Abstract
Aptamers are nucleic acid oligomers with distinct conformational shapes that allow them to bind targets with high affinity and specificity. Aptamers are selected from a random oligonucleotide library by their capability to bind a certain molecular target. A variety of targets ranging from small molecules like amino acids to complex targets and whole cells have been used to select aptamers. These characteristics and the ability to create specific aptamers against virtually any cell type in a process termed “systematic evolution by exponential enrichment” make them interesting tools for flow cytometry. In this contribution, we review the application of aptamers as probes for flow cytometry, especially cell-phenotyping and detection of various cancer cell lines and virus-infected cells and pathogens. We also discuss the potential of aptamers combined with nanoparticles such as quantum dots for the generation of new multivalent detector molecules with enhanced affinity and sensitivity. With regard to recent advancements in aptamer selection and the decreasing costs for oligonucleotide synthesis, aptamers may rise as potent competitors for antibodies as molecular probes in flow cytometry.
Similar content being viewed by others
References
Armengol CE, Schlager TA, Hendley JO (2004) Sensitivity of a rapid antigen detection test for group A streptococci in a private pediatric office setting: answering the Red Book’s request for validation. Pediatrics 113:924–926
Avci-Adali M, Metzger M, Perle N, Ziemer G, Wendel HP (2010) Pitfalls of cell-systematic evolution of ligands by exponential enrichment (SELEX): existing dead cells during in vitro selection anticipate the enrichment of specific aptamers. Oligonucleotides 20:317–323
Avci-Adali M, Wilhelm N, Perle N, Stoll H, Schlensak C, Wendel HP (2013) Absolute quantification of cell-bound DNA aptamers during SELEX. Nucleic Acid Ther 23:125–130
Bagalkot V, Zhang L, Levy-Nissenbaum E, Jon S, Kantoff PW, Langer R, Farokhzad OC (2007) Quantum dot—aptamer conjugates for synchronous cancer imaging, therapy, and sensing of drug delivery based on bi-fluorescence resonance energy transfer. Nano Lett 7:3065–3070
Baird GS (2010) Where are all the aptamers? Am J Clin Pathol 134:529–531
Blank M (2001) Systematic evolution of a DNA aptamer binding to rat brain tumor microvessels. Selective targeting of endothelial regulatory protein pigpen. J Biol Chem 276:16464–16468
Boltz A, Piater B, Toleikis L, Guenther R, Kolmar H, Hock B (2011) Bi-specific aptamers mediating tumor cell lysis. J Biol Chem 286:21896–21905
Burzlaff A, Kalesse M, Kasper C, Scheper T (2003) Multi parameter in vitro testing of ratjadone using flow cytometry. Appl Microbiol Biotechnol 62:174–179
Burzlaff A, Brethauer S, Kasper C, Jackisch B, Scheper T (2004) Flow cytometry: interesting tool for studying binding behavior of DNA on inorganic layered double hydroxide (LDH). Cytom A 62:65–69
Cao X, Li S, Chen L, Ding H, Xu H, Huang Y, Li J, Liu N, Cao W, Zhu Y, Shen B, Shao N (2009) Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus. Nucleic Acids Res 37:4621–4628
Carlson R (2009) The changing economics of DNA synthesis. Nat Biotechnol 27:1091–1094
Chattopadhyay PK, Price DA, Harper TF, Betts MR, Yu J, Gostick E, Perfetto SP, Goepfert P, Koup RA, de Rosa SC, Bruchez MP, Roederer M (2006) Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry. Nat Med 12:972–977
Chattopadhyay PK, Hogerkorp C, Roederer M (2008) A chromatic explosion: the development and future of multiparameter flow cytometry. Immunology 125:441–449
Chen CB, Chernis GA, van Hoang Q, Landgraf R (2003) Inhibition of heregulin signaling by an aptamer that preferentially binds to the oligomeric form of human epidermal growth factor receptor-3. Proc Natl Acad Sci U S A 100:9226–9231
