Nucleic acid aptamers for living cell analysis.

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Annual Review of Analytical Chemistry 2014.7. Downloaded from www.annualreviews.org by Rutgers University Libraries on 06/28/14. For personal use only.

Nucleic Acid Aptamers for Living Cell Analysis Xiangling Xiong,1,2,3,∗ Yifan Lv,1,∗ Tao Chen,2,3 Xiaobing Zhang,1 Kemin Wang,1 and Weihong Tan1,2,3 1

Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Bio-Sensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Collaborative Innovation Center for Molecular Engineering and Theranostics, Hunan University, Changsha 410082, China

2 Department of Chemistry and 3 Department of Physiology and Functional Genomics, Shands Cancer Center, Center for Research at Bio/Nano Interface, University of Florida, Gainesville, Florida 32611-7200; email: [email protected]fl.edu

Annu. Rev. Anal. Chem. 2014. 7:18.1–18.22

Keywords

The Annual Review of Analytical Chemistry is online at anchem.annualreviews.org

aptamer, cell membrane analysis, cell detection and isolation, real-time monitoring

This article’s doi: 10.1146/annurev-anchem-071213-015944 c 2014 by Annual Reviews. Copyright All rights reserved ∗

X.X. and Y.L. contributed equally to this article.

Abstract Cells as the building blocks of life determine the basic functions and properties of a living organism. Understanding the structure and components of a cell aids in the elucidation of its biological functions. Moreover, knowledge of the similarities and differences between diseased and healthy cells is essential to understanding pathological mechanisms, identifying diagnostic markers, and designing therapeutic molecules. However, monitoring the structures and activities of a living cell remains a challenging task in bioanalytical and life science research. To meet the requirements of this task, aptamers, as “chemical antibodies,” have become increasingly powerful tools for cellular analysis. This article reviews recent advances in the development of nucleic acid aptamers in the areas of cell membrane analysis, cell detection and isolation, real-time monitoring of cell secretion, and intracellular delivery and analysis with living cell models. Limitations of aptamers and possible solutions are also discussed.

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1. INTRODUCTION


Living organisms function properly by establishing well-organized hierarchical structures, beginning with the cell, the smallest structural and functional unit. Multiple populations of cells form ordered tissue architectures to regulate life activities. For example, nerve cells are responsible for processing and transmitting information, whereas white blood cells are capable of defending the body against pathogens. Understanding the structural, functional, and biological traits of a cell is critical in biological sciences, particularly developmental biology, as well as cancer research, which is based on cellular abnormalities and metastasis. Elucidating the differences between healthy and cancerous cells will pave way for new diagnostic and therapeutic approaches to combat this dreadful disease (1). More specifically, such differences are usually caused by abnormal protein expression, which is a reflection of genetic alterations. Therefore, highly sensitive tools capable of detecting gene or protein expression, activity, and function, preferably in real time, are key to improvements in cell analysis. Although polymer chain reaction (PCR) is a powerful and sensitive biochemical method for gene detection, it involves multiple steps, including cell lysis, which cannot provide real-time analysis. The development of new high-throughput sequencing technologies capable of deciphering large amounts of genetic data has been indispensable for basic biological research. However, more emphasis on processing and interpreting the sequencing data is needed to make better use of genetic information in cell analysis. Immunostaining, an antibody-based method to detect a specific protein, is commonly used in a variety of molecular and cellular assays. Typically, a signal transduction moiety, such as a fluorescent tag or an enzyme, is attached to an antibody for detection, and the antibody provides specific recognition of the protein of interest. Although they have been developed into a variety of immunoassays and play an important role in cell analysis, antibodies present several drawbacks. For instance, antibodies are usually large, which restricts internalization by cells for intracellular detection. In addition, because antibodies can only be generated for immunogenic substances, their target range is limited. In response, several types of antibody mimicry have been developed, most notably aptamers, which are usually single-stranded oligonucleotides generated by the iterative screening method Systematic Evolution of Ligands by EXponential enrichment (SELEX). Aptamers were first reported independently by two research groups in 1990. Ellington & Szostak (2) reported a selection of RNA molecules with specific binding to organic dyes, and Tuerk & Gold (3) found RNA ligands to bacteriophage T4 DNA polymerase. The former group gave these RNA molecules the name “aptamer,” from the Latin “aptus,” meaning “to fit” (2), and Tuerk & Gold defined the process for identifying aptamers as SELEX (3). The discovery of aptamers and the invention of SELEX gave rise to a new field that is active in both academia and industry. Since 1990, aptamers have been explored extensively as specific, high-affinity probes for diagnostic and therapeutic applications (4). In this review, we focus on recent advances in the development of nucleic acid aptamers, with emphasis on DNA aptamers, for living cell analysis, including cell membrane analysis, cell detection and isolation, real-time monitoring of cell secretion, and intracellular delivery and analysis.

1.1. Generation of Aptamers The invention of PCR by Kary Mullis in 1983 tremendously revolutionized biomedical research, and SELEX is one example that takes advantage of PCR technology. Since its invention in 1990, SELEX has been applied to a variety of targets, ranging from small molecules, such as inorganic dyes, to large moieties like proteins, cells, and entire tumors (2, 3, 5–33). The process starts by 18.2

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generating a randomized RNA or DNA (and later even peptide) sequence library. By permutation and combination of four basic nucleotides into sequences 20–40 nucleotides in length, this library normally consists of 1014 –1015 different candidate aptamers that arguably can recognize virtually any target molecule (34). Each candidate shares a forward and a reverse primer region for PCR amplification. An RNA library also contains binding sequences for reverse transcription. To identify aptamer(s) specific to the target of interest, the library is incubated with target for a certain period of time, and the binding sequences are eluted and amplified by PCR. This process is iterated 5–25 times until the pool is enriched with repeated binding sequences. To improve target specificity, counterselection (or negative selection) with a molecule that shares similar motifs with the target molecule, e.g., a target protein isoform, is incorporated in some selection processes. The aim of counterselection is to remove sequences that bind to both target and nontarget molecules, leaving only the most specific sequences. Negative selection can be performed before and/or after positive selection, depending on the similarity between the target and nontarget molecules. Finally, the enriched library is sequenced to identify potential aptamers, which are synthesized and validated with the original target.

