RNA and DNA aptamers in cytomics analysis

Ulrich, Henning; Martins, Ana; Pesquero, João Bosco

doi:10.1002/cyto.a.20056

Cited by 79 publications

(65 citation statements)

References 73 publications

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“…Chemical modifications that enhance aptamer stability for in vivo applications and possible therapy include substitutions of the 2 0 OH group of the ribose backbone of pyrimidines by amino, fluoride, or O-methylene groups. With such modifications, the half-life of RNA aptamers in biological fluids such as human serum or urine increases from $8 s for unmodified RNA to 86 h for fluoroor amino-modified RNA (7). Various aptamer lead compounds identified as inhibitors of protein functions implicated in disease are currently under evaluation in preclinical and clinical tests.…”

Section: The Selex Technologymentioning

confidence: 99%

“…Aptamers binding to Mycobacterium tuberculosis (19), kinetoplastid membrane protein-11 (KMP-11) of Leishmania infantum (20), or to specific cell surface antigens of T. cruzi (21) or T. brucei (22) may be used for diagnosis of bacterial or parasite infection. Like antibodies, aptamers can be easily tagged with fluorescence reporters or nanoparticles for localization-or pull-down-experiments of target proteins (7). In addition to cell sorting for the enrichment of target-cell specific aptamers during the SELEX process (23), detection of cultured precursor T-cell acute lymphoblastic leukemia CCRF-CEM cells in whole blood samples was performed by flow cytometry (24,25).…”

Section: The Selex Technologymentioning

confidence: 99%

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Disease‐specific biomarker discovery by aptamers

Ulrich

Wrenger

2009

Cytometry Pt A

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RNA and DNA aptamers developed by an in vitro selection process, Systematic Evolution of Ligands by EXponential enrichment (SELEX), comprise a novel class of highaffinity and specific capture agents, which can be easily modified for cytometry and in vivo applications. A novel application of this technique (Cell SELEX) explores the expression of cell surface epitopes that differ between two given cell types or between healthy and diseased cells. Using whole cells as targets, aptamer libraries can be identified that bind to biomarkers expressed by target cells and not by any other cells. Aptamers have been developed that specifically interact with cell surface epitopes of trypanosomes or distinguish between the differences in molecular signature of somatic and cancer cells. Aside from its use for target cell identification by image and flow cytometry and laser-scanning microscopy, aptamers can be used for ligand-mediated purification and identification of their binding proteins in target cell membranes. In this review, we discuss an approach for the development of aptamers targeting parasitederived surface proteins of Trypanosoma and Plasmodium. ' 2009 International Society for Advancement of CytometryKey terms aptamers; Plasmodium falciparum; deconvolution SELEX; cell and target protein identification; fluorescence-labeled aptamers for clinical and cytomics application THE SELEX TECHNOLOGYThe SELEX technique (Systematic Evolution of Ligands by EXponential enrichment) initially developed by the laboratories of Larry Gold (1) and Jack Szostak (2) uses iterative in vitro selection of combinatorial RNA or DNA pools against a target molecule for the identification of high-affinity oligonucleotide ligands, known as aptamers. This methodology has been extensively used to isolate aptamers for a wide variety of peptides and proteins of therapeutic importance, including growth and coagulation factors (3). This novel class of oligonucleotide-based ligands recognizes their targets with binding specificities and affinities comparable to those of monoclonal antibodies. Moreover, aptamers are able to distinguish between protein isoforms and different conformational forms of the same protein (4,5). In most cases, aptamer binding to its target protein inhibits its biological activity, either resulting from aptamer interference with the catalytic site of an enzyme or with sites involved in ligandreceptor recognition. However, it is also possible that aptamer-target protein binding induces allosteric effects, such as changes in conformational states resulting in loss of biological activity of the target protein.Aptamers can be identified by in vitro selection against almost any target, including toxins and antigens which do not induce immune responses in host animals for antibody production. As a result, this novel class of ligands is highly promising for the development of therapeutics and biotechnological tools. The potential utility of aptamers for in vivo applications and as therapeutic agents is considerably enhanced ...

