• BMB Reports
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• v.48 no.4
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• pp.234-237
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• 2015

Aptamers, composed of single-stranded DNA or RNA oligonucleotides that interact with target molecules through a specific three-dimensional structure, are selected from pools of combinatorial oligonucleotide libraries. With their high specificity and affinity for target proteins, ease of synthesis and modification, and low immunogenicity and toxicity, aptamers are considered to be attractive molecules for development as anticancer therapeutics. Two aptamers - one targeting nucleolin and a second targeting CXCL12 - are currently undergoing clinical trials for treating cancer patients, and many more are under study. In this mini-review, we present the current clinical status of aptamers and aptamer-based cancer therapeutics. We also discuss advantages, limitations, and prospects for aptamers as cancer therapeutics. [BMB Reports 2015; 48(4): 234-237]

• Journal of Microbiology and Biotechnology
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• v.16 no.10
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• pp.1634-1639
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• 2006

Hepatitis C virus (HCV)-encoded nonstructural protein 5B (NS5B) possesses RNA-dependent RNA polymerase activity, which is considered essential for viral proliferation. Thus, HCV NS5B is a good therapeutic target protein for the development of anti-HCV agents. In this study, we isolated two different kinds of nuclease-resistant RNA aptamers with 2'-fluoro pyrimidines against the HCV NS5B from a combinatorial RNA library with 40 nucleotide random sequences, using SELEX technology. The isolated RNA aptamers were observed to specifically and avidly bind the HCV NS5B with an apparent $K_d$ of 5 nM and 18 nM, respectively, in contrast with the original RNA library that hardly bound the target protein. Moreover, these aptamers could partially inhibit RNA synthesis of the HCV subgenomic replicon when transfected into Huh-7 hepatoma cell lines. These results suggest that the RNA aptamers selected in vitro could be useful not only as therapeutic agents of HCV infection but also as a powerful tool for the study of the HCV RNA-dependent RNA polymerase mechanism.

• Journal of Microbiology and Biotechnology
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• v.25 no.3
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• pp.393-398
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• 2015

Aptamers are composed of single-stranded oilgonucleotides that can selectively bind desired molecules. It has been reported that RNA or DNA could act as not only a genetic messenger but also a catalyst in metabolic pathways. RNA aptamers (average sizes 40-50 bp) are smaller than antibodies and have strong binding capacities to target molecules, similar to antigenantibody interactions. Once an aptamer was selected, it can be readily produced in large quantities at low cost. The objectives of this study are to screen and develop aptamers specific to oral pathogens such as Porphyromonas gingivalis, Treponema denticola, and Streptococcus mutans. The bacterial cell pellet was fixed with formaldehyde as a target molecule for the screening of aptamers. The SELEX method was used for the screening of aptamers and a modified western blot analysis was used to verify their specificities. Through SELEX, 40 kinds of aptamers were selected and the specificity of the aptamers to the bacterial cells was confirmed by modified western blot analysis. Through the SELEX method, 40 aptamers that specifically bind to oral pathogens were screened and isolated. The aptamers showed possibility as effective candidates for the detection agents of oral infections.

• Biomolecules & Therapeutics
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• v.27 no.2
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• pp.201-209
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• 2019

Mixed lineage leukemia proteins (MLL) are the key histone lysine methyltransferases that regulate expression of diverse genes. Aberrant activation of MLL promotes leukemia as well as solid tumors in humans, highlighting the urgent need for the development of an MLL inhibitor. We screened and isolated MLL1-binding ssRNAs using SELEX (${\underline{S}}ystemic$ ${\underline{E}}volution$ of ${\underline{L}}igands$ by ${\underline{E}}xponential$ enrichment) technology. When sequences in sub-libraries were obtained using next-generation sequencing (NGS), the most enriched aptamers-APT1 and APT2-represented about 30% and 26% of sub-library populations, respectively. Motif analysis of the top 50 sequences provided a highly conserved sequence: 5'-A[A/C][C/G][G/U][U/A]ACAGAGGG[U/A]GG[A/C] GAGUGGGU-3'. APT1, APT2, and APT5 embracing this motif generated secondary structures with similar topological characteristics. We found that APT1 and APT2 have a good binding activity and the analysis using mutated aptamer variants showed that the site information in the central region was critical for binding. In vitro enzyme activity assay showed that APT1 and APT2 had MLL1 inhibitory activity. Three-dimensional structure prediction of APT1-MLL1 complex indicates multiple weak interactions formed between MLL1 SET domain and APT1. Our study confirmed that NGS-assisted SELEX is an efficient tool for aptamer screening and that aptamers could be useful in diagnosis and treatment of MLL1-mediated diseases.

• Bulletin of the Korean Chemical Society
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• v.27 no.5
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• pp.699-704
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• 2006

Escherichia coli RNase P is a ribonucleoprotein composed of M1 RNA and C5 protein. Previously, analysis of RNA aptamers selected for C5 protein from a synthetic RNA library showed that C5 protein could bind various RNA molecules as an RNA binding protein. In this study, we searched cellular RNA motifs that could be recognized by C5 protein by a genomic SELEX approach. We found various C5 protein-binding RNA motifs derived from E. coli genomic sequences. Our results suggest that C5 protein interacts with various cellular RNA species in addition to M1 RNA.

