Rapid Complexation of Aptamers by Their Specific Antidotes.

Nucleic acid ligands, aptamers, harbor the unique characteristics of small molecules and antibodies. The specificity and high affinity of aptamers enable their binding to different targets, such as small molecules, proteins, or cells. Chemical modifications of aptamers allow increased bioavailability. A further great benefit of aptamers is the antidote (AD)-mediated controllability of their effect. In this study, the AD-mediated complexation and neutralization of the thrombin binding aptamer NU172 and Toll-like receptor 9 (TLR9) binding R10-60 aptamer were determined. Thereby, the required time for the generation of aptamer/AD-complexes was analyzed at 37 °C in human serum using gel electrophoresis. Afterwards, the blocking of aptamers' effects was analyzed by determining the activated clotting time (ACT) in the case of the NU172 aptamer, or the expression of immune activation related genes IFN-1ß, IL-6, CXCL-10, and IL-1ß in the case of the R10-60 aptamer. Gel electrophoresis analyses demonstrated the rapid complexation of the NU172 and R10-60 aptamers by complementary AD binding after just 2 min of incubation in human serum. A rapid neutralization of anticoagulant activity of NU172 was also demonstrated in fresh human whole blood 5 min after addition of AD. Furthermore, the TLR9-mediated activation of PMDC05 cells was interrupted after the addition of the R10-60 AD. Using these two different aptamers, the rapid antagonizability of the aptamers was demonstrated in different environments; whole blood containing numerous proteins, cells, and different small molecules, serum, or cell culture media. Thus, nucleic acid ADs are promising molecules, which offer several possibilities for different in vivo applications, such as antagonizing aptamer-based drugs, immobilization, or delivery of oligonucleotides to defined locations.

Generation of Large-Scale DNA Hydrogels with Excellent Blood and Cell Compatibility.

Hemocompatibility and cytocompatibility of biomaterials codetermine the success of tissue engineering applications. DNA, the natural component of our cells, is an auspicious biomaterial for the generation of designable scaffolds with tailorable characteristics. In this study, a combination of rolling circle amplification and multiprimed chain amplification is used to generate hydrogels at centimeter scale consisting solely of DNA. Using an in vitro rotation model and fresh human blood, the reaction of the hemostatic system on DNA hydrogels is analyzed. The measurements of hemolysis, platelets activation, and the activation of the complement, coagulation, and neutrophils using enzyme-linked immunosorbent assays demonstrate excellent hemocompatibility. In addition, the cytocompatibility of the DNA hydrogels is tested by indirect contact (agar diffusion tests) and material extract experiments with L929 murine fibroblasts according to the ISO 10993-5 specifications and no negative impact on the cell viability is detected. These results indicate the promising potential of DNA hydrogels as biomaterials for versatile applications in the field of regenerative medicine.

Microfluidic chip system for the selection and enrichment of cell binding aptamers.

Aptamers are promising cell targeting ligands for several applications such as for the diagnosis, therapy, and drug delivery. Especially, in the field of regenerative medicine, stem cell specific aptamers have an enormous potential. Using the combinatorial chemistry process SELEX (Systematic Evolution of Ligands by Exponential enrichment), aptamers are selected from a huge oligonucleotide library consisting of approximately 10(15) different oligonucleotides. Here, we developed a microfluidic chip system that can be used for the selection of cell specific aptamers. The major drawbacks of common cell-SELEX methods are the inefficient elimination of the unspecifically bound oligonucleotides from the cell surface and the unspecific binding/uptake of oligonucleotides by dead cells. To overcome these obstacles, a microfluidic device, which enables the simultaneous performance of dielectrophoresis and electrophoresis in the same device, was designed. Using this system, viable cells can be selectively assembled by dielectrophoresis between the electrodes and then incubated with the oligonucleotides. To reduce the rate of unspecifically bound sequences, electrophoretic fields can be applied in order to draw loosely bound oligonucleotides away from the cells. Furthermore, by increasing the flow rate in the chip during the iterative rounds of SELEX, the selection pressure can be improved and aptamers with higher affinities and specificities can be obtained. This new microfluidic device has a tremendous capability to improve the cell-SELEX procedure and to select highly specific aptamers.

Importance of rigorous in vitro evaluation of prospective cell binding aptamers.

