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783 KiB

Open AccessArticle

Structural and Affinity Analyses of G-Quadruplex DNA Aptamers for Camptothecin Derivatives

by Hiroto Fujita, Yuri Imaizumi, Yuuya Kasahara, Shunsuke Kitadume, Hiroaki Ozaki, Masayasu Kuwahara and Naoki Sugimoto

Pharmaceuticals 2013, 6(9), 1082-1093; https://doi.org/10.3390/ph6091082 - 29 Aug 2013

Cited by 21 | Viewed by 9122

Abstract

We recently selected DNA aptamers that bind to camptothecin (CPT) and CPT derivatives from a 70-mer oligodeoxyribonucleotide (ODN) library using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method. The target-binding activity of the obtained 70-mer CPT-binding DNA aptamer, termed CA-70, which [...] Read more.

We recently selected DNA aptamers that bind to camptothecin (CPT) and CPT derivatives from a 70-mer oligodeoxyribonucleotide (ODN) library using the Systematic Evolution of Ligands by EXponential enrichment (SELEX) method. The target-binding activity of the obtained 70-mer CPT-binding DNA aptamer, termed CA-70, which contains a 16-mer guanine (G)-core motif (G₃TG₃TG₃T₂G₃) that forms a three-tiered G-quadruplex, was determined using fluorescence titration. In this study, truncated fragments of CA-70 that all have the G-core motif, CA-40, -20, -19, -18A, -18B, -17, and -16, were carefully analyzed. We found that CA-40 retained the target-binding activity, whereas CA-20, -19, and -18B exhibited little or no binding activities. Further, not only CA-18A but also the shorter length fragments CA-17 and -16 clearly retained the binding activity, indicating that tail strands of the G-quadruplex structure can significantly affect the target binding of G-quadruplex DNA aptamers. Further analyses using circular dichroism (CD) spectroscopy and fluorescence polarization (FP) assay were conducted to investigate the structure and affinity of G-quadruplex DNA aptamers. Full article

(This article belongs to the Special Issue Aptamer-Based Therapeutics)

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1590 KiB

Open AccessArticle

The Inhibition of Stat5 by a Peptide Aptamer Ligand Specific for the DNA Binding Domain Prevents Target Gene Transactivation and the Growth of Breast and Prostate Tumor Cells

by Axel Weber, Corina Borghouts, Christian Brendel, Richard Moriggl, Natalia Delis, Boris Brill, Vida Vafaizadeh and Bernd Groner

Pharmaceuticals 2013, 6(8), 960-987; https://doi.org/10.3390/ph6080960 - 20 Aug 2013

Cited by 19 | Viewed by 10899

Abstract

The signal transducer and activator of transcription Stat5 is transiently activated by growth factor and cytokine signals in normal cells, but its persistent activation has been observed in a wide range of human tumors. Aberrant Stat5 activity was initially observed in leukemias, but [...] Read more.

The signal transducer and activator of transcription Stat5 is transiently activated by growth factor and cytokine signals in normal cells, but its persistent activation has been observed in a wide range of human tumors. Aberrant Stat5 activity was initially observed in leukemias, but subsequently also found in carcinomas. We investigated the importance of Stat5 in human tumor cell lines. shRNA mediated downregulation of Stat5 revealed the dependence of prostate and breast cancer cells on the expression of this transcription factor. We extended these inhibition studies and derived a peptide aptamer (PA) ligand, which directly interacts with the DNA-binding domain of Stat5 in a yeast-two-hybrid screen. The Stat5 specific PA sequence is embedded in a thioredoxin (hTRX) scaffold protein. The resulting recombinant protein S5-DBD-PA was expressed in bacteria, purified and introduced into tumor cells by protein transduction. Alternatively, S5-DBD-PA was expressed in the tumor cells after infection with a S5-DBD-PA encoding gene transfer vector. Both strategies impaired the DNA-binding ability of Stat5, suppressed Stat5 dependent transactivation and caused its intracellular degradation. Our experiments describe a peptide based inhibitor of Stat5 protein activity which can serve as a lead for the development of a clinically useful compound for cancer treatment. Full article

