Dae-Ro Ahn

Vertical silicon nanowires as a universal platform for delivering biomolecules into living cells

A generalized platform for introducing a diverse range of biomolecules into living cells in high-throughput could transform how complex cellular processes are probed and analyzed. Here, we demonstrate spatially localized, efficient, and universal delivery of biomolecules into immortalized and primary mammalian cells using surface-modified vertical silicon nanowires. The method relies on the ability of the silicon nanowires to penetrate a cell’s membrane and subsequently release surface-bound molecules directly into the cell’s cytosol, thus allowing highly efficient delivery of biomolecules without chemical modification or viral packaging. This modality enables one to assess the phenotypic consequences of introducing a broad range of biological effectors (DNAs, RNAs, peptides, proteins, and small molecules) into almost any cell type. We show that this platform can be used to guide neuronal progenitor growth with small molecules, knock down transcript levels by delivering siRNAs, inhibit apoptosis using peptides, and introduce targeted proteins to specific organelles. We further demonstrate codelivery of siRNAs and proteins on a single substrate in a microarray format, highlighting this technology’s potential as a robust, monolithic platform for high-throughput, miniaturized bioassays.

Modulation of p300 binding by posttranslational modifications of the C-terminal activation domain of hypoxia-inducible factor-1α

Posttranslational modifications of hypoxia‐inducible factor‐1α (HIF‐1α) influence HIF‐mediated transcription, likely by affecting binding to p300/cAMP‐response element‐binding protein (CBP). To systematically analyze the HIF‐1α–p300/CBP interaction, we developed a fluorescence polarization‐based binding assay, employing fluorescein‐labeled peptides derived from the C‐terminal transactivation domain (C‐TAD) of HIF‐1α. After optimized for effectively capturing p300/CBP, the assay was utilized for evaluating direct effects of posttranslational modifications of the HIF‐1α C‐TAD on p300 binding. The results demonstrated that asparagine hydroxylation and S‐nitrosylation of HIF‐1α decrease p300 binding, while its phosphorylation does not affect p300 binding, which was reconfirmed by competitive inhibition analyses using mutant peptides.

Nitric Oxide Donor, (±)-S-Nitroso-N-acetylpenicillamine, Stabilizes Transactive Hypoxia-Inducible Factor-1α by Inhibiting von Hippel-Lindau Recruitment and Asparagine Hydroxylation

We have confirmed that the NO donor (±)-S-nitroso-N-acetylpenicillamine (SNAP) stabilizes the transactive form of hypoxia-inducible factor-1α (HIF-1α), leading to the induction of HIF-1α target genes such as vascular endothelial growth factor and carbonic anhydrase 9. Activation of HIF-1α should require inhibition of the dual system that keeps it inactive. One is ubiquitination, which is triggered by hydroxylation of HIF-1α-proline and the subsequent binding of E3 ubiquitin ligase, the von Hippel Lindau (VHL) protein. The other is hydroxylation of HIF-1α-asparagine, which reduces the affinity of HIF-1α for its coactivator, cAMP responsive element binding protein/p300. We examined the effects of the NO donor SNAP on proline and asparagine hydroxylation of HIF-1α peptides by measuring the activities of the corresponding enzymes, HIF-1α-specific proline hydroxylase 2 (PHD2) and the HIF-1α-specific asparagine hydroxylase, designated factor inhibiting HIF-1α (FIH-1), respectively. We found that the SNAP did not prevent PHD2 from hydroxylating the proline of HIF-1α. Instead, it blocked the interaction between VHL and the proline-hydroxylated HIF-1α, but only when the reducing agents Fe(II) and vitamin C were limiting. The fact that the absence of cysteine 520 of HIF-1α abolishes its responsiveness to SNAP suggests that this residue mediates the inhibition by SNAP of the interaction between VHL and HIF-1α, presumably by S-nitrosylation of HIF-1α. Un-like PHD2, asparagine hydroxylation by FIH-1 was directly inhibited by SNAP, but again only when reducing agents were limiting. Substitution of cysteine 800 of HIF-1α with alanine failed to reverse the inhibitory effects of SNAP on asparagine hydroxylation, implying that FIH-1, not its substrate HIF-1α, is inhibited by SNAP.