Chen F, Zhou J, Luo F, Mohammed A, Zhang X (2007) Aptamer from whole-bacterium SELEX as new therapeutic reagent against virulent Mycobacterium tuberculosis. Biochem Biophys Res Commun 357:743–748
Chen HW, Medley CD, Sefah K, Shangguan D, Tang Z, Meng L, Smith JE, Tan W (2008) Molecular recognition of small-cell lung cancer cells using aptamers. ChemMedChem 3:991–1001
Chen X, Estévez M, Zhu Z, Huang Y, Chen Y, Wang L, Tan W (2009) Using aptamer-conjugated fluorescence resonance energy transfer nanoparticles for multiplexed cancer cell monitoring. Anal Chem 81:7009–7014
Chu TC, Shieh F, Lavery LA, Levy M, Richards-Kortum R, Korgel BA, Ellington AD (2006) Labeling tumor cells with fluorescent nanocrystal-aptamer bioconjugates. Biosens Bioelectron 21:1859–1866
Czechowska K, Johnson DR, van der Meer JR (2008) Use of flow cytometric methods for single-cell analysis in environmental microbiology. Curr Opin Microbiol 11:205–212
Davis KA, Abrams B, Lin Y, Jayasena SD (1996) Use of a high affinity DNA ligand in flow cytometry. Nucleic Acids Res 24:702–706
Davis KA, Lin Y, Abrams B, Jayasena SD (1998) Staining of cell surface human CD4 with 2'-F-pyrimidine-containing RNA aptamers for flow cytometry. Nucleic Acids Res 26:3915–3924
Di Giusto DA, King GC (2004) Construction, stability, and activity of multivalent circular anticoagulant aptamers. J Biol Chem 279:46483–46489
Di Giusto DA, Wlassoff WA, Gooding JJ, Messerle BA, King GC (2005) Proximity extension of circular DNA aptamers with real-time protein detection. Nucleic Acids Res 33:e64
Di Giusto DA, Knox SM, Lai Y, Tyrelle GD, Aung MT, King GC (2006) Multitasking by multivalent circular DNA aptamers. ChemBioChem 7:535–544
Drolet DW, Moon-McDermott L, Romig TS (1996) An enzyme-linked oligonucleotide assay. Nat Biotechnol 14:1021–1025
Duan N, Wu S, Chen X, Huang Y, Wang Z (2012) Selection and identification of a DNA aptamer targeted to Vibrio parahemolyticus. J Agric Food Chem 60:4034–4038
Duan N, Wu S, Chen X, Huang Y, Xia Y, Ma X, Wang Z (2013) Selection and characterization of aptamers against Salmonella Typhimurium using whole-bacterium systemic evolution of ligands by exponential enrichment (SELEX). J Agric Food Chem 61:3229–3234
Dwivedi HP, Smiley RD, Jaykus L (2010) Selection and characterization of DNA aptamers with binding selectivity to Campylobacter jejuni using whole-cell SELEX. Appl Microbiol Biotechnol 87:2323–2334
Dwivedi HP, Smiley RD, Jaykus L (2013) Selection of DNA aptamers for capture and detection of Salmonella Typhimurium using a whole-cell SELEX approach in conjunction with cell sorting. Appl Microbiol Biotechnol 97:3677–3686
Ellington AD, Szostak JW (1990) In vitro selection of RNA molecules that bind specific ligands. Nature 346:818–822
Funakoshi T, Tachibana I, Hoshida Y, Kimura H, Takeda Y, Kijima T, Nishino K, Goto H, Yoneda T, Kumagai T, Osaki T, Hayashi S, Aozasa K, Kawase I (2003) Expression of tetraspanins in human lung cancer cells: frequent downregulation of CD9 and its contribution to cell motility in small cell lung cancer. Oncogene 22:674–687
Glaser V (2009) Oligo market benefits from RNAi focus. Genet Eng Biotechnol News 29
Hamula CLA, Zhang H, Guan LL, Li X, Le XC (2008) Selection of aptamers against live bacterial cells. Anal Chem 80:7812–7819
Hamula CL, Le XC, Li X (2011a) DNA aptamers binding to multiple prevalent M-types of Streptococcus pyogenes. Anal Chem 83:3640–3647
Hamula CL, Zhang H, Li F, Wang Z, Le Chris X, Li X (2011b) Selection and analytical applications of aptamers binding microbial pathogens. TrAC Trends Anal Chem 30:1587–1597
Heine F, Stahl F, Sträuber H, Wiacek C, Benndorf D, Repenning C, Schmidt F, Scheper T, von Bergen M, Harms H, Müller S (2009) Prediction of flocculation ability of brewing yeast inoculates by flow cytometry, proteome analysis, and mRNA profiling. Cytom A 75:140–147
