White Paper Aptamer Applications


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White Paper Aptamer Applications

  1. 1. WHITE PAPER: “Aptamers and their applications at IceNine Bio” (excerpts from various proposals) April 2011 Aptamer development has been limited to one-target at a time Aptamers are single-stranded DNA or RNA (ssDNA or ssRNA)molecules that can bind to pre-selected targets including proteins andpeptides with high affinity and specificity. These molecules can assume avariety of shapes due to their propensity to form helices and single-stranded loops, explaining their versatility in binding to dverse targets.They are used as sensors [1], and therapeutic tools [2], and to regulatecellular processes [3], as well as to guide drugs to their specific cellular Amplify elutedtargets [4-7]. Contrary to the actual genetic material, their specificity and binderscharacteristics are not directly determined by their primary sequence, butinstead by their tertiary structure [8]. Aptamers are generated from large random libraries by an iterativeprocess often called Systematic Evolution of Ligands by ExponentialEnrichment (SELEX) [9, 10]. The conventional SELEX technique (Figure1) starts with a large library of random single stranded nucleotides oraptamers (ca. 1015 unique sequences). A typical library will contain a Figure 1. Schematic of conventional,randomized region of ca. 40 nucleotides flanked by two constant regions single-target DNA aptamer selection.for PCR priming. The library is exposed to a target and the boundaptamers are partitioned and amplified for the next round. With each round the stringency of the bindingconditions is increased until the only remaining aptamers in the pool are highly specific for, and bind with highaffinity to, the target. Once multiple (typically 10-15) rounds of SELEX are completed, the DNA sequences areusually identified by conventional cloning and sequencing. While in general the accepted process for selecting aptamers, so-called “Systematic Evolution of Ligands byEXponential enrichment (SELEX)” [9, 10] is quite effective, SELEX is commonly performed against only asingle protein target at a time. Because the process is tedious and time consuming, the yield of just one or,at best, several aptamer candidates for a single target greatly limits throughput. IceNine has addressed this severe limitation by successfully parallel-izing the conventionalaptamer selection process. Additional Documented Applications of Aptamers So far, aptamers are best known as ligands to proteins, rivaling antibodies in both affinity and specificity[11-14], and the first aptamer-based therapeutics were recently FDA-approved (Macugen) [15-17]. Morerecently, however, aptamers have also been developed to bind small organic molecules and cellular toxins [18-26], viruses [27, 28], and even targets as small as heavy metal ions [29-33]. While aptamers are analogous toantibodies in their range of target recognition and variety of applications, they possess several key advantagesover their protein counterparts [34]: They are self-refolding, single-chain, and redox-insensitive. They also lack the large hydrophobic cores of proteins and thus do not aggregate. They tolerate (or recover from) pH and temperatures that proteins do not. They are easier and more economical to produce (especially at the affinity reagent scale). In stark contrast to peptides, proteins and to some small chemicals, oligonucleotides ( = DNA aptamers) are made through chemical synthesis, a process that is well defined, highly reproducible, sequence independent and can be readily and predictably scaled up. Their production does not depend on bacteria, cell cultures or animals. In contrast to antibodies, toxicity and low immunogenicity of particular antigens do not interfere with the aptamer selection. Further, using the technology proposed, highly custom or “orphaned” targets can be address rapidly and cheaply. 1