Baicalein Induces Functional Hypoxia-Inducible Factor-1α and Angiogenesis

Targeting the oxygen-sensing mechanisms of the hypoxiainducible factor (HIF) pathway provides pharmacological ways of manipulating the HIF response. Because HIF-1α-specific prolyl-4 hydroxylases (PHDs) prime degradation of HIF-1α, we have made an effort to find a small molecule capable of up-regulating the HIF pathway by inhibiting prolyl hydroxylation. Through an in vitro high-throughput screen, we have discovered a PHD2 inhibitor baicalein, which is also found to abrogate asparaginyl hydroxylation of HIF-1α. Such inhibitory effects are reversed by the addition of excess 2-oxoglutarate and iron(II), suggesting the involvement of baicaleins binding at the enzyme active sites, which has also been corroborated by spectroscopic binding assays between baicalein and enzyme. In addition, baicalein suppresses ubiquitination of HIF-1α, which works in concert with the inhibition of the HIF-specific hydroxylases to increase the HIF-1α content, leading to induction of HIF-1-mediated reporter gene activity and target gene transcription in tissue culture cells, whereas it induces HIF-independent activation of other genes. Furthermore, in vivo organ models based on the chick chorioallantoic membrane assay demonstrate that baicalein promotes new blood vessel formation. Together, our results indicate that baicalein possesses a proangiogenic potential and thus might have the therapeutic utility in the treatment of ischemic diseases.

Sentinel lymph node imaging by a fluorescently labeled DNA tetrahedron.

Sentinel lymph nodes (SLNs) are the first lymph nodes which cancer cells reach after traveling through lymphatic vessels from the primary tumor. Evaluating the nodal status is crucial in accurate staging of human cancers and accordingly determines prognosis and the most appropriate treatment. The commonly used methods for SLN identification in clinics are based on employment of a colloid of radionuclide or injection of a small dye. Although these methods have certainly contributed to improve surgical practice, new imaging materials are still required to overcome drawbacks of the techniques such as inconvenience of handling radioactive materials and short retention time of small dyes in SLNs. Here, we prepare a fluorescence-labeled DNA tetrahedron and perform SLN imaging by using the DNA nanoconstruct. With a successful identification of SLNs by the DNA nanoconstruct, we suggest that DNA tetrahedron hold great promises for clinical applications.

Highly sensitive detection of a bio-threat pathogen by gold nanoparticle-based oligonucleotide-linked immunosorbent assay

Francisella (F.) tularensis causes the zoonotic disease tularemia and categorized as one of the highest-priority biological agents. The sensing approaches utilized by conventional detection methods, including enzyme-linked immunosorbent assay (ELISA), are not sensitive enough to identify an infectious dose of this high-risk pathogen due to its low infective dose. As an attempt to detect F. tularensis with high sensitivity, we utilized the highly sensitive immunoassay system named gold nanoparticle-based oligonucleotide-linked immunosorbent assay (GNP-OLISA) which uses antibody-gold nanoparticles conjugated with DNA strands as a signal generator and RNA oligonucleotides appended with a fluorophore as a quencher for signal amplification. We modified the GNP-OLISA for the detection F. tularensis to utilize one antibody for both the capture of the target and for signal generation instead of using two different antibodies, which are usually employed to construct the antibody sandwich in the ELISA. The GNP-OLISA showed 37-fold higher sensitivity compared with ELISA and generated very consistent detection results in the sera. In addition, the detection specificity was not affected by the presence of non-target bacteria, suggesting that GNP-OLISA can be used as a sensitive detection platform for monitoring high-risk pathogens thereby overcoming the limit of the conventional assay system.

An approach to multiplexing an immunosorbent assay with antibody-oligonucleotide conjugates.

Early detection of cancer biomarkers provides clinically valuable information. While the conventional enzyme-linked immunosorbent assay (ELISA) has been routinely used for individual cancer markers, methods for simultaneous determination of multiple markers within a single sample are still in demand. Here, we present a novel oligonucleotide-linked immunosorbent assay (OLISA) with a multiplexing capability on the same microwell plate-based system as in ELISA. Employing a DNA oligonucleotide that is covalently conjugated to the detection antibody and a complementary RNA oligonucleotide which is appended with a fluorophore and a quencher, degradation of the RNA in the DNA-RNA duplex by RNase H is exploited for fluorescent signal generation. Iterative cycles of DNA-RNA duplexation and subsequent degradation of the RNA in the duplex by RNase H further lead to amplification of the detection signal in OLISA. Moreover, the use of antibody-oligonucleotide conjugates enables multiplexing of OLISA, which is successfully demonstrated by tethering DNA molecules to detection antibodies and by performing assays for three common cancer markers including α-fetoprotein, prostate-specific antigen, and carcinoembryonic antigen. With the simple procedure and reliable detection performance, the developed multiplex OLISA has a wide potential for use in analysis of a panel of biomarkers in clinical diagnostics.