(This article belongs to the Special Issue Aptamer-Based Therapeutics)

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456 KiB

Open AccessArticle

Anti-HCV RNA Aptamers Targeting the Genomic cis-Acting Replication Element

by Soledad Marton, Beatriz Berzal-Herranz, Eva Garmendia, Francisco J. Cueto and Alfredo Berzal-Herranz

Pharmaceuticals 2012, 5(1), 49-60; https://doi.org/10.3390/ph5010049 - 28 Dec 2011

Cited by 13 | Viewed by 8210

Abstract

Hepatitis C virus (HCV) replication is dependent on the existence of several highly conserved functional genomic RNA domains. The cis-acting replication element (CRE), located within the 3' end of the NS5B coding region of the HCV genome, has been shown essential for [...] Read more.

Hepatitis C virus (HCV) replication is dependent on the existence of several highly conserved functional genomic RNA domains. The cis-acting replication element (CRE), located within the 3' end of the NS5B coding region of the HCV genome, has been shown essential for efficient viral replication. Its sequence and structural features determine its involvement in functional interactions with viral RNA-dependent RNA polymerase and distant RNA domains of the viral genome. This work reports the use of an in vitro selection strategy to select aptamer RNA molecules against the complete HCV-CRE. After six selection cycles, five potential target sites were identified within this domain. Inhibition assays using a sample of representative aptamers showed that the selected RNAs significantly inhibit the replication (>80%) of a subgenomic HCV replicon in Huh-7 cell cultures. These results highlight the potential of aptamer RNA molecules as therapeutic antiviral agents. Full article

(This article belongs to the Special Issue Aptamer-Based Therapeutics)

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550 KiB

Open AccessArticle

RNA Detection in Live Bacterial Cells Using Fluorescent Protein Complementation Triggered by Interaction of Two RNA Aptamers with Two RNA-Binding Peptides

by Hung-Wei Yiu, Vadim V. Demidov, Paul Toran, Charles R. Cantor and Natalia E. Broude

Pharmaceuticals 2011, 4(3), 494-508; https://doi.org/10.3390/ph4030494 - 10 Mar 2011

Cited by 24 | Viewed by 12149

Abstract

Many genetic and infectious diseases can be targeted at the RNA level as RNA is more accessible than DNA. We seek to develop new approaches for detection and tracking RNA in live cells, which is necessary for RNA-based diagnostics and therapy. We recently [...] Read more.

Many genetic and infectious diseases can be targeted at the RNA level as RNA is more accessible than DNA. We seek to develop new approaches for detection and tracking RNA in live cells, which is necessary for RNA-based diagnostics and therapy. We recently described a method for RNA visualization in live bacterial cells based on fluorescent protein complementation [1-3]. The RNA is tagged with an RNA aptamer that binds an RNA-binding protein with high affinity. This RNA-binding protein is expressed as two split fragments fused to the fragments of a split fluorescent protein. In the presence of RNA the fragments of the RNA-binding protein bind the aptamer and bring together the fragments of the fluorescent protein, which results in its re-assembly and fluorescence development [1-3]. Here we describe a new version of the RNA labeling method where fluorescent protein complementation is triggered by paired interactions of two different closely-positioned RNA aptamers with two different RNA-binding viral peptides. The new method, which has been developed in bacteria as a model system, uses a smaller ribonucleoprotein complementation complex, as compared with the method using split RNA-binding protein, and it can potentially be applied to a broad variety of RNA targets in both prokaryotic and eukaryotic cells. We also describe experiments exploring background fluorescence in these RNA detection systems and conditions that improve the signal-to-background ratio. Full article

(This article belongs to the Special Issue Aptamer-Based Therapeutics)

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Review

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567 KiB

Open AccessReview

Aptamer-Based Therapeutics: New Approaches to Combat Human Viral Diseases

by Ka-To Shum, Jiehua Zhou and John J. Rossi

Pharmaceuticals 2013, 6(12), 1507-1542; https://doi.org/10.3390/ph6121507 - 25 Nov 2013

Cited by 62 | Viewed by 14281

Abstract

Viruses replicate inside the cells of an organism and continuously evolve to contend with an ever-changing environment. Many life-threatening diseases, such as AIDS, SARS, hepatitis and some cancers, are caused by viruses. Because viruses have small genome sizes and high mutability, there is [...] Read more.