  2. 2. They are capable of greater specificity and affinity than antibodies [35]. They can easily be modified chemically to yield improved, custom tailored properties. For instance, reporter and functional groups and PEG can easily be attached to the aptamer in a deterministic way. In fact, they can even be combined with antibodies [36, 37]. Similarly, their ADME properties can be readily tuned by conjugation to other groups (PEG, etc). Their small size leads to a high number of moles of target bound per gram, and they may have improved transport properties allowing cell specific targeting and improved tissue penetration [38- 42]. They are much more stable at ambient temperature than antibodies yielding a much higher shelf life, and they can tolerate transportation without any special requirements for cooling, eliminating the need for a continuous cold chain. . The fact that, after selection, aptamers can be produced by chemical synthesis eliminates batch-to-batchvariation which complicates production of therapeutic proteins [43] and the variability of diagnostic antibodyreagents. Aptamers identified by SELEX can also be easily analyzed and manipulated to characterize theminimum sequence requirements for aptamer:target recognition. Sequences can be inspected for primary andsecondary structure motifs [44-48], and single base perturbations of identified aptamers can be studied for theresulting affects on binding affinity. This sort of rational/directed optimization would be much more challengingusing the antibody approach. In most applications, aptamers have been successfully employed as direct replacements for antibodies.Aptamer dot-blots [49] and aptamer-westerns [50, 51] are well supported in the literature. With respect to theintegration of aptamers into biosensors, numerous publications support the feasibility of this approach. Forinstance, aptamers have been incorporated into lead sensors [30-32], drug sensors [24, 26], and estrogenmeasuring devices [52]. They can be used in single-target measuring devices or even in arrays [38], furtherextending their versatility. Recently, aptamers have also been used as chimeric conjugates to siRNAs toimprove delivery [53]. Ongoing aptamer development and applications at BioTex The principal scientist at IceNine, Dr. Bill Jackson, has considerable experience in the development andnovel application of nucleic acid aptamers, and these molecules have enjoyed recent increasing acceptance inboth diagnostic and therapeutic settings – (in 2010, Dr. Jackson authored a 200+ page market research reporton the use of aptamers in both diagnostic and therapeutic applications) [54]. In addition to the aptamer selection services offered by IceNine, our group maintains an active researchprogram involving novel uses of aptamers. The PI and his colleagues have considerable experience indeveloping novel molecular diagnostics using a variety of analytical techniques including mass spectrometry[55-59] and DNA microarrays [56, 60]. More recently the PI has been involved in a variety of projects involvingaptamers [61-64]. Recent funding has included an EPA SBIR Phase I project for aptamer-based detection ofthe cyanobacterial toxin, anatoxin-a, a project to develop aptamer-mimetics to proteins such as stem cell factor(SCF) to replace peptide agonists with inexpensive aptamers, and the aforementioned project to develop aplatform for massively parallel SELEX. We have also developed recombinant techniques to express smallforeign RNA’s (aptamers and siRNA) within the ribosome of E. coli [62-65]. Currently we are developingaptamer affinity reagents to high priority cancer biomarker proteins under Contract SBIR Phase Ifunding from the National Cancer Institute. 2