Herr JK, Smith JE, Medley CD, Shangguan D, Tan W (2006) Aptamer-conjugated nanoparticles for selective collection and detection of cancer cells. Anal Chem 78:2918–2924
Hicke BJ, Marion C, Chang YF, Gould T, Lynott CK, Parma D, Schmidt PG, Warren S (2001) Tenascin-C aptamers are generated using tumor cells and purified protein. J Biol Chem 276:48644–48654
Huang Y, Chang H, Tan W (2008) Cancer cell targeting using multiple aptamers conjugated on nanorods. Anal Chem 80:567–572
Hyeon J, Chon J, Choi I, Park C, Kim D, Seo K (2012) Development of RNA aptamers for detection of Salmonella Enteritidis. J Microbiol Methods 89:79–82
Iliuk AB, Hu L, Tao WA (2011) Aptamer in bioanalytical applications. Anal Chem 83:4440–4452
Jackman DM, Johnson BE (2005) Small-cell lung cancer. Lancet 366:1385–1396
Jiménez E, Sefah K, López-Colón D, van Simaeys D, Chen HW, Tockman MS, Tan W (2012) Generation of lung adenocarcinoma DNA aptamers for cancer studies. PLoS One 7:e46222
Kang WJ, Chae JR, Cho YL, Lee JD, Kim S (2009) Multiplex imaging of single tumor cells using quantum-dot-conjugated aptamers. Small 5:2519–2522
Keefe AD, Cload ST (2008) SELEX with modified nucleotides. Curr Opin Chem Biol 12:448–456
Kökpinar O, Walter J, Shoham Y, Stahl F, Scheper T (2011) Aptamer-based downstream processing of his-tagged proteins utilizing magnetic beads. Biotechnol Bioeng 108:2371–2379
Kruth HS (1982) Flow cytometry: rapid biochemical analysis of single cells. Anal Biochem 125:225–242
Kulbachinskiy AV (2007) Methods for selection of aptamers to protein targets. Biochemistry (Mosc) 72:1505–1518
Kunii T, Ogura S, Mie M, Kobatake E (2011) Selection of DNA aptamers recognizing small cell lung cancer using living cell-SELEX. Analyst 136:1310
Lao Y, Peck K, Chen L (2009) Enhancement of aptamer microarray sensitivity through spacer optimization and avidity effect. Anal Chem 81:1747–1754
Li N, Ebright JN, Stovall GM, Chen X, Nguyen HH, Singh A, Syrett A, Ellington AD (2009) Technical and biological issues relevant to cell typing with aptamers. J Proteome Res 8:2438–2448
Lübbecke M, Walter J, Stahl F, Scheper T (2012) Aptamers as detection molecules on reverse phase protein microarrays for the analysis of cell lysates. Eng Life Sci 12:144–151
Lupold SE, Hicke BJ, Lin Y, Coffey DS (2002) Identification and characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostate-specific membrane antigen. Cancer Res 62:4029–4033
Mallikaratchy P, Tang Z, Kwame S, Meng L, Shangguan D, Tan W (2007) Aptamer directly evolved from live cells recognizes membrane bound immunoglobin heavy mu chain in Burkitt’s lymphoma cells. Mol Cell Proteomics 6:2230–2238
Mallikaratchy PR, Ruggiero A, Gardner JR, Kuryavyi V, Maguire WF, Heaney ML, McDevitt MR, Patel DJ, Scheinberg DA (2011) A multivalent DNA aptamer specific for the B cell receptor on human lymphoma and leukemia. Nucleic Acids Res 39:2458–2469
Mayer G, Ahmed ML, Dolf A, Endl E, Knolle PA, Famulok M (2010) Fluorescence-activated cell sorting for aptamer SELEX with cell mixtures. Nat Protoc 5:1993–2004
McKeague M, Derosa MC (2012) Challenges and opportunities for small molecule aptamer development. J Nucleic Acids 2012:748913
Medley CD, Bamrungsap S, Tan W, Smith JE (2011) Aptamer-conjugated nanoparticles for cancer cell detection. Anal Chem 83:727–734
Moretti P, Behr L, Walter JG, Kasper C, Stahl F, Scheper T (2010) Characterization and improvement of cell line performance via flow cytometry and cell sorting. Eng Life Sci:NA
Morris KN, Jensen KB, Julin CM, Weil M, Gold L (1998) High affinity ligands from in vitro selection: complex targets. Proc Natl Acad Sci U S A 95:2902–2907