• Bulletin of the Korean Chemical Society
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• v.30 no.8
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• pp.1827-1831
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• 2009

Ligand molecules that can recognize and interact with cancer cell surface marker proteins with high affinity and specificity should greatly aid the development of novel cancer diagnostics and therapeutics. HER-2/ErbB2/Neu (HER-2), a member of the epidermal growth factor receptor family, is specifically overexpressed on the surface of breast cancer cells and serves as both a useful biomarker and a therapeutic target for breast cancer. In this study, we aimed to isolate RNA aptamers that specifically bind to a HER-2-overexpressing human breast cancer cell line, SK-BR-3, using Cell-SELEX strategy. The selected aptamers showed strong affinity to SK-BR-3, but not to MDAMB- 231, a HER-2-underexpressing breast cancer cell line. In addition, we confirmed the specific targeting of HER-2 receptor by aptamers using an unrelated mouse cell line overexpressing human HER-2 receptor. The HER-2-targeting RNA aptamers could become a useful reagent for the development of breast cancer diagnostics and therapeutics.

• Journal of Life Science
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• v.12 no.1
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• pp.14-18
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• 2002

Subpopulations of nucleotides that bind specifically to a variety of proteins have been isolated from a population of random sequence RNA/DNA molecules. Roughly one in $10^{13}$ random sequence RNA/DNA molecules folds in such a way as to create a specific binding site for small ligands. Since the development of in vitro selection procedure, more than 50 nucleic acid ligands (aptamers) have been isolated. These molecules are very useful for the study of molecular recognition between nucleic acid and protein/organic compound. In addition to these basic studies this method gives us a dream to produce new drugs against several diseases. We focused on several aptamers which specifically binds to trypsin-like serine proteases (thrombin, human neutrophil elastase, activated protein C and NS3 protease of human hepatitis C virus) and want to introduce their structural characteristics and some functions.

• Molecules and Cells
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• v.42 no.10
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• pp.721-728
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• 2019

Non-structural protein 1 (NS1) of influenza virus has been shown to inhibit the innate immune response by blocking the induction of interferon (IFN). In this study, we isolated two single-stranded RNA aptamers specific to NS1 with $K_d$ values of $1.62{\pm}0.30nM$ and $1.97{\pm}0.27nM$, respectively, using a systematic evolution of ligand by exponential enrichment (SELEX) procedure. The selected aptamers were able to inhibit the interaction of NS1 with tripartite motif-containing protein 25 (TRIM25), and suppression of NS1 enabled retinoic acid inducible gene I (RIG-I) to be ubiquitinated regularly by TRIM25. Additional luciferase reporter assay and quantitative real-time PCR (RT-PCR) experiments demonstrated that suppression of NS1 by the selected aptamers induced IFN production. It is noted that viral replication was also inhibited through IFN induction in the presence of the selected aptamers. These results suggest that the isolated aptamers are strongly expected to be new therapeutic agents against influenza infection.

• Bulletin of the Korean Chemical Society
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• v.35 no.5
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• pp.1279-1284
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• 2014

The binding of TATA-binding protein (TBP) to the TATA-box containing promoter region is aided by many other transcriptional factors including TFIIA and TFIIB. The mechanistic insight into the assembly of RNA polymerase II preinitation complex (PIC) has been gained by either directly altering a function of target protein or perturbing molecular interactions using drugs, RNAi, or aptamers. Aptamers have been found particularly useful for studying a role of a subset of PIC on transcription for their ability to inhibit specific molecular interactions. One major hurdle to the wide use of aptamers as specific inhibitors arises from the difficulty with traditional assays to validate and determine specificity, affinity, and binding epitopes for aptamers against targets. Here, using a technique called the bio-layer interferometry (BLI) designed for a label-free, real-time, and multiplexed detection of molecular interactions, we studied the assembly of a subset of PIC, TBP binding to TATA DNA, and two distinct classes of aptamers against TPB in regard to their ability to inhibit TBP binding to TFIIA or TATA DNA. Using BLI, we measured not only equilibrium binding constants ($K_D$), which were overall in close agreement with those obtained by electrophoretic mobility shift assay, but also kinetic constants of binding ($k_{on}$ and $k_{off}$), differentiating aptamers of comparable KDs by their difference in binding kinetics. The assay developed in this study can readily be adopted for high throughput validation of candidate aptamers for specificity, affinity, and epitopes, providing both equilibrium and kinetic information for aptamer interaction with targets.

• KSBB Journal
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• v.20 no.2 s.91
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• pp.88-92
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• 2005

Phosphorylation of amino acid residues in proteins, plays a major role in biological mechanism. Phosphorylation acts as a process regulating the protein activity in variable pathways such as metabolism, signal transduction and cell division. Therefore the development of ligands for phosphoamino acids are an important work for protein analysis and proteomics studies. In this study, RNA aptamers for o-phosphoserine, o-phosphotyrosine and o-phosphotyrosine which appears frequently in nature were developed by in vitro evolution method. We could obtain similar sequences from random RNAs of 40 mer by SELEX method through 10 cycles. As result, the aptamers for o-phosphoserine and o-phosphothreonine among phosphoamino acids aptamers showed high affinity of Kd=2.60 nM and 2.65 nM for their target molecules, respectively. In addition, these aptamers could be confirmed the high selectivity for their target.