptazyme sensors for hepatitis C virus replicase and helicase without blank signal.

The fusion molecule (i.e. aptazyme) of aptamer and hammerhead ribozyme was developed as in situ sensor. Previously, the hammerhead ribozyme conjugated with aptamer through its stem II module showed a significant blank signal by self-cleavage. To reduce or remove its self-cleavage activity in the absence of target molecule, rational designs were attempted by reducing the binding affinity of the aptazyme to its RNA substrate, while maintaining the ribonuclease activity of the aptazyme. Interestingly, the bis-aptazymes which comprise the two aptamer-binding sites at both stem I and stem III of the hammerhead ribozyme showed very low blank signals, and their ratios of reaction rate constants, i.e. signal to noise ratios, were several tens to hundred times higher than those of the stem II-conjugated bis-aptazymes. The reduction in the blank signals seems to be caused by a higher dissociation constant between the main strand of the bis-aptazyme and its substrate arising from multi-point base-pairing of the bis-aptazymes. The bis-aptazymes for HCV replicase and helicase showed high selectivity against other proteins, and a linear relationship existed between their ribozyme activities and the target concentrations. In addition, a bis-aptazyme of dual functions was designed by inserting both aptamers for HCV replicase and helicase into the stem I and stem III of hammerhead ribozyme, respectively, and it also showed greater sensitivity and specificity for both proteins without blank signal.

Aptamers, Nucleotide↗

PubMed2005

Microaffinity purification of proteins based on photolytic elution: toward an efficient microbead affinity chromatography on a chip.

A bead affinity chromatography system, which was based on the photolytic elution method, was integrated into a glass-silicon microchip to purify specific target proteins. CutiCore beads, which were coupled with a photo-cleavable ligand, such as biotin and an RNA aptamer, were introduced into a filter chamber in the microchip. The protein mixture containing target protein labeled with fluorescein isothiocyanate (FITC) was then passed through the packed affinity beads in the microchamber by pressure-driven flow. During the process, the adsorbed protein on the bead was monitored by fluorescence. The concentrated target protein on the affinity bead was released by simple irradiation with UV light at a wavelength of 360 nm, and subsequently eluted with the phosphate buffer flow. The eluted target protein was quantitatively detected via the fluorescence intensity measurements at the downstream of the capillary connected to the outlet of the microchip. The microaffinity purification allowed for a successful method for the identification of specific target proteins from a protein mixture. In addition, the feasibility of this system for use as a diagnosis chip was demonstrated.

Biotin↗

PubMed2004

Microbead-based affinity chromatography chip using RNA aptamer modified with photocleavable linker.

A microbead-based affinity chromatography chip (micro-BACC) controlling hundreds of nanoliters of reaction volume was developed to separate and analyze hepatitis C virus (HCV) RNA polymerase protein by immobilization of an RNA aptamer on beads. A photocleavable linker was conjugated in between the beads and the aptamer to elute the bound RNA polymerase from the RNA aptamer in one step by UV irradiation, resulting in an efficient method to elute and identify the target molecule bound on RNA using a mass spectrometer. This linker showed a cleavage activity over 70% upon UV irradiation at 1050 mW/cm2 for more than 5 min. The photoelution method could prevent the target molecule from contaminations in affinity chromatography caused by elution solutions of high salt concentration, extreme pH and detergent, respectively. In this chip, sample reagents up to 800 nL could be metered quantitatively into the bead chamber using a nanoliter dispenser working, based on surface-guided flow control and pneumatic control by external air pressure on the chip. RNA polymerase eluted after UV irradiation was successfully analyzed by trypsin treatment without additional purification. As a result, using the aptamer, we could detect RNA polymerase from 800 nL hepatitis C patient serum containing 96 fmol HCV RNA polymerase. The detection limit of this system was estimated to be 9.6 fmol HCV RNA polymerase.

Chromatography, Affinity↗