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1.  Nucleic Acid Aptamers as Potential Therapeutic and Diagnostic Agents for Lymphoma 
Journal of cancer therapy  2013;4(4):872-890.
Lymphomas are cancers that arise from white blood cells and usually present as solid tumors. Treatment of lymphoma often involves chemotherapy, and can also include radiotherapy and/or bone marrow transplantation. There is an un-questioned need for more effective therapies and diagnostic tool for lymphoma. Aptamers are single stranded DNA or RNA oligonucleotides whose three-dimensional structures are dictated by their sequences. The immense diversity in function and structure of nucleic acids enable numerous aptamers to be generated through an iterative in vitro selection technique known as Systematic Evolution of Ligands by EXponential enrichment (SELEX). Aptamers have several biochemical properties that make them attractive tools for use as potential diagnostic and pharmacologic agents. Isolated aptamers may directly inhibit the function of target proteins, or they can also be formulated for use as delivery agents for other therapeutic or imaging cargoes. More complex aptamer identification methods, using whole cancer cells (Cell-SELEX), may identify novel targets and aptamers to affect them. This review focuses on recent advances in the use of nucleic acid aptamers as diagnostic and therapeutic agents and as targeted delivery carriers that are relevant to lymphoma. Some representative examples are also discussed.
PMCID: PMC4104705  PMID: 25057429
Lymphoma; Nucleic Acid Aptamer; SELEX; Cell-Type Specific Drug Delivery; Biosensor; Nanotechnology
2.  Chimeric aptamers in cancer cell-targeted drug delivery 
Aptamers are single-stranded structured oligonucleotides (DNA or RNA) that can bind to a wide range of targets ("apatopes") with high affinity and specificity. These nucleic acid ligands, generated from pools of random-sequence by an in vitro selection process referred to as systematic evolution of ligands by exponential enrichment (SELEX), have now been identified as excellent tools for chemical biology, therapeutic delivery, diagnosis, research, and monitoring therapy in real-time imaging. Today, aptamers represent an interesting class of modern Pharmaceuticals which with their low immunogenic potential mimic extend many of the properties of monoclonal antibodies in diagnostics, research, and therapeutics. More recently, chimeric aptamer approach employing many different possible types of chimerization strategies has generated more stable and efficient chimeric aptamers with aptamer-aptamer, aptamer-nonaptamer biomacromolecules (siRNAs, proteins) and aptamer-nanoparticle chimeras. These chimeric aptamers when conjugated with various biomacromolecules like locked nucleic acid (LNA) to potentiate their stability, biodistribution, and targeting efficiency, have facilitated the accurate targeting in preclinical trials. We developed LNA-aptamer (anti-nucleolin and EpCAM) complexes which were loaded in iron-saturated bovine lactofeerin (Fe-blf)-coated dopamine modified surface of superparamagnetic iron oxide (Fe3O4) nanoparticles (SPIONs). This complex was used to deliver the specific aptamers in tumor cells in a co-culture model of normal and cancer cells. This review focuses on the chimeric aptamers, currently in development that are likely to find future practical applications in concert with other therapeutic molecules and modalities.
PMCID: PMC3233271  PMID: 21955150
Aptamer; SELEX; chimera; targeted drug delivery; si RNA; locked nucleic acid; nanoparticles
3.  Aptamer-Gated Nanoparticles for Smart Drug Delivery 
Pharmaceuticals  2011;4(8):1137-1157.
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.
PMCID: PMC4058663
aptamers; nanoparticles; drug delivery; molecular gating; nanovalves
4.  Oligonucleotide Aptamers: New Tools for Targeted Cancer Therapy 
Aptamers are a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets. Similar to antibodies, aptamers interact with their targets by recognizing a specific three-dimensional structure and are thus termed “chemical antibodies.” In contrast to protein antibodies, aptamers offer unique chemical and biological characteristics based on their oligonucleotide properties. Hence, they are more suitable for the development of novel clinical applications. Aptamer technology has been widely investigated in various biomedical fields for biomarker discovery, in vitro diagnosis, in vivo imaging, and targeted therapy. This review will discuss the potential applications of aptamer technology as a new tool for targeted cancer therapy with emphasis on the development of aptamers that are able to specifically target cell surface biomarkers. Additionally, we will describe several approaches for the use of aptamers in targeted therapeutics, including aptamer-drug conjugation, aptamer-nanoparticle conjugation, aptamer-mediated targeted gene therapy, aptamer-mediated immunotherapy, and aptamer-mediated biotherapy.
PMCID: PMC4221593  PMID: 25093706
cell surface biomarker; nanomedicine; oligonucleotide aptamer; SELEX; targeted cancer therapy
5.  Rationally Manipulating Aptamer Binding Affinities in a Stem-Loop Molecular Beacon 
Bioconjugate Chemistry  2014;25(10):1769-1776.