1.2. Characteristics of Aptamers The development of NX1838 (brand name Macugen) (35, 36), which is currently the most commercially successful aptamer, revealed several advantages of aptamers. First, aptamers are highly specific in recognizing closely related proteins or chemical structures. For instance, Macugen can discriminate vascular endothelial growth factor-165 (VEFG165 ) from the VEFG family. Aptamers that differentiate subtle chemical structural variations, such as methyl or hydroxyl groups or the D- versus L-enantiomer, have been reported (5–9). The high specificity may be attributed to the repeated selection process, in which the highest binders (potential aptamers) are retained. Moreover, during the target recognition process, nucleic acid aptamers undergo target-induced conformational changes to better fit into the binding pocket, further improving the selectivity of aptamers (37–41). The dissociation constants (Kd s) of aptamers reported in the literature are in the low nanomolar to high picomolar range, comparable to the Kd s of antibodies (42). In addition, measurement of the rupture forces between a protein and both its aptamer and its antibody by single-molecule atomic force microscopy showed that they were equal, proving that synthetic aptamers bind as robustly as natural antibodies (43). Moreover, aptamers have the advantage of being smaller than antibodies. Depending on the number of nucleotides, aptamers usually weigh 8–15 kDa. Antibodies, however, normally have weights of approximately 150 kDa (44). Smaller size leads to better tissue penetration and faster blood clearance. Aptamers can be chemically synthesized and modified rather easily. Phosphoramidite chemistry complemented with solid-phase synthesis has made oligonucleotide synthesis rapid and inexpensive. Modifications of nucleotides on both the phosphate/ribose backbone and the nucleobases have been studied to improve resistance to enzymatic degradation and reduce off-target events (45). Some of these modified nucleotides are compatible with enzymatic steps, such as PCR amplification (46), thereby diversifying the selection library. Moreover, with the development of phosphoramidite chemistry, most of the modifications can be incorporated into the programming of a DNA synthesizer, which makes them easy to perform and readily accessible. Moreover, nucleotides can be tagged to various functional molecules or moieties, such as fluorescent molecules (47–51) and nanoparticles (NPs) (52–55), for diagnostic purposes, thus advancing the applications of aptamers in bioanalytical fields. The chemical nature of aptamers also indicates that they are cost-effective and easy to produce. Antibodies rely heavily on cell-based production systems, require complicated manufacturing www.annualreviews.org • Nucleic Acid Aptamers for Living Cell Analysis


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processes, and often suffer batch-to-batch variation (34). Aptamers, however, can be reproduced chemically with little or no variation. In addition, aptamers are essentially nonimmunogenic (56, 57), which makes them suitable for in vivo applications.

2. APTAMERS FOR CELL MEMBRANE ANALYSIS


The cell membrane holds thousands of different proteins, carbohydrates, and lipids, which retain specific structures and play crucial roles in cellular activities. The composition, structure or orientation of these biomolecules may vary among cell types and between healthy and diseased cells. Unveiling the differences is critical to our understanding of tumor biology and the discovery of pharmacological targets.

2.1. Cell-SELEX as a way to Study Cell Membranes Cell membrane proteins comprise an important but understudied group, because of technical difficulties in their isolation and characterization (58). SELEX may provide an alternative way to demystify membrane proteins by first probing them with high-affinity aptamers. Compared with pure target, such as an organic dye molecule or a protein, a cell membrane is a very complex system. The Gold group (10) first reported the use of a cell membrane as a selection target. In this study, they used red blood cell ghosts as a positive selection target and demonstrated that SELEX could be applied not only to pure compounds, but also complex targets. One advantage of cell-SELEX is simultaneous generation of high-affinity probes for multiple targets (10). After the introduction by Gold and coworkers, selection on whole cells (11, 13), especially tumor cells (12, 25), was reported, and, hence, SELEX for cancer research became popular. Because of the counterselection process, cell-SELEX is particularly useful for identifying subtle differences between normal and cancerous cells or different subtypes of cancerous cells. Positive selection on target cells (cancer cells) and negative selection on nontarget cells (normal cells or another type of cancer cell) can reveal molecular differences among membrane proteins and may lead to biomarkers for specific cancer types. The general procedures of cell-SELEX are demonstrated in Figure 1, and selections on cancer cells reported in the literature are summarized in Table 1. To date, most cell-target aptamers used are DNA aptamers because DNA aptamers, in contrast to RNA aptamers, in addition to forming complex secondary and tertiary structures, also are much easier to handle and less expensive to generate.

2.2. Aptamers in Biomarker Discovery Conventional methods of biomarker discovery, such as mass spectrometric (MS) approaches, enable investigators to analyze the entire cell proteome and identify cell-specific proteins. However, the most under-represented group in proteome analysis remains membrane proteins, which are difficult to tackle (58). Aptamers generated from cell-SELEX profile membrane protein differences among different cells and simultaneously act as affinity probes for protein extraction, presenting unique advantages in identifying protein molecules on cell membrane surfaces (60). The common practice in aptamer-assisted biomarker discovery includes the following steps. First, target cells are expanded, collected, and lysed or physically homogenized. The cell debris or membrane protein components are solubilized and incubated with biotinylated aptamers with or without priory extraction proteins captured by nonbinding sequences. The protein-aptamer complex is isolated by streptavidin-coated magnetic beads (MBs), and then the bound proteins are eluted and analyzed by SDS/PAGE and silver staining. The aptamer-purified protein band 18.4

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Wash to remove unbound sequences


Control cell

Target cell

Positive selection

Extracted bound sequences

ssDNA library Cell-SELEX

Negative selection PCR amplification

Sequencing and screening potential DNA aptamer

Retain unbound sequences Remove bound sequences Figure 1 Scheme of the cell-SELEX process. Abbreviations: PCR, polymer chain reaction; SELEX, Systematic Evolution of Ligands by EXponential enrichment. Adapted with permission from Reference 59.

is excised, digested, and analyzed by LC-MS/MS for target identification. Finally, the identified target protein is confirmed with an existing antibody. One concern is that membrane proteins may be denatured during the extraction process. Therefore, chemical modification for crosslinking the aptamer with its target protein before processing the cells is a valid alternative. To date, three membrane proteins with aptamers generated from cell-SELEX have been elucidated (12, 61, 62). Although the methodology has proven to be effective, it may need to be further optimized to accelerate target identification. More importantly, the identified proteins need to be validated with a large amount of clinical samples before they can be called biomarkers, even though some aptamers have been found effective with patient samples (63).