  3. 3. Application of aptamers in flow cytometry In addition to validation of aptamers via SPR, we have successfully employed aptamers influorescence activated cell sorting (FACS) with collaborators at nearby M.D. Anderson Cancer Center(Houston, TX). Specifically, our collaborator, Dr. Laurence Cooper at MDACC has been using our aptamersto label and differentiate a panel of recombinant proteins expressed on desired T-cell subsets using flowcytometry. The availability of an inexpensive and efficacious alternative to monoclonal antibodies (mAbs) inthis application is important as a method to produce cellular reagents to be used in compliance with currentgood manufacturing practice (cGMP). Obtaining monoclonal antibodies (mAbs) as reagents to validatethe manufacture of cell-based therapeutics for clinical application is tedious, labor intensive andexpensive as applied to generation of clinical-grade reagents. This is chiefly because of the concern ofadventitious virus that may accompany the production of mAbs. Aptamer technology based on in vitro DNAsynthesis will avoid this issue and greatly simplify and reduce the costs associated with producing materials foruse in compliance with cGMP. Figure 2. Demonstration of the feasibility of FACS using fluorescent aptamers selected against cell surface proteins. While some cross-reactivity is observed, screening and better selection of recombinant aptamer targets should alleviate these problems. Figure 2 above shows some of the preliminary fluorescence activated cell sorting (FACS) data utilizing ouraptamers as replacements to fluorescently labeled mAb reagents. Briefly, cells were first transfected with adefined membrane-bound interleukins (IL7, IL15, or IL21). Cells were then washed to remove serum andculture media and incubated at 37° for 60 minutes with an aptamer developed to either IL-7, IL-15, or IL-21. CAptamers were synthesized with a 3’-biotin for facile labeling in situ with streptavidin-FITC conjugate.Following binding, aptamer-bound cells were washed once with PBS and then stained with streptavidin-FITCfollowed by an additional wash with PBS. Finally, cells were sorted by conventional FACS based on FITCfluorescence; cell counts to the right of the vertical blue line represent positive binding. As expected from aninitial screen, there is some cross-reactivity of the tested aptamers. This is due to the use of a commonrecombinant Fc region for presenting these three cytokines on the cell surface. Nevertheless, some degree ofspecificity/orthogonality of the aptamers is seen, and the experiment demonstrates that in principal, expensivemAbs could be replaced by aptamers for the FACS application. 3
  4. 4. Development of Aptamers and Sensing Chemistry for the Hormone, Thyroxine (T4) and the Protein, Insulin Researchers at BioTex/IceNine have also employed aptamers in competitive sensing chemistriesfor detection of environmental and clinical analytes. For instance, a sensor for the thyroid hormone (smallmolecule), thyroxine (so-called “free-T4”) was developed. Figure 3 below depicts the modular,aptamer/quantum-dot-based sensing scheme employed. To select a DNA aptamer to the molecule, thyroxine was covalently immobilized via its primary amine to asolid phase gel by standard chemistries. After 10 rounds of conventional (e.g. single-target) selection,individual aptamers were cloned and sequenced. Evolution of a unique sequence, characterized by a high GCcontent, as typical for known thyroxine aptamers [66] was clearly observed (sequence not shown). Figure 4 shows the performance of the FRET-aptamer sensor which has been described in detailelsewhere [67]. The sensor was found non-responsive to several structurally similar chemicals and was thus,specific for the analyte. BioTex has considerable fluorescent biosensor expertise [67-70] especially throughco-investigator, Dr. Ralph Ballerstadt. The fluorescence emitted from