Müller S, Bley T (2011) High resolution microbial single cell analytics. Springer-Verlag Berlin Heidelberg, Berlin, Heidelberg
Ninomiya K, Kaneda K, Kawashima S, Miyachi Y, Ogino C, Shimizu N (2013) Cell-SELEX based selection and characterization of DNA aptamer recognizing human hepatocarcinoma. Bioorg Med Chem Lett 23:1797–1802
Nitsche A, Kurth A, Dunkhorst A, Pänke O, Sielaff H, Junge W, Muth D, Scheller F, Stöcklein W, Dahmen C, Pauli G, Kage A (2007) One-step selection of vaccinia virus-binding DNA aptamers by MonoLEX. BMC Biotechnol 7:48
Nurse P, Hayles J (2011) The cell in an era of systems biology. Cell 144:850–854
Parekh P, Tang Z, Turner PC, Moyer RW, Tan W (2010) Aptamers recognizing glycosylated hemagglutinin expressed on the surface of vaccinia virus-infected cells. Anal Chem 82:8642–8649
Paul A, Avci-Adali M, Ziemer G, Wendel HP (2009) Streptavidin-coated magnetic beads for DNA strand separation implicate a multitude of problems during cell-SELEX. Oligonucleotides 19:243–254
Preffer F, Dombkowski D (2009) Advances in complex multiparameter flow cytometry technology: applications in stem cell research. Cytometry B Clin Cytom 76:295–314
Raddatz ML, Dolf A, Endl E, Knolle P, Famulok M, Mayer G (2008) Enrichment of cell–targeting and population–specific aptamers by fluorescence–activated cell sorting. Angew Chem Int Ed 47:5190–5193
Rieseberg M, Kasper C, Reardon KF, Scheper T (2001) Flow cytometry in biotechnology. Appl Microbiol Biotechnol 56:350–360
Ringquist S, Parma D (1998) Anti-l-selectin oligonucleotide ligands recognize CD62L-positive leukocytes: binding affinity and specificity of univalent and bivalent ligands. Cytometry 33:394–405
Roederer M, Bigos M, Nozaki T, Stovel RT, Parks DR, Herzenberg LA (1995) Heterogeneous calcium flux in peripheral T cell subsets revealed by five-color flow cytometry using log-ratio circuitry. Cytometry 21:187–196
Schütze T, Wilhelm B, Greiner N, Braun H, Peter F, Mörl M, Erdmann VA, Lehrach H, Konthur Z, Menger M, Arndt PF, Glökler J (2011) Probing the SELEX process with next generation sequencing. PLoS One 6:e29604
Sefah K, Tang ZW, Shangguan DH, Chen H, Lopez-Colon D, Li Y, Parekh P, Martin J, Meng L, Phillips JA, Kim YM, Tan WH (2009) Molecular recognition of acute myeloid leukemia using aptamers. Leukemia 23:235–244
Sefah K, Shangguan D, Xiong X, O’Donoghue MB, Tan W (2010) Development of DNA aptamers using Cell-SELEX. Nat Protoc 5:1169–1185
Sefah K, Bae K, Phillips JA, Siemann DW, Su Z, McClellan S, Vieweg J, Tan W (2013) Cell-based selection provides novel molecular probes for cancer stem cells. Int J Cancer 132:2578–2588
Shamah SM, Healy JM, Cload ST (2008) Complex target SELEX. Acc Chem Res 41:130–138
Shangguan D, Li Y, Tang Z, Cao ZC, Chen HW, Mallikaratchy P, Sefah K, Yang CJ, Tan W (2006) Aptamers evolved from live cells as effective molecular probes for cancer study. Proc Natl Acad Sci 103:11838–11843
Shangguan D, Cao ZC, Li Y, Tan W (2007) Aptamers evolved from cultured cancer cells reveal molecular differences of cancer cells in patient samples. Clin Chem 53:1153–1155
Shangguan D, Meng L, Cao ZC, Xiao Z, Fang X, Li Y, Cardona D, Witek RP, Liu C, Tan W (2008) Identification of liver cancer-specific aptamers using whole live cells. Anal Chem 80:721–728
Shapiro HM (1983) Multistation multiparameter flow cytometry: a critical review and rationale. Cytometry 3:227–243
Shigdar S, Lin J, Yu Y, Pastuovic M, Wei M, Duan W (2011) RNA aptamer against a cancer stem cell marker epithelial cell adhesion molecule. Cancer Sci 102:991–998
Shigdar S, Qiao L, Zhou S, Xiang D, Wang T, Li Y, Lim LY, Kong L, Li L, Duan W (2013) RNA aptamers targeting cancer stem cell marker CD133. Cancer Lett 330:84–95
Smith JE, Medley CD, Tang Z, Shangguan D, Lofton C, Tan W (2007) Aptamer-conjugated nanoparticles for the collection and detection of multiple cancer cells. Anal Chem 79:3075–3082