Single-stranded DNA sequences that are highly specific for a target ligand are called aptamers. While the incorporation of aptamer sequences into stem-loop molecular beacons has become an essential tool in optical biosensors, the design principles that determine the magnitude of binding affinity and its relationship to placement of the aptamer sequence in the stem-loop architecture are not well defined. By controlled placement of the aptamer along the loop region of the molecular beacon, it is observed that the binding affinity can be tuned over 4 orders of magnitude (1.3 nM – 203 μM) for the Huizenga and Szostak ATP DNA aptamer sequence. It is observed that the Kd is enhanced for the fully exposed sequence, with reduced binding affinity when the aptamer is part of the stem region of the beacon. Analysis of the ΔG values indicate a clear correlation between the aptamer hybridized length in the stem and its observed Kd. The use of a nanometal surface energy transfer probe method for monitoring ATP binding to the aptamer sequence allows the observation of negative cooperativity between the two ATP binding events. Maintenance of the high binding affinity of this ATP aptamer and the observation of two separate Kd’s for ATP binding indicate NSET as an effective, nonmanipulative, optical method for tracking biomolecular changes.
PMCID: PMC4198099  PMID: 25170558
6.  Current Progress of RNA Aptamer-Based Therapeutics 
Frontiers in Genetics  2012;3:234.
Aptamers are single-stranded nucleic acids that specifically recognize and bind tightly to their cognate targets due to their stable three-dimensional structure. Nucleic acid aptamers have been developed for various applications, including diagnostics, molecular imaging, biomarker discovery, target validation, therapeutics, and drug delivery. Due to their high specificity and binding affinity, aptamers directly block or interrupt the functions of target proteins making them promising therapeutic agents for the treatment of human maladies. Additionally, aptamers that bind to cell surface proteins are well suited for the targeted delivery of other therapeutics, such as conjugated small interfering RNAs (siRNA) that induce RNA interference (RNAi). Thus, aptamer-siRNA chimeras may offer dual-functions, in which the aptamer inhibits a receptor function, while the siRNA internalizes into the cell to target a specific mRNA. This review focuses on the current progress and therapeutic potential of RNA aptamers, including the use of cell-internalizing aptamers as cell-type specific delivery vehicles for targeted RNAi. In particular, we discuss emerging aptamer-based therapeutics that provide unique clinical opportunities for the treatment various cancers and neurological diseases.
PMCID: PMC3486975  PMID: 23130020
RNA aptamers; systematic evolution of ligands by exponential enrichment; RNA interference; small interfering RNA; targeted delivery
7.  Use of Cell-SELEX to Generate DNA Aptamers as Molecular Probes of HPV-Associated Cervical Cancer Cells 
PLoS ONE  2012;7(4):e36103.
Disease-specific biomarkers are an important tool for the timely and effective management of pathological conditions, including determination of susceptibility, diagnosis, and monitoring efficacy of preventive or therapeutic strategies. Aptamers, comprising single-stranded or double-stranded DNA or RNA, can serve as biomarkers of disease or biological states. Aptamers can bind to specific epitopes on macromolecules by virtue of their three dimensional structures and, much like antibodies, aptamers can be used to target specific epitopes on the basis of their molecular shape. The Systematic Evolution of Ligands by EXponential enrichment (SELEX) is the approach used to select high affinity aptamers for specific macromolecular targets from among the >1013 oligomers comprising typical random oligomer libraries. In the present study, we used live cell-based SELEX to identify DNA aptamers which recognize cell surface differences between HPV-transformed cervical carcinoma cancer cells and isogenic, nontumorigenic, revertant cell lines.
Methodology/Principal Findings
Whole-cell SELEX methodology was adapted for use with adherent cell lines (which we termed Adherent Cell-SELEX (AC-SELEX)). Using this approach, we identified high affinity aptamers (nanomolar range Kd) to epitopes specific to the cell surface of two nontumorigenic, nontumorigenic revertants derived from the human cervical cancer HeLa cell line, and demonstrated the loss of these epitopes in another human papillomavirus transformed cervical cancer cell line (SiHa). We also performed preliminary investigation of the aptamer epitopes and their binding characteristics.
Using AC-SELEX we have generated several aptamers that have high affinity and specificity to the nontumorigenic, revertant of HPV-transformed cervical cancer cells. These aptamers can be used to identify new biomarkers that are related to carcinogenesis. Panels of aptamers, such as these may be useful in predicting the tumorigenic potential and properties of cancer biopsies and aid in the effective management of pathological conditions (diagnosis, predicted outcome, and treatment options).