the sensor was measured in a portable,inexpensive Qubit™ fluorometer (Invitrogen). Thus, using a sensing cocktail that can be readily lyophilizedand reconstituted by the sample, such an assay could be taken to the field or bedside for environmental orclinical use. This sort of sensing scheme can be readily devised for proteins as well. We have developed ananalogous sensing chemistry using aptamers specific for insulin evolved at BioTex. Figure 5 (next page)shows that result. These data demonstrate not only the ability of BioTex researchers to select DNAaptamers to novel targets, but also the broad, modular applicability of DNA aptamers in numerousapplications. A QD Nanoshell B Immobilized analyte- Fluorescence Emission analog Aptamer with quencher dye analyte EX. hν EX. hν ~ 10 nm PEG coating Figure 3. Schematic of Modular, Aptamer-based QD-FRET sensing chemistry (approximate scale). In a competitive-binding fluorescence resonance energy transfer (FRET) assay, quantum dots (QDs) are conjugated to an immobilized version of the intended analyte (T4 in the text example). Aptamers which bind T4 with high specificity are identified by in vitro selection or "SELEX". Panel (A): Aptamers synthesized with a terminal fluorophore for quenching of the QD are bound to immobilized T4. Panel (B) When free T4 in the sample is exposed to this reagent mixture, QD-quenching aptamers are released from the QDs resulting in a fluorescence signal proportional to the T4 concentration. Qubit Reading (5 min values) 200 180 160 140 120 AFUs 100 80 60 40 20 0 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Thyroxine Conc. [ug/ul] Figure 4. Quantitative detection of the small molecule thyroxine (T4) via FRET-aptamer detection. Insets show structure of T4 and portable (4.5 x 6.5 x 1.8 inch) Qubit™ fluorometer (Invitrogen) used to acquire data. 4
  5. 5. B A Figure 5. Response curve of quantum-dot-based sensor (Figure 4) to insulin as formulated with the insulin-specific aptamer #43 selected at BioTex. (Panel A) The increase in fluorescence with increasing insulin concentrations is due to FRET. The putative secondary structure of insulin- aptamer #43 is shown in panel B.Whole cell SELEX for aptamers competing with ‘Stem Cell Factor’ (SCF) In addition to selection of aptamers against purified targets, we also have significant experience inselection of aptamers against the surface of whole cells. Specifically, under NSF funding, we collaboratedwith our literal neighbor, Synthecon Inc. (Houston, TX) in an attempt to develop an inexpensive DNA aptamermimetic for so-called stem cell factor (SCF or “kit ligand”). Recombinant SCF is a rather expensive, butcommonly used reagent in stem cell culture. To achieve this goal, we enriched our randomized DNA library foraptamers binding whole stem cells and then selectively displaced the desired aptamers from the c-Kit receptorusing SCF itself. Although we were unable to evolve an agonist mimetic (to effect stem cell proliferation inculture), did successfully develop a number of candidate c-Kit specific antagonists. We are currentlyquantifying their affinity. 5
  6. 6. Additional Aptamer Applications:Aptamer-Western blots of 2D gels and Affinity Depletion of Abundant Proteins It has already been demonstrated that aptamers can perform well in a Western-blot application [50, 51].Similarly they have functioned as direct replacements for antibody reporters in ELISAs and dot blots [49]. Anumber of aptamer conjugation/functionalization schemes are likely accepted for this purpose. Most simply,aptamers can synthesized to contain either a 5’- or 3’-fluorphore of choice. Alternatively, as described inPreliminary Work, we have considerable experience in conjugating aptamers to quantum dots with theadvantage of higher quantum yield and larger Stokes’ shifts of fluorescence. Finally, for additional signal,aptamers