Vascular endothelial cells are essential to vascular function and maintenance. Dysfunction of these cells can lead to the development of cardiovascular disease or contribute to tumorigenesis. As such, the therapeutic modulation and monitoring of vascular endothelial cells are of significant clinical interest, and several endothelial-specific ligands have been developed for drug delivery and the monitoring of endothelial function. However, the application of these ligands has been limited by their high cost and tendency to induce immune responses, highlighting a need for alternate methods of targeting vascular endothelial cells. In the present study, we explore the therapeutic potential of DNA aptamers. Using cell-SELEX technology, we identified two aptamers with specific binding affinity for vascular endothelial cells and propose that these molecules show potential for use as new ligands for drug and biomarker research concerning vascular endothelial cells.

The effects of modified deoxyuridine triphosphates (mod-dUTPs) with different substituents at the C5 position of the pyrimidine cycle on the kinetics of PCR with Taq and Vent (exo-) DNA polymerases are studied. Substituents in mod-dUTP include carboxamide group and groups that are part of the side chains of alanine, valine, leucine, phenylalanine, tryptophan, or tyrosine. For each mod-dUTP, the yields of the target product are measured with the full substitution of dTTP. A fragment of bacterial DNA with a certain nucleotide sequence and a synthetic combinatorial DNA library of random nucleotide sequences are used as templates for amplification. For each mod-dUTP-template-polymerase combination, the correlation between the amplification efficiencies and yields of the target product are investigated. PCR product accumulation curves are influenced by both the template used and the presence of a modified substrate. The catalytic activity of Taq polymerase is higher when mod-dUTPs with short aliphatic substituents are used and decreases when the derivatives with long aliphatic, phenyl, and indole substituents are utilized. Vent (exo-) polymerase is less sensitive to the chemical structure of mod-dUTP. The dynamic measuring of DNA accumulation may be useful for optimizing the temperature-time PCR profiles individually for each of the mod-dUTP. The derivatives may be used in combination with Vent (exo-) polymerase to obtain modified DNA sequences for the method of selection of modified aptamers (mod-SELEX).

The delivery of therapeutics across the blood-brain barrier remains a considerable challenge in investigating central nervous system related processes. In this work, a liposome vehicle was surface-modified with an aptamer that binds to the transferrin receptor and was loaded with two different dopamine-binding aptamer payloads. This system was effectively used to promote the delivery of the aptamer cargo from the peripheral injection site into the brain. The effect of these delivered aptamers on behavior was investigated in vivo in a locomotor task. The first dopamine binding aptamer assessed was a DNA aptamer, the binding of which had been previously validated through the aptamer-based biosensor development reported by several independent research groups. The second aptamer investigated was the result of a novel in vitro selection experiment described herein. Our data suggest that systemic administration of the modified liposomes led to delivery of the dopamine aptamers into the brain. Fluorescence microscopy revealed differential distribution of fluorescence based on the presence or absence of the transferrin receptor aptamer on the surface of fluorescently modified liposomes. In a behavioral experiment using cocaine administration to induce elevated concentrations of neural dopamine, systemic pretreatment with the dopamine aptamer-loaded liposomes reduced cocaine-induced hyperlocomotion. Multiple controls including a transferrin-negative liposome control and transferrin-positive liposomes loaded with either a nonbinding, base-substituted dopamine aptamer or a random oligonucleotide were investigated. None of these controls altered cocaine-induced hyperlocomotion. Chronic systemic administration of the modified liposomes produced no deleterious neurobehavioral or neural degenerative effects. Importantly, this work is one example of an application for this versatile multiaptamer payload/targeting system. Its general application is limited only by the availability of aptamers for specific neural targets.

Early identification of neoplastic diseases is essential to achieve timely therapeutic interventions and significantly reduce the mortality of patients. A well-known biomarker is the Cancer Antigen 125 (CA125) or 16 mucin (MUC 16), a glycoprotein of the human family of mucins, already used for the diagnostic and prognostic evaluation of ovarian cancer. Therefore, the detection of CA125 to now remains a promising tool in the early diagnosis of this tumor. In this paper, we describe the development of RNA aptamers that bind with high affinity the tumor antigen CA125. We performed eight cycles of selection against CA125 purified protein. The selected aptamers were cloned and sequenced and the binding properties of the most promising sequences were studied by Real Time PCR and Surface Plasmon Resonance (SPR) to evaluate their ability in targeting CA125 protein with perspective applications in aptamer-based bioassays.

RNA-binding proteins (RBPs) play an important role in regulating gene expression at the posttranscriptional level, including the steps of pre-mRNA splicing, polyadenylation, mRNA stabilization, mRNA export from the nucleus to the cytoplasm, mRNA localization, and translation. RBPs regulate these processes primarily by binding to specific sequence elements in newly synthesized or mature transcripts. While many RPBs are known to recognize certain nucleotide sequences in RNA, information is insufficient for others. In particular, RBPs often compete for RNA binding or interact with RNA cooperatively. Hence, it is of importance to study the RNA-protein interactions in vivo. Numerous methods have been developed to identify the target nucleotide sequences of RBPs. The methods include the electrophoretic mobility shift assay (EMSA), systematic evolution of ligands by exponential enrichment (SELEX), RNA pull-down assay, RNA footprinting, RNA immunoprecipitation (RIP), UV-induced crosslinking immunoprecipitation (CLIP) and its variants, and measurement of the level for newly synthesized transcripts. Each of the methods has its limitation, and several methods supplementing each other should be employed in order to detect the RNA sequence to which a protein binds.