PMCID: PMC3335032  PMID: 22536456
8.  Selective Uptake and Imaging of Aptamer- and Antibody-Conjugated Hollow Nanospheres Targeted to Epidermal Growth Factor Receptors Overexpressed in Head and Neck Cancer 
ACS Nano  2014;8(5):4530-4538.
The purpose of this study was to compare the binding affinity and selective targeting of aptamer- and antibody-coated hollow gold nanospheres (HAuNS) targeted to epidermal growth factor receptors (EGFR). EGFR-targeting aptamers were conjugated to HAuNS (apt-HAuNS) by attaching a thiol-terminated single-stranded DNA to the HAuNS and then adding the complementary RNA targeted to EGFR. Apt-HAuNS was characterized in terms of size, surface charge, absorption, and number of aptamers per particle. The in vivo pharmacokinetics, in vivo biodistribution, and micro-SPECT/CT imaging of 111In-labeled apt-HAuNS and anti-EGFR antibody (C225)-conjugated HAuNS were evaluated in nude mice bearing highly malignant human OSC-19 oral tumors. 111In-labeled PEG-HAuNS was used as a control (n = 5/group). Apt-HAuNS did not have an altered absorbance profile or size (λmax = 800 nm; diameter = 55 nm) compared to C225-HAuNS or PEG-HAuNS. The surface charge became more negative upon conjugation of the aptamer (−51.4 vs −19.0 for PEG-HAuNS and −25.0 for C225-HAuNS). The number of aptamers/particle was ∼250. In vitro cell binding and in vivo biodistribution showed selective binding of the apt-HAuNS to EGFR. μSPECT/CT imaging confirmed that there was more tumor uptake of apt-HAuNS than C225-HAuNS. Aptamer is a promising ligand for image-guided delivery of nanoparticles for treatment of tumor cells overexpressing EGFR.
PMCID: PMC4046795  PMID: 24754567
hollow gold nanospheres; SPECT/CT; biodistribution; epidermal growth factor receptor; head and neck cancer; aptamer
9.  Dual Functional RNA Nanoparticles Containing Phi29 Motor pRNA and Anti-gp120 Aptamer for Cell-type Specific Delivery and HIV-1 Inhibition 
Methods (San Diego, Calif.)  2011;54(2):284-294.
The potent ability of small interfering RNA (siRNA) to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for diseases including HIV. However, efficient delivery of siRNAs remains a key obstacle to successful application. A targeted intracellular delivery approach for siRNAs to specific cell types is highly desirable. HIV-1 infection is initiated by the interactions between viral glycoprotein gp120 and cell surface receptor CD4, leading to fusion of the viral membrane with the target cell membrane. Once HIV infects a cell it produces gp120 which is displayed at the cell surface. We previously described a novel dual inhibitory anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. We also demonstrated that gp120 can be used for aptamer mediated delivery of anti-HIV siRNAs.
Here we report the design, construction and evaluation of chimerical RNA nanoparticles containing a HIV gp120-binding aptamer escorted by the pRNA of bacteriophage phi29 DNA packaging motor. We demonstrate that pRNA-aptamer chimeras specifically bind to and are internalized into cells expressing HIV gp120. Moreover, the pRNA-aptamer chimeras alone also provide HIV inhibitory function by blocking viral infectivity. The Ab′ pRNA-siRNA chimera with 2′-F modified pyrimidines in the sense strand not only improved the RNA stability in serum, but also was functionally processed by Dicer, resulting in specific target gene silencing. Therefore, this dual functional pRNA-aptamer not only represents a potential HIV-1 inhibitor, but also provides a cell-type specific siRNA delivery vehicle, showing promise for systemic anti-HIV therapy.
PMCID: PMC3107903  PMID: 21256218
RNAi; Anti-gp120 aptamer; nanobiotechnology; bionanotechnology; nanotechnology; AIDS Treatment; viral DNA packaging; nanomotors
10.  Developing aptamer probes for acute myelogenous leukemia detection and surface protein biomarker discovery 
The majority of patients with acute myelogenous leukemia (AML) still die of their disease. In order to improve survival rates in AML patients, new strategies are necessary to discover biomarkers for the detection and targeted therapy of AML. One of the advantages of the aptamer-based technology is the unique cell-based selection process, which allows us to efficiently select for cell-specific aptamers without knowing which target molecules are present on the cell surface.
The NB4 AML cell line was used as the target cell population for selecting single stranded DNA aptamers. After determining the affinity of selected aptamers to leukocytes, the aptamers were used to phenotype human bone marrow leukocytes and AML cells in clinical specimens. Then a biotin-labelled aptamer was used to enrich and identify its target surface protein.