can be synthesized with a 5’- or 3’-biotin and detected by a streptavidin/horseradish peroxidaseconjugate (strep-HRP). Numerous commercial sources for both fluorescent and chromagenic HRP substratesare available. Such an approach is often referred to as an “ELONA” [71, 72] or aptamer-ELISA (“ELASA”) [73].Aptamer-bead precipitation or enrichment As mentioned elsewhere, one of the attractions/advantages of aptamers is that they are readilysynthesized with modifications for conjugation, covalent coupling, fluorescent reporting, etc. Specifically,aptamers can be readily coupled to beads or nanoparticles by synthesizing them with a terminal biotin oramino-group. Such functionalized particles have been used to concentrate transcription factors [74], or, in theform of magnetic beads, have been employed to enrich bacterial pathogens [75] or cancer cells [76]. Similarly,it should be feasible to develop highly multiplexed bead-based assays such as Luminex/Magpix.Immunohistochemistry Employing aptamers in conventional histology and molecular imaging is another logical extension for theiruse in lieu of antibodies. In recent years, directly, or indirectly labeled aptamers have, for instance, been usedto detect individual receptors [77] or to differentiate cancerous from non-cancerous cells [78]. SUMMARY: Aptamers represent a promising form of synthetic, inexpensive affinity reagent which can beemployed in numerous applications. Despite their promise, however, they have generally only beendeveloped or ‘evolved’ against a single target at a time. High costs for aptamer selection (as well asnow-expiring intellectual property constraints) have until now hindered their more widespread use.IceNine’s novel approach to multiplex aptamer selection can inexpensively provide aptamer affinityreagents to a revolutionary breadth of targets. Finally, our team has the expertise to demonstrate andsupport our customers in a variety of novel aptamer-based application areas. 6
  7. 7. REFERENCES CITED:1. Holthoff EL, Bright FV: Molecularly templated materials in chemical sensing. Anal. Chim. Acta 2007,594:147-161.2. Kaur G, Roy I: Therapeutic applications of aptamers. Expert opinion on investigational drugs 2008, 17:43-60.3. Toulme JJ, Di Primo C, Boucard D: Regulating eukaryotic gene expression with aptamers. FEBS letters2004, 567:55-62.4. Chu TC, Marks 3rd JW, Lavery LA, Faulkner S, Rosenblum MG, Ellington AD, Levy M: Aptamer:toxinconjugates that specifically target prostate tumor cells. Cancer Res. 2006, 66:5989-92.5. Chu TC, Twu KY, Ellington AD, Levy M: Aptamer mediated siRNA delivery. Nucl. Acids Res. 2006,34:e73-.6. Chu TC, Shieh F, Lavery LA, Levy M, Richards-Kortum R, Korgel BA, Ellington AD: Labeling tumor cellswith fluorescent nanocrystal-aptamer bioconjugates. Biosens Bioelectron. 2006, Feb 20.7. Cao Z, Tong R, Mishra A, Xu W, Wong GCL, Cheng J, Lu Y: Reversible Cell-Specific Drug Delivery withAptamer-Functionalized Liposomes. Angew. Chem. Int. Ed. 2009, 48:6494-6498.8. Breaker RR: Natural and engineered nucleic acids as tools to explore biology. Nature 2004, 432:838-45.9. Tuerk C, Gold L: Systematic evolution of ligands by exponential enrichment: RNA ligands tobacteriophage T4 DNA polymerase. Science 1990, 249:505-10.10. Ellington AD, Szostak JW: In vitro selection of RNA molecules that bind specific ligands. Nature 1990,346:818-22.11. Tuerk C, MacDougal S, Gold L: RNA pseudoknots that inhibit human immunodeficiency virus type 1reverse transcriptase. Proceedings of the National Academy of Sciences 1992, 89:6988-92.12. Lee JF, Hesselberth JR, Meyers LA, Ellington AD: Aptamer Database. Nucleic Acids Res 2004, 32:D95-100.13. Ruckman J, Green LS, Beeson J, Waugh S, Gillette WL, Henninger DD, Claesson-Welsh L, Janjic N: 2’-Fluoropyrimidine RNA-based Aptamers to the 165-Amino Acid Form of Vascular Endothelial GrowthFactor (VEGF165), Inhibition Of Receptor Binding And Vegf-Induced Vascular Permeability ThroughInteractions Requiring The Exon 7-Encoded Domain. J Biol Chem 1998, 273:20556-7.14. Jayasena SD: Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clin.Chem. 1999, 45:1628-1650.15. Lee J-H, Canny MD, De Erkenez A, Krilleke D, Ng Y-S, Shima DT, Pardi A, Jucker F: A therapeuticaptamer inhibits angiogenesis by specifically targeting the heparin binding domain of VEGF165. Proc.Natl. Acad. Sci. 2005, 102:18902-18907.16. Ng EWM, Shima DT, Calias P, Cunningham ET, Guyer DR, Adamis AP: Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease. Nature Reviews Drug Discovery 2006, 5:123-132.17. Siddiqui MAA, Keating GM: Pegaptanib: in exudative age-related macular degeneration. Drugs 2005,65:1571-1577.18. Sazani PL, Larralde R, Szostak JW: A Small Aptamer with Strong and Specific Recognition of theTriphosphate of ATP. J Am Chem Soc 2004, 126:8371. 7
  8. 8. 19. Tang J, Breaker RR: Mechanism for allosteric inhibition of an ATP-sensitive ribozyme. Nucleic acidsresearch 1998, 26:4214-21.20. Yang Q, Goldstein IJ, Mei H-Y, Engelke DR: DNA ligands that bind tightly and selectively tocellobiose. Proc Natl Acad Sci U S A 1998, 95:5462-5467.21. Babendure JR, Adams SR, Tsien RY: Aptamers Switch on Fluorescence of Triphenylmethane Dyes. JAm Chem Soc 2003, 125:14716-7.22. Hirao I, Yoshinari S, Yokoyama S, Endo Y, Ellington AD: In vitro selection of aptamers that bind toribosome-inactivating toxins. Nucleic Acids Symp Ser 1997, 37:283-4.23. Hirao I, Harada Y, Nojima T, Osawa Y, Masaki H, Yokoyama S: In Vitro Selection of RNA Aptamers thatBind to Colicin E3 and Structurally Resemble the Decoding Site of 16S Ribosomal RNA†. Biochemistry2004, 43:3214-3221.24. Liu J, Lu Y: Fast Colorimetric Sensing of Adenosine and Cocaine Based on a General Sensor DesignInvolving Aptamers and Nanoparticles. Angew. Chem., Int. Ed. 2006, 117:90-94.25. Stojanovic MN, de Prada P, Landry DW: Aptamer-Based Folding Fluorescent Sensor for Cocaine. JAm Chem Soc 2001, 123:4928-4931.26. Stojanovic MN, Landry DW: Aptamer-Based Colorimetric Probe for Cocaine. J Am Chem Soc 2002,124:9678-9679.27. Gopinath SCB, Misono TS, Kawasaki K, Mizuno T, Imai M, Odagiri T, Kumar PKR: An RNA aptamer thatdistinguishes between closely related human influenza viruses and inhibits haemagglutinin-mediatedmembrane fusion. J Gen Virol 2006, 87:479-87.28. Gopinath SCB, Sakamaki Y, Kawasaki K, Kumar PKR: An Efficient RNA Aptamer against HumanInfluenza B Virus Hemagglutinin. Journal of Biochemistry 2006 139(5):837-846 2006, 139:837-46.29. Liu J, Lu Y: A Colorimetric Lead Biosensor Using DNAzyme-Directed Assembly of GoldNanoparticles. Journal of the American Chemical Society 2003, 125:6642-6643.30. Chang I-H, Tulock J, Liu J, Kim W-S, Cannon Jr. D, Lu Y, Bohn P, Sweedler J, Cropek D: MiniaturizedLead Sensor Based on Lead-Specific DNAzyme in a Nanocapillary Interconnected Microfluidic Device.Environ. Sci. Technol. 2005, 39:3756.31. Swearingen CB, Wernette DP, Cropek DM, Lu Y, Sweedler JV, Bohn PW: Immobilization of a CatalyticDNA Molecular Beacon on Au for Pb(II) Detection. Anal. Chem. 2005, 77:442-8.32. Wernette DP, Kim H-K, Liu J, Swearingen CB, Yue Z, Zavareh M, Ingram CW, Economy J, Shannon MA,Bohn PW, Lua Y: New Catalytic DNA Biosensors for Trace Contaminants in Water.33. Wrzesinski J, Ciesiolka J: Characterization of structure and metal ions specificity of Co2+-bindingRNA aptamers. Biochemistry 2005, 44:6257-68.34. Stoltenburg R, Reinemann C, Strehlitz B: SELEX--a (r)evolutionary method to generate high-affinitynucleic acid ligands. Biomolecular engineering 2007, 24:381-403.35. Jayasena SD: Aptamers: An Emerging Class of Molecules That Rival Antibodies in Diagnostics.Clinical Chemistry 1999, 45:1628–1650.36. Ferreira CS, Papamichael K, Guilbault G, Schwarzacher T, Gariepy J, Missailidis S: DNA aptamersagainst the MUC1 tumour marker: design of aptamer-antibody sandwich ELISA for the early diagnosisof epithelial tumours. Analytical and bioanalytical chemistry 2008, 390:1039-50. 8