2.3. Aptamers as Molecular Tools to Probe Membrane Proteins Aptamers are proven to be useful molecular tools for studying membrane proteins directly on live cells. In one example, Chen et al. (64) used fluorescently labeled aptamers, together with fluorescence correlation spectroscopy (FCS), to map receptor densities on live cell membranes. The applied aptamers targeted certain cell-surface receptors with high affinity and specificity, and that interaction could be sensitively monitored by FCS in a femtoliter-sized observation volume. The small volume allowed the detection of fluorescent-aptamer down to two molecules. The binding www.annualreviews.org • Nucleic Acid Aptamers for Living Cell Analysis


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Table 1 Summary of cancer cell–SELEX Target cell

Dissociation Cancer type


line

constant (nM)a

Aptamer targetb

Patient sample tested

Reference

HL60

Acute myeloid leukemia

4.5 ± 1.6

NA

Bone marrow

17

CEM

Acute T lymphoblastic leukemia

0.8 ± 0.09

Tyrosine-protein kinase-like 7

Bone marrow

25, 61, 66, 132

Ramos

Burkitt’s lymphoma

ND

NA

Primary human chronic lymphocytic leukemia B cells

133

Ramos


49.6 ± 5.5

NA

NA

134

Ramos


0.76 ± 0.13

IgM heavy chain

Bone marrow

15, 65

DLD-1

Colon cancer

32.1 ± 3.4

NA

Tissue section

22

HCT 116

Colon cancer

3.9 ± 0.4

NA

Tissue section

22

U251

Glioblastoma

150

Tenascin-C

NA

12

A172

Glioblastoma

61.82 ± 6.37

NA

NA

24

MEAR

Mouse liver hepatoma

4.51 ± 0.39

NA

Human hepatocarcinoma cell line

62, 135

A549

Non-small cell lung cancer

28.2 ± 5.5

NA

Tissue section

19

TOV21G

Ovarian clear cell adenocarcinoma

0.25 ± 0.08

Stress-inducedphosphoprotein 1

NA

23, 136

CAO-3

Ovarian serous adenocarcinoma

39 ± 20

NA

NA

23

NCI-H69

Small cell lung cancer

38

NA

Blood sample

16

Abbreviations: NA: not available; ND: not determined. a Best Kd among all aptamers reported in the paper(s). b Target of one reported aptamer.

affinity of the aptamer with its target membrane receptor was determined first by FCS, and the result was consistent with a previous finding. Then this aptamer-receptor interaction was applied to a cellular model to estimate receptor densities and distribution on the cell surface. The study revealed different expression levels of a receptor on two different types of cancer cells. The densityestimation approach was validated with competition studies using excess unlabeled aptamers and proteinase treatment studies. Besides ligand-density distribution patterns, this method was used for a ligand-receptor kinetics study, both of which provided knowledge key to understanding cellular behavior and biological function, as well as developing novel diagnostic and therapeutic agents. Aptamers can also be used to elucidate protein structural components. For example, aptamer Sgc8 was conjugated to gold NPs for the construction of a surface energy transfer (SET) nanoruler to monitor the distance of two binding sites of a membrane receptor on a living cell membrane (65). In this SET ruler system, the donor and the acceptor were brought separately to two independent binding pockets on one protein by a fluorophore-labeled monoclonal antibody and an aptamermodified gold NP, respectively. The distance from the dye molecule to the metal NP could be manipulated by varying NP sizes, and the change in energy transfer efficiency was evaluated by both flow cytometry and confocal imaging. Fluorescence quenching from the donor on the 18.6

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antibody binding site was observed in the presence of the aptamer-gold NP conjugates on the aptamer binding site, and the quenching efficiency was plotted against the diameter of the NPs and adopted in the calculations to determine the distance between the two binding sites. The potential factors that may influence the quenching efficiency were excluded by control experiments. In this work, aptamers functioning as high-affinity probes played an important role in immobilizing the SET nanoruler on a living cell membrane and enabled the resolution of protein structure in its natural state.


3. CELL DETECTION AND ISOLATION Modern molecular medicine has increasingly focused on developing novel target-specific molecular probes for improving the prognosis and diagnosis of diseases and developing the best treatment regimens. For example, in contrast to traditional cancer diagnostics, which rely on morphological definition and histological examination, new method development focuses on molecular profiles of cancerous and normal cells. The heterogeneity of cancer (66) complicates the diagnosis, so analysis at a single-cell level may be required in some circumstances. In addition, the identification of circulating tumor cells (CTCs), along with their clinical value, calls for the development of efficient extraction methods to separate rare CTCs from the massive number of blood cells. Aptamers directly selected against cancer cell surfaces, or against target-specific tumor-associated antigens, are attractive molecular tools for sensitive cell detection and isolation.

3.1. Aptamer-Based Cell Detection Aptamers chemically conjugated with various signal-generating components have been developed for acoustic (67–69), electrochemical (70–73), and optical (74–76) sensing. Crucial to detection is having high signal-to-noise ratio. To accomplish this, several methods are employed to either increase signals or decrease backgrounds. Because aptamers and their conjugates used for cell imaging have been extensively reviewed elsewhere (77), cell imaging is excluded from this section. Because of their base-pairing capability, aptamers can be adapted for various amplification systems to enhance signals, which is not possible in antibody-based detection systems. For example, the exponential signal amplification power of PCR can be used, in which DNA aptamers function as recognition elements for detection and, at the same time, as templates for PCR amplification. Song et al. (78) specifically and sensitively detected acute myeloid leukemia cells with target cell– specific aptamers by the PCR approach. After incubation with aptamers, cells were collected, lysed, and used as a PCR template. DNA aptamers that bound to target cells were amplified with fluorescein-labeled dGTP and dUTP in the substrates to achieve enhanced fluorescence signals. In addition, a cationic-conjugated polymer was added for additional signal amplification via Forster resonance energy transfer (FRET). Similarly, RNA-based aptamers conjugated to ¨ RNA polymerase promoters can be used to monitor cancer cells (79). Although this method was sensitive for single-cell detection, the off-line PCR step complicated the detection process and prolonged detection time. In addition, cells needed to be lysed before PCR, which made them unavailable for further analysis. Another approach includes aptamer recognition into rolling circle amplification (RCA) reaction. In one system, the binding of DNA aptamers to target cancer cells triggered the self-assembly of a polycatenated DNA scaffold, which initiated the release of circular DNAs (80). The circular DNAs containing DNAzyme information served as the templates for RCA reaction, and as such, several DNAzymes were produced for the generation of chemiluminescence signals. The cascade signal amplification steps led to a limit of detection (LOD) of 137 cells per mL. www.annualreviews.org • Nucleic Acid Aptamers for Living Cell Analysis