Three new aptamers were characterized from the selected aptamer pools (JH6, JH19, and K19). All of them can selectively recognize myeloid cells with Kd in the low nanomole range (2.77 to 12.37 nM). The target of the biotin-labelled K19 aptamer probe was identified as Siglec-5, a surface membrane protein in low abundance whose expression can serve as a biomarker of granulocytic maturation and be used to phenotype AML. More importantly, Siglec-5 expression can be used to detect low concentrations of AML cells in human bone marrow specimens, and functions as a potential target for leukemic therapy.
We have demonstrated a pipeline approach for developing single stranded DNA aptamer probes, phenotyping AML cells in clinical specimens, and then identifying the aptamer-recognized target protein. The developed aptamer probes and identified Siglec-5 protein may potentially be used for leukemic cell detection and therapy in our future clinical practice.
PMCID: PMC3895837  PMID: 24405684
Acute myeloid leukemia; Aptamer; Biomarker; Cell-SELEX; Siglec-5
11.  DNA-Aptamers Binding Aminoglycoside Antibiotics 
Sensors (Basel, Switzerland)  2014;14(2):3737-3755.
Aptamers are short, single stranded DNA or RNA oligonucleotides that are able to bind specifically and with high affinity to their non-nucleic acid target molecules. This binding reaction enables their application as biorecognition elements in biosensors and assays. As antibiotic residues pose a problem contributing to the emergence of antibiotic-resistant pathogens and thereby reducing the effectiveness of the drug to fight human infections, we selected aptamers targeted against the aminoglycoside antibiotic kanamycin A with the aim of constructing a robust and functional assay that can be used for water analysis. With this work we show that aptamers that were derived from a Capture-SELEX procedure targeting against kanamycin A also display binding to related aminoglycoside antibiotics. The binding patterns differ among all tested aptamers so that there are highly substance specific aptamers and more group specific aptamers binding to a different variety of aminoglycoside antibiotics. Also the region of the aminoglycoside antibiotics responsible for aptamer binding can be estimated. Affinities of the different aptamers for their target substance, kanamycin A, are measured with different approaches and are in the micromolar range. Finally, the proof of principle of an assay for detection of kanamycin A in a real water sample is given.
PMCID: PMC3958260  PMID: 24566637
aptamer; Capture-SELEX; aminoglycoside antibiotic; kanamycin
12.  Aptamers as Therapeutics in Cardiovascular Diseases 
Current medicinal chemistry  2011;18(27):4169-4174.
With many advantages over other therapeutic agents such as monoclonal antibodies, aptamers have recently emerged as a novel and powerful class of ligands with excellent potential for diagnostic and therapeutic applications. Typically generated through Systematic Evolution of Ligands by EXponential enrichment (SELEX), aptamers have been selected against a wide range of targets such as proteins, phospholipids, sugars, nucleic acids, as well as whole cells. DNA/RNA aptamers are single-stranded DNA/RNA oligonucleotides (with a molecular weight of 5–40 kDa) that can fold into well-defined 3D structures and bind to their target molecules with high affinity and specificity. A number of strategies have been adopted to synthesize aptamers with enhanced in vitro/in vivo stability, aiming at potential therapeutic/diagnostic applications in the clinic. In cardiovascular diseases, aptamers can be developed into therapeutic agents as anti-thrombotics, anti-coagulants, among others. This review focuses on aptamers that were selected against various molecular targets involved in cardiovascular diseases: von Willebrand factor (vWF), thrombin, factor IX, phospholamban, P-selectin, platelet-derived growth factor, integrin αvβ3, CXCL10, vasopressin, among others. With continued effort in the development of aptamer-based therapeutics, aptamers will find their niches in cardiovascular diseases and significantly impact clinical patient management.
PMCID: PMC3205281  PMID: 21848510
Aptamers; cardiovascular diseases; von Willebrand factor (vWF); thrombin; factor IX; DNA; RNA; peptide aptamer
13.  Pharmacokinetics on a microscale: visualizing Cy5-labeled oligonucleotide release from poly(n-butylcyanoacrylate) nanocapsules in cells 
For successful design of a nanoparticulate drug delivery system, the fate of the carrier and cargo need to be followed. In this work, we fluorescently labeled poly(n-butylcyanoacrylate) (PBCA) nanocapsules as a shell and separately an oligonucleotide (20 mer) as a payload. The nanocapsules were formed by interfacial anionic polymerization on aqueous droplets generated by an inverse miniemulsion process. After uptake, the PBCA capsules were shown to be round-shaped, endosomal structures and the payload was successfully released. Cy5-labeled oligonucleotides accumulated at the mitochondrial membrane due to a combination of the high mitochondrial membrane potential and the specific molecular structure of Cy5. The specificity of this accumulation at the mitochondria was shown as the uncoupler dinitrophenol rapidly diminished the accumulation of the Cy5-labeled oligonucleotide. Importantly, a fluorescence resonance energy transfer investigation showed that the dye-labeled cargo (Cy3/Cy5-labeled oligonucleotides) reached its target site without degradation during escape from an endosomal compartment to the cytoplasm. The time course of accumulation of fluorescent signals at the mitochondria was determined by evaluating the colocalization of Cy5-labeled oligonucleotides and mitochondrial markers for up to 48 hours. As oligonucleotides are an ideal model system for small interfering RNA PBCA nanocapsules demonstrate to be a versatile delivery platform for small interfering RNA to treat a variety of diseases.