  9. 9. 37. Burbulis I, Yamaguchi K, Yu R, Resnekov O, Brent R: Quantifying small numbers of antibodies with a“near-universal” protein-DNA chimera. Nature Methods 2007, 4:1011-3.38. McCauley TG, Hamaguchi N, Stanton M: Aptamer-based biosensor arrays for detection andquantification of biological macromolecules. Analytical biochemistry 2003, 319:244-50.39. Cao Z, Tong R, Mishra A, Xu W, Wong GCL, Cheng J, Lu Y: Reversible Cell-Specific Drug Delivery withAptamer-Functionalized Liposomes. Angew. Chem. Int. Ed. 2009, 48:6494-6498.40. De Rosa G, La Rotonda MI: Nano and Microtechnologies for the Delivery of Oligonucleotides withGene Silencing Properties. Molecules 2009, 14:2801-2823.41. Ferreira CSM, Cheung MC, Missailidis S, Bisland S, Gariepy J: Phototoxic aptamers selectively enterand kill epithelial cancer cells. Nucl. Acids Res. 2009, 37:866-876.42. Yan AC, Levy M: Aptamers and aptamer targeted delivery. rnabiology 2009, 6:316-320.43. Cload S, McCauley T, Keefe A, Healy J, Wilson C: Properties of Therapeutic Aptamers. In AptamerHandbook, The. Wiley-VCH Verlag GmbH & Co. KGaA; 2006.44. Mathews DH, Sabina J, Zuker M, Turner DH: Expanded Sequence Dependence of ThermodynamicParameters Improves Prediction of RNA Secondary Structure. J Mol Biol 1999, 288:911-40.45. Markham NR, Zuker M: UNAFold: software for nucleic acid folding and hybridization. Methods Mol.Biol 2008, 453:3-31.46. Zuker M: Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res2003, 31:3406-15.47. Zuker M and S: Optimal computer folding of large RNA sequences using thermodymanics andauxiliary information. Nucleic Acids Res. 1981, 9:133-148.48. Hofacker IL: Vienna RNA secondary structure server. Nucleic Acids Res 2003, 31:3429-3431.49. Zhu J, Li T, Hu J, Wang E: A novel dot-blot DNAzyme-linked aptamer assay for protein detection.Anal Bioanal Chem 2010, 397:2923-2927.50. Tombelli S, Mascini M: Aptamers as molecular tools for bioanalytical methods. Curr. Opin. Mol. Ther2009, 11:179-188.51. Shin S, Kim I-H, Kang W, Yang JK, Hah SS: An alternative to Western blot analysis using RNAaptamer-functionalized quantum dots. Bioorg. Med. Chem. Lett 2010, 20:3322-3325.52. Kim YS, Jung HS, Matsuura T, Lee HY, Kawai T, Gu MB: Electrochemical detection of 17beta-estradiolusing DNA aptamer immobilized gold electrode chip. Biosensors & bioelectronics 2007, 22:2525-31.53. Neff CP, Zhou J, Remling L, Kuruvilla J, Zhang J, Li H, Smith DD, Swiderski P, Rossi JJ, Akkina R: AnAptamer-siRNA Chimera Suppresses HIV-1 Viral Loads and Protects from Helper CD4+ T Cell Declinein Humanized Mice. Sci Transl Med 2011, 3:66ra6.54. Jackson GW, Strych U: Report #BIO071A - Nucleic Acid Aptamers for Diagnostics and Therapeutics:Global Markets. BCC Research; 2010.55. Jackson GW: Microbial Identification by MALDI-TOF Mass Spectrometry of Ribosomal RNA. 2006. 9
  10. 10. 56. Jackson GW, Karouia F, Putonti C, Fofanov Y, Fox GE, Willson RC: Microarray Designs and MALDI-TOF Mass Spectrometry for Rapid Microbial Identification. In 229th National Meeting of the AmericanChemical Society. San Diego, CA: 2005.57. Jackson GW, McNichols RJ, Fox GE, Willson RC: Bacterial genotyping by 16S rRNA mass cataloging.BMC bioinformatics 2006, 7:321.58. Jackson GW, McNichols RJ, Fox GE, Willson RC: Universal Bacterial Identification by MassSpectrometry of 16S Ribosomal RNA Cleavage Products. Intl J Mass Spectrometry 2007, 261:218-26.59. Jackson GW, McNichols RJ, Fox GE, Willson RC: Toward universal flavivirus identification by masscataloging. J Mol Diagn 2008, 10:135-41.60. Jackson GW, Fox GE, Willson RC: Novel Microarray Designs With Real-Time HybridizationMonitoring For Microbial Identification. In 22nd Annual Meeting of the Houston