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hv

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n

c

n

Selective binding

FRET n

Insulin

n

n

In situ self-assembly

n

Insulin-binding aptamer

Single-walled carbon nanotube

Selfassembly

Pancreatic INS-1 cell


Cell surface

b

Pillar Cell

Blood in

Aptamer

Collagen Extracellular matrix

d Cellular uptake

Blood out

Figure 2 Aptamers used for cell analysis. (a) Demonstration of in situ self-assembly of fluorescent DNA on target living cell surfaces. (blue) Aptamer, (red ) initiator sequence, and (light blue and green) two partially complimentary hairpin monomers. (b) Microfluidic device consisting of one inlet, one outlet, and eight channels connected through bifurcation. Enlargement shows the optical micrograph of a portion of a micropillar array in a channel modified with cancer cell capture aptamers. (c) Schematic of optical nanosensor architecture for real-time measurement of insulin secreted from pancreatic INS-1 cells in situ. (d ) Working principle of switchable aptamer micelle flares. FRET, Forster resonance energy transfer. ¨

The third type employs hybridization chain reaction of DNA. In general, aptamers were engineered with initiator sequences that could activate hybridization of two partially complementary hairpin monomers (Figure 2a) (81). The self-assembled DNA sequences formed a long linear nanostructure, which enabled the loading of multiple fluorophores for enhanced fluorescence via either chemical conjugation or physical association. The cascading polymerization of monomeric DNA building blocks could take place in site and selectively on target living cell surfaces in cell mixtures, indicating the potential of this method in cell detection. Unlike PCR-based detection, this method is nondestructive, allowing cells to be characterized by other types of assays. However, DNA-based amplification methods usually require an extra 1–2 h of reaction time and washing steps, thereby making real-time monitoring impractical. The vastly increased surface-area-to-volume ratio presented in nanomaterials makes them ideal scaffolds for molecular assembly. Moreover, numerous physical properties are altered when the materials are decreased to the nanoscale level, and these can be applied for sensitive signal transduction. Aptamers have been conjugated to different kinds of nanomaterials for enhanced cell detection via the multivalency effect of aptamers, as well as physical properties of nanomaterials. A 18.8

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simple colorimetric assay was designed using aptamer-conjugated gold NPs (82). In the presence of target cells, aptamers acted as selective biorecognition probes signaled by gold NPs, which underwent a color change upon assembly around the cell surface. With optimized gold NP size and concentration, the assay could detect approximately 3,000 cells per mL by the naked eye or 300 cells per mL by spectrophotometry. This principle has been used in a strip biosensor device for rapid cancer cell detection with a sensitivity comparable to the above-mentioned assays (83). Overall, gold NP-based detection is simple, rapid, and straightforward, albeit with compromised sensitivity. Because of its low toxicity, high sensitivity, low background, and capacity for multiplex monitoring, fluorescence is a widely used method in the life sciences for nondestructive sensing of living cells. Some nanomaterials, such as quantum dots (QDs), are naturally fluorescent as a function of the particle diameter. Others can be doped with or conjugated to fluorescent molecules to obtain optical properties. In both cases, the fluorescent signals are greatly improved compared with a single fluorescent molecule. Huang et al. (84) report the use of nanomaterials as a nanoplatform to assemble multiple fluorescently labeled aptamers for enhanced affinity and sensitivity. The authors determined that the molecular assembly of aptamers on gold-silver nanorods improved the intrinsic affinity of the original aptamer, as well as the fluorescence signal, 26- and 300-fold, respectively. NPs can be used to encapsulate hundreds to thousands of dye molecules and then applied to aptamer-labeled cells for ultrasensitive detection (85). Silica NPs doped with three FRET dyes were prepared and conjugated with three different aptamers for multiplexed cancer cell monitoring in cell mixtures (86). Multiplexing detection is an advantage of fluorescent-based assays, although caution must be taken to avoid signal overlapping, as well as possible cross-reactions among probes. Moreover, DNA aptamers can serve as a template and stabilizer for the formation of silver nanoclusters (AgNCs), which display strong, robust, and tunable fluorescence (87). The aptamerAgNC complexes were prepared in one step without chemical conjugation, which required the design of one single-stranded oligonucleotide capable of both in situ AgNC synthesis and target biorecognition. However, further studies are needed to compare the sensitivity of this probe with other fluorescent probes and to determine stability in a more complex biological environment. Most fluorescent-based detection is designed in a so-called always-on pattern, which usually causes a high background problem. In response, probes that are turned on only in the presence of targets have been designed based on the FRET principle. Taking advantage of adaptive recognition, aptamers were designed into analyte-responsive biosensors with a quencher and a fluorophore on the probe (88). The fluorescence was quenched until the probe recognized its target cells (89). Using the same principle, nanomaterials, such as graphene oxide (GO), were used as a superquencher in cellular detection. In addition to its optical property, GO can effectively absorb DNA strands on its surface via π-π stacking. Cao et al. (90) have incorporated a GO-based FRET biosensor into a miniature multiplex chip for cancer cell detection. They reported a low LOD of approximately 25 cells per mL and the ability to quantitatively analyze seven different samples in a relatively short period of time. However, the small detection volume of only 40 μL may be a concern when handling a large amount of sample. Electrochemical and electrochemiluminescence (ECL) assays are powerful analytical tools because they have limited background optical signal. Different types of NPs in combination with aptamers have been adapted for electrochemical—or ECL—based cell detection. Ding et al. (91) reported the detection of Ramos cells using both types of assays. In the electrochemical assay, multiple CdS QDs were anchored on one aptamer-modified AuNP for signal amplification, while the entire Au-CdS complex was immobilized on MBs for easy separation. Target cancer cells competitively disassociated the Au-CdS complexes from the MBs, changing the concentration of Cd2+ in the solution, which was reflected in the differential pulse voltammograms (DPVs). The www.annualreviews.org • Nucleic Acid Aptamers for Living Cell Analysis