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Section: The Selex Technologymentioning

confidence: 99%

Section: The Selex Technologymentioning

confidence: 99%

Disease‐specific biomarker discovery by aptamers

Ulrich

Wrenger

2009

Cytometry Pt A

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“…While this class of reagents is very diverse, including nucleic acid aptamers, affinity peptides and artificial binders based on scaffold molecules (reviewed in: Rimmele, 2003;Ulrich et al, 2004;Nygren and Skerra, 2004), here, we focus only on antibodies, as they are used in almost all currently ongoing programs. The use of antibodies will be a valuable asset for relating function and pathology to genomic sequence in biology and medicine.…”

Section: Antibodies For Functional Genomicsmentioning

confidence: 99%

Perspectives for systematic in vitro antibody generation

Konthur

Hust

Dübel

2005

Gene

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After the completion and refinement of the human genome, the characterization of individual gene products in respect of their functions, their modifications, their cellular localization and regulation in both space and time has generated an increased demand for antibodies for their analysis. Taking into account that the human genome contains¨25,000 genes, and that their products are found in different splice variants and produce proteins with post-translational modifications, it can be estimated that at least 100,000 different protein products have to be investigated to gain a complete picture of what's going on in the proteome of a cell.Antibodies are preferred tools helping with the characterization and detection of proteins as well as with elucidating their individual functions. The generation of antibodies to all available human protein products by immunization and/or the hybridoma technology is not only logistically and financially enduring, but may prove to be a difficult task, as quite a number of interesting targets may evade the immune response of experimental animals, for example, allosteric variants dependent on fragile interactions to cofactors, highly conserved antigens etc. For this reason, alternative methods for the generation of antibodies have to supplement these approaches. In vitro methods for antibody generation are seen to offer this capability. In addition, they may provide a cost effective and large scale production alternative for detection reagents for the research community in their own right. Among in vitro techniques, phage display has been evolved as the most efficient option for tackling this problem and approaches optimised for automation are emerging.Maximum benefit for proteomic research could be generated by judicious and preferably international coordination of the ongoing efforts to combine the strengths of the well established animal based approaches and the novel opportunities offered by in vitro methods. D

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“…Aptamers have great potential to function as molecular recognition elements in analytical systems for detection, separation, or purification of target molecules. Aptamers can substitute antibodies or be used in conjunction with antibodies in different assays and play increasingly important role in medical diagnosis and therapy (Brody and Gold, 2000), in cytomic research (Ulrich et al, 2004), and also in the wide area of environmental analysis.…”

Section: Introductionmentioning

confidence: 99%

Design and Characterization of DNA Aptamer for Breast Tumor Marker by an Advantageous Method

Agnihotri¹,

Dubey²,

Bhide³

2014

IJIRSET

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ABSTRACT:Aptamers are agents able to bind tightly and selectively to disease markers, offering great potential for applications in disease diagnosis and therapy. Functionally they are ssDNA or RNA oligonucleotides which are highly specific and selective to their target along with highest affinity. We have selected DNA aptamer against breast tumor marker (CA 15-3) by using a modified SELEX methodology. For the selection round CA 15-3 were immobilized on magnetic beads and to check the affinity of selected aptamers for their target fluorescent labeled aptamers were used. Aptamers were selected from an initial library containing 25 base long variable regions for their ability to bind to the CA 15-3. After 12 rounds of the selection and amplification we found 90% pool of DNA sequences which are able to bind with CA 15-3. These aptamers were cloned, sequenced and labeled by fluorescent molecule. The binding affinity of these aptamers (K D value) was quantified by using FAM labeled aptamers. Our results aim to develop new diagnostic assays against breast tumor marker for the early diagnosis of breast cancer.

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RNA and DNA aptamers in cytomics analysis

Cited by 79 publications

References 73 publications

Disease‐specific biomarker discovery by aptamers

Disease‐specific biomarker discovery by aptamers

Perspectives for systematic in vitro antibody generation

Design and Characterization of DNA Aptamer for Breast Tumor Marker by an Advantageous Method

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