Discovery of a non-cationic cell penetrating peptide derived from membrane-interacting human proteins and its potential as a protein delivery carrier

Cell penetrating peptides (CPPs) are peptides that can be translocated into cells and used as a carrier platform for the intracellular uptake of cargo molecules. Subject to the source of CPP sequences and their positively charged nature, the cytotoxicity and immunogenicity of conventional CPPs needs to be optimized to expand their utility for biomedical applications. In addition to these safety issues, the stability of CPPs needs to be addressed since their positively charged residues are prone to interact with the biological milieu. As an effort to overcome these limitations of the current CPP technology, we isolated CPP candidate sequences and synthesized peptides from twelve isoforms of annexin, a family of membrane-interacting human proteins. The candidate screen returned a CPP rich in hydrophobic residues that showed more efficient cellular uptake than TAT-CPP. We then investigated the uptake mechanism, subcellular localization, and biophysical properties of the newly found CPP, verifying low cytotoxicity, long-term serum stability, and non-immunogenicity. Finally, model proteins conjugated to this peptide were successfully delivered into mammalian cells both in vitro and in vivo, indicating a potential use of the peptide as a carrier for the delivery of macromolecular cargos.

An RNase H-Assisted Fluorescent Biosensor for Aptamers

Aptamers are nucleic acid species that are routinely selected in vitro through SELEX (systematic evolution of ligands by exponential enrichment). Since their introduction by the Gold and Szostak groups, aptamers have been exploited as molecular-recognition elements to detect virtually any target of interest, ranging from small molecules to proteins to even cells and tissues. Aptamers, which rival antibodies in sensitivity and specificity, are readily reproduced by chemical synthesis with low cost. Furthermore, they possess desirable storage properties and elicit little or no immunogenicity in a biological context. Owing to their advantages in comparison to antibodies, their utility in therapuetics and diagnostics has significantly expanded. Recently, the lack of inherent signaling properties of aptamers has prompted development of various strategies for transducing target-binding events into readily measurable signals for biotechnological and biomedical applications. Methods that employ fluorescent reporters have proven to be particularly useful in generation of aptamer-based biosensors ; these include monochromophore approaches, aptamerbeacon engineering, structure-switching signaling, in situ labeling, allosteric chimeras, dye-staining approaches, and polymer-conjugate based fluorescent chemosensors. While these systems generally produce signals in a stoichiometric manner, attempts have been made to amplify signals by incorporation of a proximity-ligation assay or an exoACHTUNGTRENNUNGnuclease-protection assay into aptamer-based sensing. Although ultrasensitive detection of proteins has been achieved, the former assay is limited to homodimer protein targets, and the latter assay suffers from tedious multistep procedures. Very recently, a DNA-polymerase assay integrated with a molecular beacon has been employed for the amplified detection of the recognition between aptamer and target small molecule. Such techniques are in continuous demand for developing simple and easily applicable aptamer-based methods that can facilitate accurate and specific bioanalysis. Here, we report the rational construction of a novel fluorescent biosensor for aptamers. Aptamers are capable of recognizing both the cognate target molecule for which they are engineered and an antisense DNA molecule through duplex formation. Accordingly, we have exploited this nature of the aptamer to cause a duplex-to-complex structural change; this was combined with a well-established enzymatic reaction since previous studies have demonstrated successful incorporation of enzymatic reactions in signal amplification for DNA-based biosensors. Thrombin was chosen as a model target protein because of the availability of its 15-mer single-stranded DNA aptamer. As illustrated in Scheme 1, the 5’-extended DNA-based thrombin aptamer, which contained six additional

Inhibition of VEGF transcription through blockade of the hypoxia inducible factor-1α–p300 interaction by a small molecule

Vascular endothelial growth factor (VEGF) plays a pro-angiogenic role in tumor progression. Stabilization of a key regulator termed the hypoxia inducible factor (HIF)-1α under oxygen deficient environment around tumor is known to elicit expression of VEGF through binding to p300. Thus, inhibition of the HIF-1α-p300 interaction would lead to down-regulation of VEGF expression, thereby providing potential cancer therapeutics. Here, we have screened a chemical library against the interaction of the HIF-1α-derived peptide with p300 employing a fluorescence polarization-based assay. We have identified a compound as the most prominent inhibitor against the protein-protein interaction. Further, we have observed suppression of the mRNA level of VEGF upon treatment of HeLa cells with the compound, demonstrating its inhibitory effect at the cellular level.