Viruses replicate inside the cells of an organism and continuously evolve to contend with an ever-changing environment. Many life-threatening diseases, such as AIDS, SARS, hepatitis and some cancers, are caused by viruses. Because viruses have small genome sizes and high mutability, there is currently a lack of and an urgent need for effective treatment for many viral pathogens. One approach that has recently received much attention is aptamer-based therapeutics. Aptamer technology has high target specificity and versatility, i.e., any viral proteins could potentially be targeted. Consequently, new aptamer-based therapeutics have the potential to lead a revolution in the development of anti-infective drugs. Additionally, aptamers can potentially bind any targets and any pathogen that is theoretically amenable to rapid targeting, making aptamers invaluable tools for treating a wide range of diseases. This review will provide a broad, comprehensive overview of viral therapies that use aptamers. The aptamer selection process will be described, followed by an explanation of the potential for treating virus infection by aptamers. Recent progress and prospective use of aptamers against a large variety of human viruses, such as HIV-1, HCV, HBV, SCoV, Rabies virus, HPV, HSV and influenza virus, with particular focus on clinical development of aptamers will also be described. Finally, we will discuss the challenges of advancing antiviral aptamer therapeutics and prospects for future success. Full article

(This article belongs to the Special Issue Aptamer-Based Therapeutics)

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458 KiB

Open AccessReview

A Review of Therapeutic Aptamer Conjugates with Emphasis on New Approaches

by John G. Bruno

Pharmaceuticals 2013, 6(3), 340-357; https://doi.org/10.3390/ph6030340 - 19 Mar 2013

Cited by 96 | Viewed by 14022

Abstract

The potential to emulate or enhance antibodies with nucleic acid aptamers while lowering costs has prompted development of new aptamer-protein, siRNA, drug, and nanoparticle conjugates. Specific focal points of this review discuss DNA aptamers covalently bound at their 3' ends to various proteins [...] Read more.

The potential to emulate or enhance antibodies with nucleic acid aptamers while lowering costs has prompted development of new aptamer-protein, siRNA, drug, and nanoparticle conjugates. Specific focal points of this review discuss DNA aptamers covalently bound at their 3' ends to various proteins for enhanced stability and greater pharmacokinetic lifetimes in vivo. The proteins can include Fc tails of IgG for opsonization, and the first component of complement (C1q) to trigger complement-mediated lysis of antibiotic-resistant Gram negative bacteria, cancer cells and possibly some parasites during vulnerable stages. In addition, the 3' protein adduct may be a biotoxin, enzyme, or may simply be human serum albumin (HSA) or a drug known to bind HSA, thereby retarding kidney and other organ clearance and inhibiting serum exonucleases. In this review, the author summarizes existing therapeutic aptamer conjugate categories and describes his patented concept for PCR-based amplification of double-stranded aptamers followed by covalent attachment of proteins or other agents to the chemically vulnerable overhanging 3' adenine added by Taq polymerase. PCR amplification of aptamers could dramatically lower the current $2,000/gram cost of parallel chemical oligonucleotide synthesis, thereby enabling mass production of aptamer-3'-protein or drug conjugates to better compete against expensive humanized monoclonal antibodies. Full article

(This article belongs to the Special Issue Aptamer-Based Therapeutics)

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959 KiB

Open AccessReview

Selecting Molecular Recognition. What Can Existing Aptamers Tell Us about Their Inherent Recognition Capabilities and Modes of Interaction?

by Qian Zhang and Ralf Landgraf

Pharmaceuticals 2012, 5(5), 493-513; https://doi.org/10.3390/ph5050493 - 18 May 2012

Cited by 14 | Viewed by 9016

Abstract

The use of nucleic acid derived aptamers has rapidly expanded since the introduction of SELEX in 1990. Nucleic acid aptamers have demonstrated their ability to target a broad range of molecules in ways that rival antibodies, but advances have been very uneven for [...] Read more.