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DPV peak current was proportional to the cell concentration, reaching a calculated LOD of 67 cells per mL under optimal conditions in complete cell culture media. MBs were also employed in the ECL assay, with aptamers immobilized on the MB surface. In addition, a ruthenium-based ECL nanoprobe was labeled with a signal DNA sequence partially complementary to the aptamer sequence. The incubation of target cells with MB-Ru biocomplex released the ECL probes from MB into the solution. The released probes were then captured by the gold electrode for ECL detection. Although no signal amplification step was involved, the ECL assay reached an LOD comparable to that of the electrochemical assay. Later, the same group reported the integration of a DNA polymerase– and endonuclease–assisted cycling amplification into the system to lower the LOD to 8 cells per mL in blood sample (92). A reticular DNA-QD sheath constructed by self-assembly of DNA-modified CdTe QD probes and DNA nanowire frameworks functionalized with cell-binding aptamers has been reported for fluorescence microscopy imaging and the electrochemical detection of target cells with LOD of 10 cells per mL (93). Similarly, a QD-based ECL method coupled with a DNA cycle-amplifying method has been applied for cell detection with a sensitivity comparable to the above-mentioned Ru-based ECL assay (94). Because of its extraordinary electron transport property and highly specific surface area, graphene has been employed in electrochemical- (95) and ECL-based detection of cells (96), both of which have realized high sensitivity. These electrochemical- and ECL-based methods are, in general, nondestructive and sensitive, but they are time-consuming, and the analytical volume is usually small. Surface-enhanced Raman spectroscopy (SERS) with a silver or gold substrate is another useful tool for cell detection and imaging given its low autofluorescence from biological samples and no photobleaching. Because of their high SERS activity, aptamer-conjugated Ag-Au nanostructures have been applied for the detection of breast cancer cells (97). The sensing was realized by specific tagging of MCF-7 cells with Rh6G-labeled aptamer-Ag-Au nanostructures, which displayed the characteristic peaks of Rh6G compared with nontarget cells in the SERS spectrum. Because most biological samples exhibit virtually no magnetic background, magnetic nanomaterials have been explored for ultrasensitive analysis of cells, especially in a complex biological environment. By measuring the change of spin-spin relaxation times of the surrounding water protons induced by the assembly of iron oxide magnetic nanoparticles (MNPs), Bamrungsap et al. (98) could detect as few as 40 cells per mL in fetal bovine serum (FBS) containing buffer and 100 cells in a 250 μL whole blood sample. The MNPs were modified with a cell targeting aptamer and thus selectively accumulated on specific cell membranes, accelerating the spin dephasing of the surrounding water protons and resulting in a decreased T2 . Moreover, with an array of aptamerconjugated MNPs, pattern recognition of various cell types could be realized, and the expression level of different receptors on each cell type could be elucidated. Compared to other techniques, the aptamer-conjugated MNPs provided robust and sensitive detection under different sample conditions, with minimal detection time and instrumentation.


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3.2. Isolation of Cancer Cells with Aptamers CTCs originating from exfoliate cells shed from primary or metastatic cancers have substantial clinical value in early cancer diagnosis and monitoring of cancer therapy. Because only a few rare CTCs are mixed in a relatively high volume of blood, their isolation and characterization is a major technological challenge. Current CTC isolation methods, such as the CellSearchTM System, rely on one epithelial marker, EpCAM, for capture and isolation and are therefore applicable only in detecting CTCs from certain types of cancers. Aptamers that recognize cell membrane targets provide additional molecular probes for CTC capture and have been investigated in several isolation platforms. An appropriate detection platform should have high capture efficiency and 18.10

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rapid blood sample processing. Generally, two types of approaches, NPs and microfluidic devices, have been under investigation. Combining magnetic and fluorescent NPs, Herr et al. (99) developed a novel dual-NP assay for the rapid collection and detection of leukemia cells from mixed cells and whole blood samples. As the molecular recognition element, aptamers were immobilized on both types of NPs. The silica-coated iron oxide–doped magnetic NPs were used for target cell extraction and enrichment with no centrifugation or presample cleanup requirement. The collection efficiency of target cells ranged from 30% to 80%, whereas control cells ranged from 0.5% to 5%. Dye-doped fluorescent silica NPs provided sensitive microscopic and flow cytometric detection by amplifying the signal intensity corresponding to a single aptamer binding event. This assay has been extended for multiplexing cancer cell detection in cell mixtures by attaching different aptamers to spectrally different fluorescent NPs (100). Later, Medley et al. (101) studied several parameters, including the fluorophores used to construct fluorescent NPs, the size of magnetic NPs, and the conjugation chemistry, to improve assay performance. More importantly, a multiple aptamer approach was investigated for improved assay performance. Those aptamers recognized different targets on the same type of cancer cell surface. Utilizing multiple aptamers in this assay may counter the problem of heterogeneity of cancer cells found in real patient samples. The use of multiple aptamer sequences on one NP and the use of multiple NPs with distinct aptamer sequences were investigated, and the statistical data supported the superiority of the former assay. Microfluidic devices are promising platforms for detecting cancerous cells in bodily fluids because of fast analysis, low cost, and simple operation. The immobilized aptamers in a microfluidic channel bind selectively to a particular cell type to yield a new technique called cell-affinity chromatography (CAC). An aptamer-modified microfluidic device was first reported by Phillips et al. (102) for enriching rare cells. Several parameters affecting cell capture efficiency, such as aptamer surface density, cell concentration, and incubation time, were first examined with a prototype device. Then a poly(dimethylsiloxane) (PDMS) device with input and output ports was assembled and connected to a syringe pump. An optimal flow rate that enabled high capture efficiency and high selectivity was determined by comparing the cell capture results at different flow rates. The PDMS device reached greater than 80% target capture efficiency with approximately 97% purity of captured target cells when the flow rate was between 150 and 200 nL/s. This work demonstrated a rapid, inexpensive, and simple device for cell capture with aptamers. On the basis of this work, Xu et al. (103) constructed another microfluidic CAC device capable of simultaneous sorting, enriching, and detecting of multiple types of cancer cells from a complex sample. The channel was designed in a serpentine shape separated into three regions for immobilization of three different aptamers. When a cell mixture flowed through the channel, each region captured and enriched different cells based on aptamer binding. The microscopic study revealed that cells of interest were enriched more than 135-fold. Cells were then released from the channel by pumping air bubbles into it, and further analysis discovered that the collected cells remained viable as unprocessed cells. Recently, the same research group integrated micropillars into the microfluidic channel for better device performance (Figure 2b) (104). The loading capacity of aptamers was increased by the added surface area of the micropillars, and the channel geometry significantly increased the probability of cell-aptamer interaction. Under conditions for optimum trade-off between efficiency and purity, as few as 10 spiked cancer cells in 1 mL whole blood could be isolated, with capture efficiency greater than 95% and with processing efficiency of 1 mL of blood in 28 min. Besides micropillars, other types of surface topology such as microwells have been used in microfluidic channels for cell isolation (105). The size of the aptamer-coated microwells was optimized to contain a single cell, allowing for subsequent single-cell analysis. www.annualreviews.org • Nucleic Acid Aptamers for Living Cell Analysis


