Kyung Hyun Lee

Combining SELEX Screening and Rational Design to Develop Light-Up Fluorophore−RNA Aptamer Pairs for RNA Tagging

We report here a new small molecule fluorogen and RNA aptamer pair for RNA labeling. The small-molecule fluorogen is designed on the basis of fluorescently quenched sulforhodamine dye. The SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure and fluorescence screening in E. coli have been applied to discover the aptamer that can specifically activate the fluorogen with micromolar binding affinity. The systematic mutation and truncation study on the aptamer structure determined the minimum binding domain of the aptamer. A series of rationally modified fluorogen analogues have been made to probe the interacting groups of fluorogen with the aptamer. These results led to the design of a much improved fluorogen ASR 7 that displayed a 33-fold increase in the binding affinity for the selected aptamer in comparison to the original ASR 1 and an 88-fold increase in the fluorescence emission after the aptamer binding. This study demonstrates the value of combining in vitro SELEX and E. coli fluorescence screening with rational modifications in discovering and optimizing new fluorogen-RNA aptamer labeling pairs.

Inhibitory Effects of Triphlorethol-A on MMP-1 Induced by Oxidative Stress in Human Keratinocytes via ERK and AP-1 Inhibition

Oxidative stress is known to generate reactive oxygen species (ROS) in cells, which subsequently induce the synthesis of matrix metalloproteinases (MMP) and an aging phenomenon. The protective effects of triphlorethol-A, derived from Ecklonia cava, were investigated against hydrogen peroxide (H2O2)-induced damage using human skin keratinocytes. Data showed that triphlorethol-A inhibited ROS formation, induced catalase expression, inhibited DNA damage, and increased cell viability in keratinocytes. Triphlorethol-A treatment significantly reduced MMP-1 expression and production, compared to H2O2-treated cells. In addition, triphlorethol-A abrogated the activation of extracellular signal regulated protein kinase (ERK), which originates upstream of MMP-1 expression, and was induced by H2O2 treatment. Moreover, triphlorethol-A inhibited DNA binding activity of activator protein-1 (AP-1), a downstream transcription factor of ERK. Data indicate that the antioxidative properties of triphlorethol-A involve the inhibition of MMP-1 via ERK and AP-1 inhibition.

Engineering the Stereochemistry of Cephalosporin for Specific Detection of Pathogenic Carbapenemase‐Expressing Bacteria

Reported herein is the design of fluorogenic probes specific for carbapenem-resistant Enterobacteriaceae (CRE) and they were designed based on stereochemically modified cephalosporin having a 6,7-trans configuration. Through experiments using recombinant β-lactamase enzymes and live bacterial species, these probes demonstrate the potential for use in the specific detection of carbapenemases, including metallo-β-lactamases in active bacterial pathogens.

A strategy to enhance the binding affinity of fluorophore–aptamer pairs for RNA tagging with neomycin conjugation

Fluorogenic sulforhodamine-neomycin conjugates have been designed and synthesized for RNA tagging. Conjugates were fluorescently activated by binding to RNA aptamers and exhibited greater than 250-400 fold enhancement in binding affinity relative to corresponding unconjugated fluorophores.

Methylation-mediated control of aurora kinase B and Haspin with epigenetically modified histone H3 N-terminal peptides.

If multiple post-translational modifications are responsible for important biological markers, additional specificity must be present to serve as embedded combinatorial markers for phosphorylation. In this investigation, we have attempted to elucidate the specificity of AURKB and Haspin by using peptides of various lengths that contain all possible methylations, acetylations, and phosphorylations in histone H3 N-terminal peptides. The activity of AURKB is affected by a wide range of modifications from R2 to K14, while that of Haspin is affected significantly by modifications at R2 and K4. In cases where kinase activity is reduced substantially by other modifications, dimethylation at R2 and R8 totally abolishes phosphorylation at S10 promoted by AURKB and as does dimethylation at R2 on Haspin promoted phosphorylation at T3.

An RNA Aptamer That Selectively Recognizes Symmetric Dimethylation of Arginine 8 in the Histone H3 N-Terminal Peptide

Epigenetic modifications of N-terminal histone tails, especially histone H3, are important for the regulation of the target genes in chromatin. Specific methods for detection of these modifications in histone H3 N-terminal peptides are valuable tools for diagnostic and therapeutic purposes. As an alternative to antibodies, RNA aptamers display compatible binding affinities and selectivites against various biologically relevant targets. Systematic evolution of ligands by exponential enrichment (SELEX) was performed against histone H3R8Me2sym. A 14-amino acid peptide that mimics this modified histone tail was prepared in a biotinylated form and 10 selection cycles of SELEX were carried out. This produced 4 aptamers, one of which (clone 1) was observed to have low nanomolar binding affinity (K(d)=12 nM) against the cognate peptide. The affinity of this aptamer is comparable to 2 commercially available antibodies against differently modified histone H3 peptides and it displays a greater selectivity than the antibodies.

Histone H3 N-Terminal Peptide Binds Directly to Its Own mRNA: A Possible Mode of Feedback Inhibition to Control Translation

Give me some feedback: In vitro selection of aptamers against the H3 peptide provided specific hairpin RNAs that possess high homology with histone H3 mRNA. The identified H3 hairpin RNA binds specifically to the H3 peptide with micromolar affinity and dose‐dependently inhibits in vitro translation of the H3 protein. Consequently, the hairpin RNA and H3 peptide are one of the rare cis‐ and trans‐elements on coding regions found among housekeeping proteins in higher eukaryotes.