The use of nucleic acid derived aptamers has rapidly expanded since the introduction of SELEX in 1990. Nucleic acid aptamers have demonstrated their ability to target a broad range of molecules in ways that rival antibodies, but advances have been very uneven for different biochemical classes of targets, and clinical applications have been slow to emerge. What sets different aptamers apart from each other and from rivaling molecular recognition platforms, specifically proteins? What advantages do aptamers as a reagent class offer, and how do the chemical properties and selection procedures of aptamers influence their function? Do the building blocks of nucleic acid aptamers dictate inherent limitations in the nature of molecular targets, and do existing aptamers give us insight in how these challenges might be overcome? This review is written as an introduction for potential endusers of aptamer technology who are evaluating the advantages of aptamers as a versatile, affordable, yet highly expandable platform to target a broad range of biological processes or interactions. Full article

(This article belongs to the Special Issue Aptamer-Based Therapeutics)

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Open AccessReview

Coupling Aptamers to Short Interfering RNAs as Therapeutics

by Laura Cerchia, Carla Lucia Esposito, Simona Camorani, Silvia Catuogno and Vittorio de Franciscis

Pharmaceuticals 2011, 4(11), 1434-1449; https://doi.org/10.3390/ph4111434 - 27 Oct 2011

Cited by 12 | Viewed by 8266

Abstract

RNA-based approaches are among the most promising strategies aimed at developing safer and more effective therapeutics. RNA therapeutics include small non-coding miRNAs, small interfering RNA, RNA aptamers and more recently, small activating RNAs. However, major barriers exist to the use of RNAs as [...] Read more.

RNA-based approaches are among the most promising strategies aimed at developing safer and more effective therapeutics. RNA therapeutics include small non-coding miRNAs, small interfering RNA, RNA aptamers and more recently, small activating RNAs. However, major barriers exist to the use of RNAs as therapeutics such as resistance to nucleases present in biological fluids, poor chemical stability, need of specific cell targeted delivery and easy entry into the cell. Such issues have been addressed by several recent reports that show the possibility of introducing chemical modifications in small RNAs to stabilize the molecular conformation and increase by several fold their integrity, while still preserving the functional activity. Further, several aptamers have been developed as excellent candidates for the specific recognition of cell surface targets. In the last few years, by taking advantage of recent advances in the small RNA field, molecular bioconjugates have been designed that permit specific targeting and may act as cargoes for cell internalization of small RNAs acting on gene expression that will be discussed in this review. Full article

(This article belongs to the Special Issue Aptamer-Based Therapeutics)

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Open AccessReview

Methods To Identify Aptamers against Cell Surface Biomarkers

by Agnes Cibiel, Daniel Miotto Dupont and Frédéric Ducongé

Pharmaceuticals 2011, 4(9), 1216-1235; https://doi.org/10.3390/ph4091216 - 20 Sep 2011

Cited by 55 | Viewed by 10319

Abstract

Aptamers are nucleic acid-based ligands identified through a process of molecular evolution named SELEX (Systematic Evolution of Ligands by Exponential enrichment). During the last 10-15 years, numerous aptamers have been developed specifically against targets present on or associated with the surface of human [...] Read more.

Aptamers are nucleic acid-based ligands identified through a process of molecular evolution named SELEX (Systematic Evolution of Ligands by Exponential enrichment). During the last 10-15 years, numerous aptamers have been developed specifically against targets present on or associated with the surface of human cells or infectious pathogens such as viruses, bacteria, fungi or parasites. Several of the aptamers have been described as potent probes, rivalling antibodies, for use in flow cytometry or microscopy. Some have also been used as drugs by inhibiting or activating functions of their targets in a manner similar to neutralizing or agonistic antibodies. Additionally, it is straightforward to conjugate aptamers to other agents without losing their affinity and they have successfully been used in vitro and in vivo to deliver drugs, siRNA, nanoparticles or contrast agents to target cells. Hence, aptamers identified against cell surface biomarkers represent a promising class of ligands. This review presents the different strategies of SELEX that have been developed to identify aptamers for cell surface-associated proteins as well as some of the methods that are used to study their binding on living cells. Full article