PMCID: PMC4251750  PMID: 25473285
drug delivery; mitochondria; miniemulsion; colocalization
14.  Clinical applications of nucleic acid aptamers in cancer 
Molecular and Clinical Oncology  2014;2(3):341-348.
Nucleic acid aptamers are small single-stranded DNA or RNA oligonucleotide segments, which bind to their targets with high affinity and specificity via unique three-dimensional structures. Aptamers are generated by an iterative in vitro selection process, termed as systematic evolution of ligands by exponential enrichment. Owing to their specificity, non-immunogenicity, non-toxicity, easily modified chemical structure and wide range of targets, aptamers appear to be ideal candidates for various clinical applications (diagnosis or treatment), such as cell detection, target diagnosis, molecular imaging and drug delivery. Several aptamers have entered the clinical pipeline for applications in diseases such as macular degeneration, coronary artery bypass graft surgery and various types of cancer. The aim of this review was to summarize and highlight the clinical applications of aptamers in cancer diagnosis and treatment.
PMCID: PMC3999129  PMID: 24772298
aptamer; clinical application; cancer diagnosis; cancer therapy; systematic evolution of ligands by exponential enrichment
15.  Aptamer-mediated selective delivery of short RNA therapeutics in cancer cells 
RNA interference (RNAi) is an important biological process that ultimately leads to suppression of gene expression. Activators of RNAi are typically small interfering RNAs (siRNA) and microRNAs (miRNA) that offer considerable therapeutic potnetial. However, a major obstacle to take these these molecules to the clinic is the absence of safe and reliable means for their specific delivery to target cells. In this regard, a highly promising class of molecules is represented by nucleic acid aptamers. These are short, structured, single-stranded RNAs or DNAs oligonucleotides that, by binding with high specificity to target molecules, provide high affinity ligands and potential antagonists of disease-associated proteins. Further, because of the high binding specificity, aptamers represent a powerful tool for the selective delivery of therapeutic cargos, including mi/siRNAs, chemotherapeutics, toxins and nanoparticles to cancer cells or tissues, thus potentially increasing the efficacy of a given therapy as well as reducing toxicity. In this review, we will focus on recent advances in the field of aptamer-mediated mi/siRNA delivery, discussing their potential and challenges in cancer therapy.
PMCID: PMC4238741  PMID: 25414727
microRNA; siRNA; aptamer; targeted delivery; cancer
16.  An RNA Aptamer Provides a Novel Approach for the Induction of Apoptosis by Targeting the HPV16 E7 Oncoprotein 
PLoS ONE  2013;8(5):e64781.
Human papillomavirus 16 (HPV16) is a high-risk DNA tumour virus, which is a major causative agent of cervical cancer. Cellular transformation is associated with deregulated expression of the E6 and E7 oncogenes. E7 has been shown to bind a number of cellular proteins, including the cell cycle control protein pRb. In this study, RNA aptamers (small, single-stranded oligonucleotides selected for high-affinity binding) to HPV16 E7 were employed as molecular tools to further investigate these protein-protein interactions.
Methodology/Principal Findings
This study is focused on one aptamer (termed A2). Transfection of this molecule into HPV16-transformed cells resulted in inhibition of cell proliferation (shown using real-time cell electronic sensing and MTT assays) due to the induction of apoptosis (as demonstrated by Annexin V/propidium iodide staining). GST-pull down and bead binding assays were used to demonstrate that the binding of A2 required N-terminal residues of E7 known to be involved in interaction with the cell cycle control protein, pRb. Using a similar approach, A2 was shown to disrupt the interaction between E7 and pRb in vitro. Furthermore, transfection of HPV16-transformed cells with A2 appeared to result in the loss of E7 and rise in pRb levels, as observed by immunoblotting.