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Inspired by marine creatures with long tentacles containing multiple adhesive domains for effective capture, Zhao et al. (106) developed a 3D DNA network for cell isolation with repeats of adhesive aptamer domains. The multivalent DNA network was synthesized directly on a microfluidic surface by RCA. The extension of DNA strands into the 3D space increased the accessibility and frequency of cell-aptamer interactions, even at high flow rates. After tailoring DNA graft density, length, and sequences, the capture efficiency of target cells outperformed monovalent aptamers and antibodies, while maintaining high purity. When the RCA-aptamer system was integrated into a herringbone microfluidic device, 80% of cells could be captured at a flow rate of 60 μL/h. In addition, the authors demonstrated that approximately 70% of cells could be released using DNase I, whereas 60–70% of released cells remained viable. This method demonstrated the advantages of aptamers over antibodies in cell capture assays. Captured cells can be detected in situ (107); however, for more complete cellular study, cells need to be released from the channel and collected after capture. Pumping air bubbles into the channel induced shear forces for cell detachment, but the collection efficiency was less than 50% as a result of the dead volumes in the device. However, the use of DNases is only feasible for RCA-aptamers, with slightly higher efficiency, yet decreased cell viability. Attempts at rapid and nondestructive release of cells have also been made by temperature modulation (108). The binding of aptamers to their targets is strongly dependent on the secondary structures of aptamers, which are temperature sensitive. At elevated temperatures, the interaction is disrupted, allowing release and elution of viable target cells from the microfluidic device. A temperature study yielded a release efficiency of approximately 80% at 48◦ C within 2 min, without affecting cell viability. Because the temperature-induced conformational change of aptamers is reversible, the device was proven to be reusable for at least three rounds of the capture-release cycle with similar performance. The recycle feature can also decrease the fabrication cost in clinical applications. Another effective cell release design utilized aptamer-functionalized glass beads (GBs) in a Hele-Shaw microfluidic device (109). The GBs sit in an ordered array of pits in PDMS channels with exposed aptamers on the curved hemispherical surfaces. After cell capture, GBs were released from the pits by simply inverting the device. Antisense sequences were used to detach 92% of the captured cells from the beads.


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4. REAL-TIME MONITORING OF CELL SECRETION Cells communicate with each other and the surrounding environment by secreting and sensing signaling molecules. Immune cells, for example, secrete cytokines to regulate or assist the active immune response. Detection of the secreted molecules is a way to evaluate cellular function. The most popular functional assays are antibody-based immunoassays, including flow cytometry, radioimmunoassay, enzyme-linked immunosorbent assay, and enzyme-linked immunosorbent spot, which are highly sensitive and reliable. However, these methods cannot provide real-time monitoring, and most of them are not suitable for single-cell analysis. To this end, several aptamer-based assays have been developed for the detection of extracellular molecules secreted by various types of cells. An optical nanosensor based on near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWNTs) was assembled with aptamers for noninvasive detection of cell-signaling molecules in real time (Figure 2c) (110). As with GO, SWNTs possess the graphitic lattice needed to allow noncovalent absorption of DNA molecules. In addition, SWNTs’ photoluminescence (PL) is sensitive to molecular binding events, thereby providing a way to transform molecular recognition to optical signals. For example, an insulin-binding aptamer (IBA) was functionalized on the surface of SWNTs for sensing insulin secretion from mammalian pancreatic cells. The quenching of PL from IBA-functionalized SWNTs was observed upon the addition of insulin with an LOD of 18.12

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10 nM in solution. To monitor insulin secretion in real time, IBA-coated SWNTs were incorporated into a collagen extracellular matrix (ECM) where pancreatic cells were cultured. The PL quenching map demonstrated the spatial and temporal resolution of insulin secretion, thus allowing in situ monitoring of cell-signaling molecules with fairly good sensitivity. Liu et al. (111) designed a microdevice featuring miniature aptamer-modified electrodes for real-time detection of local interferon gamma (IFN-γ) released from primary human leukocytes. The hairpin-structured aptamers were assembled onto Au electrodes at the 3 end, with redox reporters covalently attached at the 5 end for electrochemical sensing of IFN-γ. The electrodes were fabricated on passivated glass slides and integrated with antibody-modified microfluidic channels. When blood was injected, leukocytes were captured by antibodies in close proximity to the aptamer sensors. IFN-γ release from captured cells was then detected by aptamers and monitored in real time by square wave voltammetry (SWV). The response could be monitored continuously to determine the production rate of IFN-γ, and signals were detected as early as 15 min after stimulation from as few as 90 T cells. This platform was extended further by the same group for simultaneous detection of two cytokines using two different aptamers (112). The specificity of individual sensors to the correct cytokine species was verified, and SWV at individual electrodes was monitored over time. The authors indicated that this microsystem may be able to detect a larger array of cytokines or biomarkers in blood analysis. Neuron cells rely on neurotransmitters for signal transmission. Adenosine triphosphate (ATP) is a pivotal neurotransmitter for extracellular signaling in various tissues and organs, including taste buds. Sensitive and specific measurements of ATP secreted from a single taste receptor cell (TRC) are essential for studying taste cell-to-cell communication mechanisms. To achieve this goal, an ATP-sensing aptamer and its DNA competitor were immobilized on the surface of a light-addressable potentiometric sensor (LAPS) to monitor local ATP secretion from a single TRC (113). The LAPS chip is a silicon-based surface potential detector that can achieve singlecell measurement if the desired cell is illuminated by a modulated laser. TRCs cultured on the surface of a LAPS chip released ATP upon stimulation. When an aptamer bound to ATP, it was released from its complementary sequences tethered on the LAPS surface, leading to a change in the surface charge and the generation of a corresponding fluctuation in the photocurrent at the illuminated local site. The fluctuation, recorded as working potential shifts of the chip, correlated with ATP concentration. To monitor local ATP secretion from a single TRC, the desired TRC was illuminated, and the working potential shifts were recorded. ATP concentration measured by the aptamer-functionalized LAPS chip was comparable to the value from a previously established method, confirming the utility of the LAPS chip in studies of taste cell communications. Another method for monitoring ATP dynamics was realized by directly anchoring ATPbinding aptamers on a glia cell surface (114). Glia cells play an essential role in the regulation of synaptic transmission in neuronal networks and are also involved in ATP release. Therefore, realtime monitoring of extracellular ATP can provide information for neurochemical studies. The neurological event usually occurs within seconds or milliseconds, allowing only a short response time for observation. Tokunaga et al. (114) employed an ATP-binding aptamer labeled with fluorescein in proximity to the binding pocket. Upon binding to ATP, the fluorescence intensity increased with sufficient speed for real-time monitoring because no large conformational change was involved. Although chemical conjugation of the DNA-based sensor on cell membrane proteins was feasible, a simpler approach using a lipophilic molecule that adheres directly to cellular membrane lipids was investigated. The lipophilic molecule, tocopherol, was covalently conjugated to the aptamer to anchor the sensor to the cell membrane. An increase in the fluorescence of the membrane-anchored ATP sensor was observed near the stimulation point, indicating the extracellular chemical transmitter dynamics. A similar approach for probing a type of growth www.annualreviews.org • Nucleic Acid Aptamers for Living Cell Analysis


