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
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