This paper includes the first characterisation of the effects of an E7 RNA aptamer in a cell line derived from a cervical carcinoma. Transfection of cells with A2 was correlated with the loss of E7 and the induction of apoptosis. Aptamers specific for a number of cellular and viral proteins have been documented previously; one aptamer (Macugen) is approved for clinical use and several others are in clinical trials. In addition to its role as a molecular tool, A2 could have further applications in the future.
PMCID: PMC3667794  PMID: 23738000
17.  Aptamers for pharmaceuticals and their application in environmental analytics 
Bioanalytical Reviews  2011;4(1):1-30.
Aptamers are single-stranded DNA or RNA oligonucleotides, which are able to bind with high affinity and specificity to their target. This property is used for a multitude of applications, for instance as molecular recognition elements in biosensors and other assays. Biosensor application of aptamers offers the possibility for fast and easy detection of environmental relevant substances. Pharmaceutical residues, deriving from human or animal medical treatment, are found in surface, ground, and drinking water. At least the whole range of frequently administered drugs can be detected in noticeable concentrations. Biosensors and assays based on aptamers as specific recognition elements are very convenient for this application because aptamer development is possible for toxic targets. Commonly used biological receptors for biosensors like enzymes or antibodies are mostly unavailable for the detection of pharmaceuticals. This review describes the research activities of aptamer and sensor developments for pharmaceutical detection, with focus on environmental applications.
PMCID: PMC3281204  PMID: 22389661
Aptamer; Small organic molecule; Pharmaceutical; Biosensor; Environmental analysis
18.  Development of Novel Single-Stranded Nucleic Acid Aptamers against the Pro-Angiogenic and Metastatic Enzyme Heparanase (HPSE1) 
PLoS ONE  2012;7(6):e37938.
Heparanase is an enzyme involved in extracellular matrix remodelling and heparan sulphate proteoglycan catabolism. It is secreted by metastatic tumour cells, allowing them to penetrate the endothelial cell layer and basement membrane to invade target organs. The release of growth factors at the site of cleaved heparan sulphate chains further enhance the potential of the tumour by encouraging the process of angiogenesis. This leads to increased survival and further proliferation of the tumour. Aptamers are single or double stranded oligonucleotides that recognise specific small molecules, peptides, proteins, or even cells or tissues and have shown great potential over the years as diagnostic and therapeutic agents in anticancer treatment. For the first time, single stranded DNA aptamers were successfully generated against the active heterodimer form of heparanase using a modified SELEX protocol, and eluted based on increasing affinity for the target. Sandwich ELISA assays showed recognition of heparanase by the aptamers at a site distinct from that of a polyclonal HPSE1 antibody. The binding affinities of aptamer to immobilised enzyme were high (7×107 to 8×107 M−1) as measured by fluorescence spectroscopy. Immunohistochemistry and immunofluorescence studies demonstrated that the aptamers were able to recognise heparanase with staining comparable or in some cases superior to that of the HPSE1 antibody control. Finally, matrigel assay demonstrated that aptamers were able to inhibit heparanase. This study provides clear proof of principle concept that nucleic acid aptamers can be generated against heparanase. These reagents may serve as useful tools to explore the functional role of the enzyme and in the future development of diagnostic assays or therapeutic reagents.
PMCID: PMC3376095  PMID: 22719856
19.  Thrombin–aptamer recognition: a revealed ambiguity 
Nucleic Acids Research  2011;39(17):7858-7867.
Aptamers are structured oligonucleotides that recognize molecular targets and can function as direct protein inhibitors. The best-known example is the thrombin-binding aptamer, TBA, a single-stranded 15-mer DNA that inhibits the activity of thrombin, the key enzyme of coagulation cascade. TBA folds as a G-quadruplex structure, as proved by its NMR structure. The X-ray structure of the complex between TBA and human α-thrombin was solved at 2.9-Å resolution, but did not provide details of the aptamer conformation and the interactions with the protein molecule. TBA is rapidly processed by nucleases. To improve the properties of TBA, a number of modified analogs have been produced. In particular, a modified TBA containing a 5′-5′ polarity inversion site, mTBA, has higher stability and higher affinity toward thrombin with respect to TBA, although it has a lower inhibitory activity. We present the crystal structure of the thrombin–mTBA complex at 2.15-Å resolution; the resulting model eventually provides a clear picture of thrombin–aptamers interaction, and also highlights the structural bases of the different properties of TBA and mTBA. Our findings open the way for a rational design of modified aptamers with improved potency as anticoagulant drugs.
PMCID: PMC3177225  PMID: 21715374
20.  Conformational plasticity of RNA for target recognition as revealed by the 2.15 Å crystal structure of a human IgG–aptamer complex 
Nucleic Acids Research  2010;38(21):7822-7829.