Song Y, Zhu Z, An Y, Zhang W, Zhang H, Liu D, Yu C, Duan W, Yang CJ (2013) Selection of DNA aptamers against epithelial cell adhesion molecule for cancer cell imaging and circulating tumor cell capture. Anal Chem 85:4141–4149
Stanlis KKH, McIntosh JR (2003) Single-strand DNA aptamers as probes for protein localization in cells. J Histochem Cytochem 51:797–808
Steiner JM, Sartor M, Sanchez AB, Messmer D, Freed A, Esener S, Messmer BT (2010) DeNAno: selectable deoxyribonucleic acid nanoparticle libraries. J Biotechnol 145:330–333
Stoltenburg R, Reinemann C, Strehlitz B (2007) SELEX—a (r)evolutionary method to generate high-affinity nucleic acid ligands. Biomol Eng 24:381–403
Tang Z, Shangguan D, Wang K, Shi H, Sefah K, Mallikratchy P, Chen HW, Li Y, Tan W (2007) Selection of aptamers for molecular recognition and characterization of cancer cells. Anal Chem 79:4900–4907
Tang Z, Parekh P, Turner P, Moyer RW, Tan W (2009) Generating aptamers for recognition of virus-infected cells. Clin Chem 55:813–822
Tracy BP, Gaida SM, Papoutsakis ET (2010) Flow cytometry for bacteria: enabling metabolic engineering, synthetic biology and the elucidation of complex phenotypes. Curr Opin Biotechnol 21:85–99
Tuerk C, Gold L (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science 249:505–510
Ulrich H, Wrenger C (2009) Disease-specific biomarker discovery by aptamers. Cytometry 75:727–733
van Simaeys D, López-Colón D, Sefah K, Sutphen R, Jimenez E, Tan W (2010) Study of the molecular recognition of aptamers selected through ovarian cancer cell-SELEX. PLoS One 5:e13770
Walter J, Kökpinar O, Friehs K, Stahl F, Scheper T (2008) Systematic investigation of optimal aptamer immobilization for protein-microarray applications. Anal Chem 80:7372–7378
Walter JG, Petersen S, Stahl F, Scheper T, Barcikowski S (2010) Laser ablation-based one-step generation and bio-functionalization of gold nanoparticles conjugated with aptamers. J Nanobiotechnology 8:21
Walter J, Heilkenbrinker A, Austerjost J, Timur S, Stahl F, Scheper T (2012a) Aptasensors for small molecule detection. Z Naturforsch B 67:976–986
Walter J, Stahl F, Scheper T (2012b) Aptamers as affinity ligands for downstream processing. Eng Life Sci 12:496–506
World Health Organization WHO (2012a) World Health Statistics 2012
World Health Organization (WHO) (2012b) World Health Statistics 2012. Noncommunicable diseases: a major health challenge of the 21st century
Zhang P, Zhao N, Zeng Z, Feng Y, Tung C, Chang C, Zu Y (2009) Using an RNA aptamer probe for flow cytometry detection of CD30-expressing lymphoma cells. Lab Invest 89:1423–1432
Zhang J, Jia X, Lv X, Deng Y, Xie H (2010a) Fluorescent quantum dot-labeled aptamer bioprobes specifically targeting mouse liver cancer cells. Talanta 81:505–509
Zhang P, Zhao N, Zeng Z, Chang C, Zu Y (2010b) Combination of an aptamer probe to CD4 and antibodies for multicolored cell phenotyping. Am J Clin Pathol 134:586–593
Zhao Z, Xu L, Shi X, Tan W, Fang X, Shangguan D (2009) Recognition of subtype non-small cell lung cancer by DNA aptamers selected from living cells. Analyst 134:1808
Zheng D, Seferos DS, Giljohann DA, Patel PC, Mirkin CA (2009) Aptamer nano-flares for molecular detection in living cells. Nano Lett 9:3258–3261
Zhu G, Lübbecke M, Walter J, Stahl F, Scheper T (2011) Characterization of optimal aptamer-microarray binding chemistry and spacer design. Chem Eng Technol 34:2022–2028
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Meyer, M., Scheper, T. & Walter, JG. Aptamers: versatile probes for flow cytometry. Appl Microbiol Biotechnol 97, 7097–7109 (2013). https://doi.org/10.1007/s00253-013-5070-z
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-013-5070-z