Society for Engineering inMedicine and Biology. Houston, TX: 2005.61. Potty AS, Kourentzi K, Fang H, Jackson GW, Zhang X, Legge GB, Willson RC: Biophysicalcharacterization of DNA aptamer interactions with vascular endothelial growth factor. Biopolymers2009, 91:145-56.62. Zhang X, Potty AS, Jackson GW, Stepanov V, Tang A, Liu Y, Kourentzi K, Strych U, Fox GE, Willson RC:Engineered 5S ribosomal RNAs displaying aptamers recognizing vascular endothelial growth factorand malachite green. J Mol Recognit 2009, 22:154-161.63. Zhang X, Potty ASR, Jackson GW, Fox GE, Willson RC: Molecular Recognition of Foreign Sequencesin Engineered 5S Ribosomal RNA. In AFFINITY 2007, The 17th Biennial Meeting of the International Societyfor Molecular Recognition. New York University, New York, NY: 2007.64. Zhang X, Potty ASR, Jackson GW, Fox GE, Willson RC: Engineered 5S Ribosomal RNAs displayingAnti-VEGF and Malachite Green Aptamers. 12th Annual Structural Biology Symposium, University of TexasMedical Branch at Galveston, May 18-19, 2007. 2007.65. Liu Y, Stepanov V, Strych U, Willson R, Jackson G, Fox G: DNAzyme-mediated recovery of smallrecombinant RNAs from a 5S rRNA-derived chimera expressed in Escherichia coli. BMC Biotechnology2010, 10:85.66. Lavesque D, Beaudoin J-D, Roy S, Perreault J-P: In vitro selection and characterization of RNAaptamers binding thyroxine hormone. Biochem. J. 2007, 403:129-138.67. Jackson GW, Strych U, Frank E, Willson RC, Ballerstadt R, McNichols RJ: Portable FRET Sensing ofProteins, Hormones, and Toxins Using DNA Aptamers and Quantum Dots. In Technical Proceedings ofthe 2009 Nanotechnology Conference and Trade Show, Nanotech 2009. Houston, TX: 2009.68. Ballerstadt R, Gowda A, McNichols RJ: In Vivo Performance Evaluation of a Transdermal Near-Infrared FRET Affinity Sensor for Continuous Glucose Monitoring. Diabetes Technology & Therapeutics2006, 8:296-311.69. Ballerstadt R, Polak A, Beuhler A, Frye J: In-vitro long-term performance study of a near-infraredfluorescence affinity sensor for glucose monitoring. Biosensors & Bioelectronics 2004, 19:905-14.70. Ballerstadt R, Gowda A, McNichols RJ: Fluorescence Resonance Energy Transfer-Based Near-Infrared Fluorescence Sensor for Glucose Monitoring. Diabetes Technology & Therapeutics 2004, 6:191-200.71. Ramos E, Piñeiro D, Soto M, Abanades DR, Martín ME, Salinas M, González VM: A DNA aptamerpopulation specifically detects Leishmania infantum H2A antigen. Lab. Invest 2007, 87:409-416. 10
  11. 11. 72. Yan XR, Gao XW, Yao LH, Zhang ZQ: [Novel methods to detect cytokines by enzyme-linkedoligonucleotide assay]. Sheng wu gong cheng xue bao = Chinese journal of biotechnology 2004, 20:679-82.73. Bruno JG, Carrillo MP, Cadieux CL, Lenz DE, Cerasoli DM, Phillips T: DNA aptamers developed againsta soman derivative cross-react with the methylphosphonic acid core but not with flanking hydrophobicgroups. J. Mol. Recognit 2009, 22:197-204.74. Oguro A, Ohtsu T, Nakamura Y: An aptamer-based biosensor for mammalian initiation factoreukaryotic initiation factor 4A. Anal. Biochem 2009, 388:102-107.75. Joshi R, Janagama H, Dwivedi HP, Senthil Kumar TMA, Jaykus L-A, Schefers J, Sreevatsan S: Selection,characterization, and application of DNA aptamers for the capture and detection of Salmonella entericaserovars. Mol. Cell. Probes 2009, 23:20-28.76. Smith JE, Medley CD, Tang Z, Shangguan D, Lofton C, Tan W: Aptamer-conjugated nanoparticles forthe collection and detection of multiple cancer cells. Anal. Chem 2007, 79:3075-3082.77. Chen Y, Munteanu AC, Huang Y-F, Phillips J, Zhu Z, Mavros M, Tan W: Mapping receptor density onlive cells by using fluorescence correlation spectroscopy. Chemistry 2009, 15:5327-5336.78. Chen HW, Medley CD, Sefah K, Shangguan D, Tang Z, Meng L, Smith JE, Tan W: Molecular recognitionof small-cell lung cancer cells using aptamers. ChemMedChem 2008, 3:991-1001. 11