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 and
peptides with high affinity and specificity. These molecules can assume a
variety 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 regulate
cellular processes [3], as well as to guide drugs to their specific cellular Amplify
eluted
targets [4-7]. Contrary to the actual genetic material, their specificity and binders
characteristics are not directly determined by their primary sequence, but
instead by their tertiary structure [8].
Aptamers are generated from large random libraries by an iterative
process often called Systematic Evolution of Ligands by Exponential
Enrichment (SELEX) [9, 10]. The conventional SELEX technique (Figure
1) starts with a large library of random single stranded nucleotides or
aptamers (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 bound
aptamers are partitioned and amplified for the next round. With each round the stringency of the binding
conditions is increased until the only remaining aptamers in the pool are highly specific for, and bind with high
affinity to, the target. Once multiple (typically 10-15) rounds of SELEX are completed, the DNA sequences are
usually identified by conventional cloning and sequencing.
While in general the accepted process for selecting aptamers, so-called “Systematic Evolution of Ligands by
EXponential enrichment (SELEX)” [9, 10] is quite effective, SELEX is commonly performed against only a
single 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 conventional
aptamer 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]. More
recently, 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 to
antibodies in their range of target recognition and variety of applications, they possess several key advantages
over 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.
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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-batch
variation which complicates production of therapeutic proteins [43] and the variability of diagnostic antibody
reagents. Aptamers identified by SELEX can also be easily analyzed and manipulated to characterize the
minimum sequence requirements for aptamer:target recognition. Sequences can be inspected for primary and
secondary structure motifs [44-48], and single base perturbations of identified aptamers can be studied for the
resulting affects on binding affinity. This sort of rational/directed optimization would be much more challenging
using 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 the
integration of aptamers into biosensors, numerous publications support the feasibility of this approach. For
instance, aptamers have been incorporated into lead sensors [30-32], drug sensors [24, 26], and estrogen
measuring devices [52]. They can be used in single-target measuring devices or even in arrays [38], further
extending their versatility. Recently, aptamers have also been used as chimeric conjugates to siRNAs to
improve delivery [53].
Ongoing aptamer development and applications at BioTex
The principal scientist at IceNine, Dr. Bill Jackson, has considerable experience in the development and
novel application of nucleic acid aptamers, and these molecules have enjoyed recent increasing acceptance in
both diagnostic and therapeutic settings – (in 2010, Dr. Jackson authored a 200+ page market research report
on 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 research
program involving novel uses of aptamers. The PI and his colleagues have considerable experience in
developing 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 involving
aptamers [61-64]. Recent funding has included an EPA SBIR Phase I project for aptamer-based detection of
the 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 a
platform for massively parallel SELEX. We have also developed recombinant techniques to express small
foreign RNA’s (aptamers and siRNA) within the ribosome of E. coli [62-65]. Currently we are developing
aptamer affinity reagents to high priority cancer biomarker proteins under Contract SBIR Phase I
funding from the National Cancer Institute.
2

3. Application of aptamers in flow cytometry
In addition to validation of aptamers via SPR, we have successfully employed aptamers in
fluorescence 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 aptamers
to label and differentiate a panel of recombinant proteins expressed on desired T-cell subsets using flow
cytometry. The availability of an inexpensive and efficacious alternative to monoclonal antibodies (mAbs) in
this application is important as a method to produce cellular reagents to be used in compliance with current
good manufacturing practice (cGMP). Obtaining monoclonal antibodies (mAbs) as reagents to validate
the manufacture of cell-based therapeutics for clinical application is tedious, labor intensive and
expensive as applied to generation of clinical-grade reagents. This is chiefly because of the concern of
adventitious virus that may accompany the production of mAbs. Aptamer technology based on in vitro DNA
synthesis will avoid this issue and greatly simplify and reduce the costs associated with producing materials for
use 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 our
aptamers as replacements to fluorescently labeled mAb reagents. Briefly, cells were first transfected with a
defined membrane-bound interleukins (IL7, IL15, or IL21). Cells were then washed to remove serum and
culture media and incubated at 37° for 60 minutes with an aptamer developed to either IL-7, IL-15, or IL-21.
C
Aptamers 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-FITC
followed by an additional wash with PBS. Finally, cells were sorted by conventional FACS based on FITC
fluorescence; cell counts to the right of the vertical blue line represent positive binding. As expected from an
initial screen, there is some cross-reactivity of the tested aptamers. This is due to the use of a common
recombinant Fc region for presenting these three cytokines on the cell surface. Nevertheless, some degree of
specificity/orthogonality of the aptamers is seen, and the experiment demonstrates that in principal, expensive
mAbs could be replaced by aptamers for the FACS application.
3

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 chemistries
for detection of environmental and clinical analytes. For instance, a sensor for the thyroid hormone (small
molecule), 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 a
solid 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 GC
content, 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 detail
elsewhere [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 through
co-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 lyophilized
and reconstituted by the sample, such an assay could be taken to the field or bedside for environmental or
clinical use.
This sort of sensing scheme can be readily devised for proteins as well. We have developed an
analogous 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 DNA
aptamers to novel targets, but also the broad, modular applicability of DNA aptamers in numerous
applications.
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. 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 in
selection of aptamers against the surface of whole cells. Specifically, under NSF funding, we collaborated
with our literal neighbor, Synthecon Inc. (Houston, TX) in an attempt to develop an inexpensive DNA aptamer
mimetic for so-called stem cell factor (SCF or “kit ligand”). Recombinant SCF is a rather expensive, but
commonly used reagent in stem cell culture. To achieve this goal, we enriched our randomized DNA library for
aptamers binding whole stem cells and then selectively displaced the desired aptamers from the c-Kit receptor
using SCF itself. Although we were unable to evolve an agonist mimetic (to effect stem cell proliferation in
culture), did successfully develop a number of candidate c-Kit specific antagonists. We are currently
quantifying their affinity.
5

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]. A
number 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 in
Preliminary Work, we have considerable experience in conjugating aptamers to quantum dots with the
advantage 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 peroxidase
conjugate (strep-HRP). Numerous commercial sources for both fluorescent and chromagenic HRP substrates
are 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 readily
synthesized 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 or
amino-group. Such functionalized particles have been used to concentrate transcription factors [74], or, in the
form 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 their
use in lieu of antibodies. In recent years, directly, or indirectly labeled aptamers have, for instance, been used
to 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 be
employed in numerous applications. Despite their promise, however, they have generally only been
developed or ‘evolved’ against a single target at a time. High costs for aptamer selection (as well as
now-expiring intellectual property constraints) have until now hindered their more widespread use.
IceNine’s novel approach to multiplex aptamer selection can inexpensively provide aptamer affinity
reagents to a revolutionary breadth of targets. Finally, our team has the expertise to demonstrate and
support our customers in a variety of novel aptamer-based application areas.
6

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