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factor in the cellular environment was reported independently by another group (115). The aptamer sensor was covalently attached to the membranes of mesenchymal stem cells for quantitative detection of high spatial and temporal resolution of platelet-derived growth factor (PDGF) secreted by neighboring cells. The authors demonstrated that the engineered stem cells could be applied for in vivo study.

5. APTAMERS IN INTRACELLULAR DELIVERY AND ANALYSIS


A daunting challenge in measurement science is the sensitive, specific, and real-time monitoring of biomolecules in living cells. Two major factors need to be considered when designing probes for intracellular sensing: effective intracellular delivery and stable, specific, and sensitive detection in the complex cellular environment. The engineering of oligonucleotides as stable and sensitive sensors for intracellular monitoring has shown great promise in recent years (116, 117). In this section, we focus on the progress of effective intracellular delivery assisted by nanotechnology or mediated by cell membrane receptors.

5.1. Aptamers Delivered by Nanomaterials for Analysis of Intracellular Targets Nanomaterials are widely used as carriers to introduce nucleic acid probes into living cells for real-time analysis. Zheng et al. (118) reported on aptamer nanoflare, a composite nanomaterial that consists of a dense monolayer of nucleic acid aptamers around a gold NP core, for intracellular ATP sensing. Previous studies have identified DNA-gold NPs with the ability to readily enter living cells. The ATP-binding aptamers were modified with fluorophores and linked to the particle by hybridizing with complementary DNA sequences directly immobilized on gold NPs. In the absence of ATP, the fluorescence signals from aptamers were effectively quenched by the gold NPs. When ATP was introduced, the aptamers dissociated from AuNPs, thereby restoring fluorescence. Using confocal microscopy and flow cytometry, the authors observed uniformly high fluorescent signals from cells treated with aptamer nanoflares compared to those treated with the mismatch DNA. A decrease in cell-associated fluorescence was observed in the presence of an ATP-depletion drug in a concentration-dependent manner, suggesting that aptamer nanoflares can be used for quantitative analysis of living cells. Another type of nanoflare using DNA micelles was developed by Wu et al. (119) (Figure 2d ). The switchable aptamer micelle flare (SAMF), formed by self-assembly of an aptamer switch probe-diacyllipid chimera, was proven to be noncytotoxic yet with high intracellular delivery efficiency, owing to the similarity between diacyllipids in the SAMFs and phospholipids in the cell membranes. Switchable aptamers were modified with a fluorophore and a quencher to bind target ATP molecules with high selectivity and specificity; this was observed as the restoration of fluorescence signal from SAMFs but not from micelles with control sequences. Confocal microscopy experiments suggested that SAMFs primarily remained in the cytoplasm, not the nucleus. Similar to the gold NP nanoflares, the fluorescence intensity of SAMFs correlated with intracellular ATP concentration. In addition to its DNA adsorbing and fluorescence quenching capacity, graphene also shows promise as a vehicle for transporting DNA sensing molecules for in situ molecular probing in living cells. Wang et al. (120) first applied a graphene oxide nanosheet (GO-nS) as transporter, protector, and quencher of an aptamer-carboxyfluorescein (FAM) sensor for real-time intracellular detection of ATP. FAM-labeled aptamers lost their fluorescence after incubation with GO-nS for 5 min, as a result of FRET between FAM and the GO-nS when DNA was physically adsorbed on the nanosheet. In the presence of ATP, aptamers competitively dissociated from the GO-nS 18.14

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for ATP binding, resulting in fluorescence recovery. The specificity of the detection system was tested with other types of triphosphates, and the fluorescence restoration was observed only with ATP. The sensitivity reached 10 μM for in vitro assay. The GO-nS protected DNA aptamers from enzymatic cleavage during cellular transit, as shown by gel electrophoresis. Recently, the same group developed this system further for simultaneous detection of multiple nucleotides by using different DNA/RNA aptamers labeled with different fluorophores (121). Additional study revealed that in the presence of high protein concentration, the adsorbed DNA can be nonspecifically desorbed from GO, resulting in a strong false-positive signal. To solve this problem, Tan et al. (122) incorporated ATP-binding aptamers into a molecular beacon structure (MB) whose fluorescence was quenched by a chemically conjugated quencher in addition to GO. The self-quenching ability of the MB-structured aptamer and control probes produced a much smaller false-positive signal, even though these DNA strains could be nonspecifically released. An assay for in situ ATP semiquantification was developed by codelivery of a nonspecific internal reference ssDNA labeled with a different dye. Because the internal reference gave almost the same fluorescence from different groups of cells, independent of both ATP concentration and probe sequences (aptamer or control probes), this simple fluorescent assay allowed semiquantification of cellular ATP. As an alternative to the restoration of fluorescence, Paige et al. (123) developed an RNA aptamer, termed Spinach, which binds and switches on the fluorescence of a small-molecule fluorophore. Later, the same group (124) fused a ligand-binding RNA aptamer to Spinach with a shared critical stem for the detection of cellular metabolites. The small-molecule binding could fold the aptamer and stabilize the stem, resulting in fluorescence. This probe could possibly be used for dynamic monitoring of cellular events on account of switched-on and -off fluorescence. Interestingly, in this platform, the small dye molecules were delivered extracellularly, and the sensor was cloned into a plasmid and expressed by Escherichia coli cells. For broader applications, however, extracellular delivery may be a better option.