(This article belongs to the Special Issue Aptamer-Based Therapeutics)

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Open AccessReview

Aptamer-Gated Nanoparticles for Smart Drug Delivery

by Veli Cengiz Ozalp, Fusun Eyidogan and Huseyin Avni Oktem

Pharmaceuticals 2011, 4(8), 1137-1157; https://doi.org/10.3390/ph4081137 - 15 Aug 2011

Cited by 66 | Viewed by 16076

Abstract

Aptamers are functional nucleic acid sequences which can bind specific targets. An artificial combinatorial methodology can identify aptamer sequences for any target molecule, from ions to whole cells. Drug delivery systems seek to increase efficacy and reduce side-effects by concentrating the therapeutic agents [...] Read more.

Aptamers are functional nucleic acid sequences which can bind specific targets. An artificial combinatorial methodology can identify aptamer sequences for any target molecule, from ions to whole cells. Drug delivery systems seek to increase efficacy and reduce side-effects by concentrating the therapeutic agents at specific disease sites in the body. This is generally achieved by specific targeting of inactivated drug molecules. Aptamers which can bind to various cancer cell types selectively and with high affinity have been exploited in a variety of drug delivery systems for therapeutic purposes. Recent progress in selection of cell-specific aptamers has provided new opportunities in targeted drug delivery. Especially functionalization of nanoparticles with such aptamers has drawn major attention in the biosensor and biomedical areas. Moreover, nucleic acids are recognized as an attractive building materials in nanomachines because of their unique molecular recognition properties and structural features. A active controlled delivery of drugs once targeted to a disease site is a major research challenge. Stimuli-responsive gating is one way of achieving controlled release of nanoparticle cargoes. Recent reports incorporate the structural properties of aptamers in controlled release systems of drug delivering nanoparticles. In this review, the strategies for using functional nucleic acids in creating smart drug delivery devices will be explained. The main focus will be on aptamer-incorporated nanoparticle systems for drug delivery purposes in order to assess the future potential of aptamers in the therapeutic area. Special emphasis will be given to the very recent progress in controlled drug release based on molecular gating achieved with aptamers. Full article

(This article belongs to the Special Issue Aptamer-Based Therapeutics)

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Open AccessReview

Metallo-β-Lactamases and Aptamer-Based Inhibition

by Sara R. Schlesinger, Mieke J. Lahousse, Taylor O. Foster and Sung-Kun Kim

Pharmaceuticals 2011, 4(2), 419-428; https://doi.org/10.3390/ph4020419 - 18 Feb 2011

Cited by 15 | Viewed by 8788

Abstract

An evolution of antibiotic-resistant bacteria has resulted in the need for new antibiotics. β-Lactam based drugs are the most predominantly prescribed antibiotics to combat bacterial infections; however, production of β-lactamases, which catalyze the hydrolysis of the β-lactam bond of this class of antibiotics, [...] Read more.

An evolution of antibiotic-resistant bacteria has resulted in the need for new antibiotics. β-Lactam based drugs are the most predominantly prescribed antibiotics to combat bacterial infections; however, production of β-lactamases, which catalyze the hydrolysis of the β-lactam bond of this class of antibiotics, by pathogenic bacteria such as Bacillus cereus, are rendering them useless. Some inhibitors of β-lactamases have been found, but there are no inhibitors against a class of β-lactamases known as metallo-β-lactamases, and it has been reported that the number of bacteria that produce metallo-β-lactamases is on the rise. Finding inhibitors of metallo-β-lactamases is thus an urgent necessity. One way to approach the problem is by employing the combinatorial method SELEX. The SELEX method is significant in discovering and producing new classes of inhibitors, as well as providing insight into the development of these inhibitors and paves the way for future aptamer applications that further novel drug discovery. Full article

(This article belongs to the Special Issue Aptamer-Based Therapeutics)

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