Hitherto, several aptamers have been selected against cell surface molecules. The use of these aptamers for in vivo applications requires the prior in-depth in vitro evaluation of cell specific binding. Here, we demonstrate the in vitro tests, which are imperatively necessary to evaluate aptamers prior to in vivo applications. Exemplarily, the target binding of a chemically synthesized model aptamer containing phosphorothioate linkages was tested after the induction of the target protein expression on the cell surface by using flow cytometry. Furthermore, different cell types were used to compare the binding of the aptamer. Different single stranded DNA oligonucleotides were selected as negative controls to evaluate sequence specific binding of the aptamer to the cells. In further experiments, the aptamer binding to the target cells was determined in a mixture containing human plasma and peripheral blood cells to simulate the binding of the aptamer to target cells in human whole blood. In this study, we demonstrated the compelling necessity of the in vitro binding tests with the selected aptamers using target and non-target cells, the use of appropriate nonsense aptamers to validate the sequence specific binding of aptamers, and the evaluation of target binding in human plasma containing blood proteins and cells. Thus, we recommend the use of described methods to validate the target specific binding of newly selected aptamers prior to in vivo applications.

New basic approach to treat non-small cell lung cancer based on RNA-interference.

BACKGROUND: To date the therapy for non-small cell lung cancer (NSCLC) is associated with severe side effects, frustrating outcomes, and does not consider different tumor characteristics. The RNA-interference (RNAi) pathway represents a potential new approach to treat NSCLC. With small interfering ribonucleic acids (siRNAs), it is possible to reduce the expression of proliferation-dependent proteins in tumor cells, leading to their apoptosis. We propose that siRNAs could be adapted to the tumor type and may cause fewer side effects than current therapy. METHODS: Four NSCLC cell lines were cultured under standard conditions and transfected with three different concentrations of siRNAs targeted against the hypoxia-inducible factors 1α and 2α (HIF1α and HIF2α) and signal transducer and activator of transcription 3 (STAT3). The expression was observed by quantitative real-time polymerase chain reaction and western blots. For the analysis of cell growth three days after transfection, the cell number was detected using a CASY cell counter system. RESULTS: The results of the silencing of the analyzed factors differ in each cell line. Cell growth was significantly reduced in all cell lines after transfection with HIF1α- and STAT3-siRNA. The silencing of HIF2α resulted in a significant effect on cell growth in squamous, and large-cell lung cancer. CONCLUSIONS: This study shows that the knockdown and viability to siRNA transfection differ in each tumor type according to the used siRNA. This implies that the tumor types differ among themselves and should be treated differently. Therefore, the authors suggest a possible approach to a more personalized treatment of NSCLC.

In vivo tissue engineering: mimicry of homing factors for self-endothelialization of blood-contacting materials.

Thrombogenicity of foreign surfaces is the major obstacle in cardiovascular interventions. Despite enormous advances in biomaterials research, the hemocompatibility of blood-contacting materials is still not satisfactory and the native endothelium still represents the ideal surface for blood contact. Circulating adult endothelial progenitor cells (EPCs) in the human blood provide an excellent source of autologous stem cells for the in vivo self-endothelialization of blood-contacting materials. For this purpose, material surfaces can be coated with capture molecules mimicking natural homing factors to attract circulating EPCs. Hitherto, several ligands, such as aptamers, monoclonal antibodies, peptides, selectins and their ligands, or magnetic molecules, are used to biofunctionalize surfaces for the capturing of EPCs directly from patient's bloodstream onto blood-contacting materials. Subsequently, attracted EPCs can differentiate into endothelial cells and generate an autologous endothelium. The in vivo self-endothelialization of blood-contacting materials prevents the recognition of them as a foreign body; this opens up revolutionary new prospects for future clinical stem-cell and tissue engineering strategies.

Absolute quantification of cell-bound DNA aptamers during SELEX.

In the fields of diagnosis, imaging, regenerative medicine, and drug targeting, aptamers are promising nucleic acid ligands for specific recognition and binding of whole living cells. These aptamers are selected by a combinatorial chemistry technique called cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment). During this iterative procedure of in vitro selection and enzymatic amplification, the enrichment of cell binding aptamers is generally monitored by flow cytometry. This method needs the use of fluorophore-labeled oligonucleotides for detection and allows only the relative evaluation of the aptamer binding compared with the control. Here, we describe the development and validation of a new quantitative real time polymerase chain reaction (qPCR) method for the absolute determination of cell bound aptamers during cell-SELEX. The method is based on SYBR Green I real-time PCR technology and uses an aptamer standard curve to determine the accurate aptamer amount on cells after the incubations. Lysates of cells with bound aptamers were used to identify the absolute amount of aptamers on cells. This method is highly sensitive and allows the detection of very small quantities of aptamers in cell lysate samples. The lower detection limit is 20 fg. The established qPCR method can be used as an additional monitoring tool during cell-SELEX to determine the enrichment of cell binding aptamers on cells, whereby the absolute quantity is determined. Furthermore, the contamination of the amplified aptamer pool with by-products can be prevented by prior determination of bound aptamer amount on cells.