Aptamers are short single-stranded nucleic acids with high affinity to target molecules and are applicable to therapeutics and diagnostics. Regardless of an increasing number of reported aptamers, the structural basis of the interaction of RNA aptamer with proteins is poorly understood. Here, we determined the 2.15 Å crystal structure of the Fc fragment of human IgG1 (hFc1) complexed with an anti-Fc RNA aptamer. The aptamer adopts a characteristic structure fit to hFc1 that is stabilized by a calcium ion, and the binding activity of the aptamer can be controlled many times by calcium chelation and addition. Importantly, the aptamer–hFc1 interaction involves mainly van der Waals contacts and hydrogen bonds rather than electrostatic forces, in contrast to other known aptamer–protein complexes. Moreover, the aptamer–hFc1 interaction involves human IgG-specific amino acids, rendering the aptamer specific to human IgGs, and not crossreactive to other species IgGs. Hence, the aptamer is a potent alternative for protein A affinity purification of Fc-fusion proteins and therapeutic antibodies. These results demonstrate, from a structural viewpoint, that conformational plasticity and selectivity of an RNA aptamer is achieved by multiple interactions other than electrostatic forces, which is applicable to many protein targets of low or no affinity to nucleic acids.
PMCID: PMC2995045  PMID: 20675355
21.  Designing Anti-Influenza Aptamers: Novel Quantitative Structure Activity Relationship Approach Gives Insights into Aptamer – Virus Interaction 
PLoS ONE  2014;9(5):e97696.
This study describes the development of aptamers as a therapy against influenza virus infection. Aptamers are oligonucleotides (like ssDNA or RNA) that are capable of binding to a variety of molecular targets with high affinity and specificity. We have studied the ssDNA aptamer BV02, which was designed to inhibit influenza infection by targeting the hemagglutinin viral protein, a protein that facilitates the first stage of the virus’ infection. While testing other aptamers and during lead optimization, we realized that the dominant characteristics that determine the aptamer’s binding to the influenza virus may not necessarily be sequence-specific, as with other known aptamers, but rather depend on general 2D structural motifs. We adopted QSAR (quantitative structure activity relationship) tool and developed computational algorithm that correlate six calculated structural and physicochemical properties to the aptamers’ binding affinity to the virus. The QSAR study provided us with a predictive tool of the binding potential of an aptamer to the influenza virus. The correlation between the calculated and actual binding was R2 = 0.702 for the training set, and R2 = 0.66 for the independent test set. Moreover, in the test set the model’s sensitivity was 89%, and the specificity was 87%, in selecting aptamers with enhanced viral binding. The most important properties that positively correlated with the aptamer’s binding were the aptamer length, 2D-loops and repeating sequences of C nucleotides. Based on the structure-activity study, we have managed to produce aptamers having viral affinity that was more than 20 times higher than that of the original BV02 aptamer. Further testing of influenza infection in cell culture and animal models yielded aptamers with 10 to 15 times greater anti-viral activity than the BV02 aptamer. Our insights concerning the mechanism of action and the structural and physicochemical properties that govern the interaction with the influenza virus are discussed.
PMCID: PMC4028238  PMID: 24846127
22.  DNA aptamers that target human glioblastoma multiforme cells overexpressing epidermal growth factor receptor variant III in vitro 
Acta Pharmacologica Sinica  2013;34(12):1491-1498.
Aptamers are oligonucleic acid or peptide molecules that bind to a specific target molecule in cells, thus may act as effective vehicles for drug or siRNA delivery. In this study we investigated the DNA aptamers that target human glioblastoma multiforme (GBM) cells overexpressing epidermal growth factor receptor variant III (EGFRvIII), which was linked to radiation and chemotherapeutic resistance of this most aggressive brain tumor.
A 73-mer ssDNA library containing molecules with 30 nt of random sequence flanked by two primer hybridization sites was chosen as the initial library. Cell systematic evolution of ligands by exponential enrichment (Cell-SELEX) method was used to select the DNA aptamers that target EGFRvIII. The binding affinity of the aptamers was measured using a cell-based biotin-avidin ELISA.
After 14 rounds of selection, four DNA aptamers (32, 41, 43, and 47) that specifically bound to the EGFRvIII-overexpressing human glioma U87Δ cells with Kd values of less than 100 nmol/L were discovered. These aptamers were able to distinguish the U87Δ cells from the negative control human glioma U87MG cells and HEK293 cells. Aptamer 32 specifically bound to the EGFRvIII protein with an affinity similar to the EGFR antibody (Kd values of aptamer 32 and the EGFR antibody were 0.62±0.04 and 0.32±0.01 nmol/L, respectively), and this aptamer was localized in the cell nucleus.
The DNA aptamers are promising molecular probes for the diagnosis and treatment of GBM.
PMCID: PMC4002567  PMID: 24304919
DNA aptamer; drug delivery; brain tumor; glioblastoma multiforme; epidermal growth factor receptor variant III; cell systematic evolution of ligands by exponential enrichment
23.  A logical molecular circuit for programmable and autonomous regulation of protein activity using DNA aptamer-protein interactions 
Journal of the American Chemical Society  2012;134(51):20797-20804.
Researchers increasingly envision an important role for artificial biochemical circuits in biological engineering, much like electrical circuits in electrical engineering. Similar to electrical circuits, which control electromechanical devices, biochemical circuits could be utilized as a type of servomechanism to control nanodevices in vitro, monitor chemical reactions in situ, or regulate gene expressions in vivo.1 As a consequence of their relative robustness and potential applicability for controlling a wide range of in vitro chemistries, synthetic cell-free biochemical circuits promise to be useful in manipulating the functions of biological molecules. Here we describe the first logical circuit based on DNA-protein interactions with accurate threshold control, enabling autonomous, self-sustained and programmable manipulation of protein activity in vitro. Similar circuits made previously were based primarily on DNA hybridization and strand displacement reactions. This new design uses the diverse nucleic acid interactions with proteins. The circuit can precisely sense the local enzymatic environment, such as the concentration of thrombin, and when it is excessively high, a coagulation inhibitor is automatically released by a concentration-adjusted circuit module. To demonstrate the programmable and autonomous modulation, a molecular circuit with different threshold concentrations of thrombin was tested as a proof of principle. In the future, owing to tunable regulation, design modularity and target specificity, this prototype could lead to the development of novel DNA biochemical circuits to control the delivery of aptamer-based drugs in smart and personalized medicine, providing a more efficient and safer therapeutic strategy.
PMCID: PMC3570058  PMID: 23194304
Aptamer; logical circuit; protein activity; thrombin
24.  Recent Progress in Nucleic Acid Aptamer-Based Biosensors and Bioassays 
Sensors (Basel, Switzerland)  2008;8(11):7050-7084.
As the key constituents of the genetic code, the importance of nucleic acids to life has long been appreciated. Despite being composed of only four structurally similar nucleotides, single-stranded nucleic acids, as in single-stranded DNAs and RNAs, can fold into distinct three-dimensional shapes due to specific intramolecular interactions and carry out functions beyond serving as templates for protein synthesis. These functional nucleic acids (FNAs) can catalyze chemical reactions, regulate gene expression, and recognize target molecules. Aptamers, whose name is derived from the Latin word aptus meaning “to fit”, are oligonucleotides that can bind their target ligands with high affinity and specificity. Since aptamers exist in nature but can also be artificially isolated from pools of random nucleic acids through a process called in vitro selection, they can potentially bind a diverse array of compounds. In this review, we will discuss the research that is being done to develop aptamers against various biomolecules, the progress in engineering biosensors by coupling aptamers to signal transducers, and the prospect of employing these sensors for a range of chemical and biological applications. Advances in aptamer technology emphasizes that nucleic acids are not only the fundamental molecules of life, they can also serve as research tools to enhance our understanding of life. The possibility of using aptamer-based tools in drug discovery and the identification of infectious agents can ultimately augment our quality of life.
PMCID: PMC3787431
Aptamers; biosensors; bioassays
25.  Aptamer-Mediated Delivery of Splice-Switching Oligonucleotides to the Nuclei of Cancer Cells 
Nucleic Acid Therapeutics  2012;22(3):187-195.
To reduce the adverse effects of cancer therapies and increase their efficacy, new delivery agents that specifically target cancer cells are needed. We and others have shown that aptamers can selectively deliver therapeutic oligonucleotides to the endosome and cytoplasm of cancer cells that express a particular cell surface receptor. Identifying a single aptamer that can internalize into many different cancer cell-types would increase the utility of aptamer-mediated delivery of therapeutic agents. We investigated the ability of the nucleolin aptamer (AS1411) to internalize into multiple cancer cell types and observed that it internalizes into a wide variety of cancer cells and migrates to the nucleus. To determine if the aptamer could be utilized to deliver therapeutic oligonucleotides to modulate events in the nucleus, we evaluated the ability of the aptamer to deliver splice-switching oligonucleotides. We observed that aptamer-splice-switching oligonucleotide chimeras can alter splicing in the nuclei of treated cells and are effective at lower doses than the splice switching oligonucleotides alone. Our results suggest that aptamers can be utilized to deliver oligonucleotides to the nucleus of a wide variety of cancer cells to modulate nuclear events such as RNA splicing.
PMCID: PMC3423875  PMID: 22703281

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