5.2. Aptamer-Assisted Specific Cellular Internalization Rather than passive internalization with nanomaterials, some aptamers can actively enter cells mainly via receptor-mediated endocytosis. These aptamers, can, in turn, bring other sensing moieties into cells for intracellular analysis. One early study was carried out by Xiao et al. (125), who reported a cell-specific internalization study of the DNA aptamer Sgc8. As previously described, aptamer Sgc8 was generated by cell-SELEX, and its target was later identified as protein kinase 7 (PTK7), a cell membrane protein. The efficiency and specificity of cellular internalization were carefully examined with the fluorescent dye-tagged Sgc8 aptamer. When both target and control cells were incubated with probes at 37◦ C, only target cells emitted fluorescence from both cell membrane and cytoplasm after 2 h of incubation. However, when cells were incubated at 4◦ C, fluorescence signals were observed only on the cell membrane, indicating that the aptamers were uptaken by endocytosis. Using an endosome-specific fluorescent probe, the authors reported that internalized aptamers were located inside the endosomes. In addition, aptamer Sgc8 shared the same intracellular distribution and internalization kinetics as the anti-PTK7 antibody. Moreover, cell viability was evaluated after internalization, and no cytotoxicity was observed, indicating that Sgc8 is a promising tool for cell type–specific intracellular delivery. Later, the same group (126) reported another aptamer identified from cell-SELEX against a breast cancer cell line for specific cell internalization. Real-time monitoring of disease-related protein dynamic processes is critical for understanding pathological process, as well as potential treatments. Chen and coworkers (127) have systematically www.annualreviews.org • Nucleic Acid Aptamers for Living Cell Analysis


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investigated aptamer-mediated NP internalization for site-specific protein labeling and cellular dynamic tracking of target proteins. The aptamer was employed not only as a ligand for protein recognition, but also as a linker to bring nanoreporters into cells. Three types of NPs, including gold NPs, silver NPs, and QDs, were successfully targeted to the membrane proteins of interest, and the receptor-dependent endocytic pathway was confirmed using different subcellular compartment markers. The movements of QDs in cells were tracked and analyzed, and three phases involved in endocytosis (membrane diffusion, vesicle transportation, and confined diffusion) were identified. Most importantly, these experiments proved the feasibility of the aptamer-directed NP-based strategy for cellular protein tracking and dynamic analysis in real time. In another example, Kim and coworkers (128) utilized an aptamer for targeted delivery of microRNA probes into cancer cells for both miRNA monitoring and cancer therapy. The aptamers and the microRNA probes were conjugated to fluorescence MNPs such that the microRNA probes were codelivered into cells with aptamer-targeted membrane proteins. The microRNA probe was linked to a dye molecule whose fluorescence was blocked by a short complementary sequence with a quencher. In addition, a disulfide linkage between the NP and the probe was introduced to facilitate the unloading of the microRNA probe once inside the cancer cells. Restoration of fluorescence was observed when the microRNA found its target, which competitively detached from the quencher sequence after incubation with target cells, indicating the successful delivery of molecular probes inside cells. The therapeutic effect of the microRNA probe was also examined.


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6. SUMMARY AND PERSPECTIVE Aptamers as chemical antibodies have become useful tools for studying living cells. The generation of aptamers by the SELEX process provides insight into the membrane features of different cells. Aptamers that recognize membrane protein targets served as high-affinity probes for unraveling receptor density and structure and for sensitive detection and isolation of cells. Depending on their cellular receptors, some aptamers are able to actively and specifically transport sensing moieties into cells. Real-time monitoring of extracellular and intracellular molecules has been realized with specific aptamer probes at the single-cell level. Various nanomaterials, such as NPs and GO, have played critical roles in accomplishing these analyses. However, aptamers are not surrogates for antibodies. One of the major concerns is that aptamers may be too specific and, as such, recognize only a specific isoform of a protein target. For example, because Macugen, the commercial name for the NX1838 aptamer, targets only the VEFG165 isoform, it is not as effective as anti-VEFG antibodies that target all isoforms of VEGF. Primarily for this reason, Macugen’s market share was overtaken by that of antibodies not long after its initial success (129). This feature is useful for studying the heterogeneity of cells, but it may cause problems in detection assays. One possible solution is the generation of aptamers for each isoform and the use of multiplexed aptamers in detection. Another concern arises from the stability of DNA and RNA aptamers, which are susceptible to nuclease degradation. Proper chemical modifications are needed, especially for cellular studies. Other than modifying aptamers after selection, non-natural bases may be incorporated into the selection process, both to improve stability and introduce library diversity (130). Moreover, aptamers selected from modified nucleotides possess higher affinities compared with normal DNA aptamers (131), further advancing the role of aptamers in measurement science.

DISCLOSURE STATEMENT The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review. 18.16

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Selective Targeting to Glioma with Nucleic Acid Aptamers.

Nucleic acid aptamers: research tools in disease diagnostics and therapeutics.

Selection of Nucleic Acid Aptamers Targeting Tumor Cell-Surface Protein Biomarkers.

Nucleic Acid aptamers: new methods for selection, stabilization, and application in biomedical science.

Nucleic Acid Aptamers: An Emerging Tool for Biotechnology and Biomedical Sensing.

Nucleic Acid Aptamers as Potential Therapeutic and Diagnostic Agents for Lymphoma.

Nucleic acid sequence analysis software for microcomputers.

In vitro evolution of chemically-modified nucleic acid aptamers: Pros and cons, and comprehensive selection strategies.

Nucleic acid aptamers stabilize proteins against different types of stress conditions.

A differential fluorescent receptor for nucleic acid analysis.

Selection, characterization and application of nucleic acid aptamers for the capture and detection of human norovirus strains.

Computer analysis of nucleic acid regulatory sequences.

Erratum: "Quick chip assay using locked nucleic acid modified epithelial cell adhesion molecule and nucleolin aptamers for the capture of circulating tumor cells" [Biomicrofluidics 9(5), 054110 (2015)].

Generating Cell Targeting Aptamers for Nanotheranostics Using Cell-SELEX.

Generation and characterization of nucleic acid aptamers targeting the capsid P domain of a human norovirus GII.4 strain.

Inhibition of Aggregation of Mutant Huntingtin by Nucleic Acid Aptamers In Vitro and in a Yeast Model of Huntington's Disease.

Nucleic Acid Ligands With Protein-like Side Chains: Modified Aptamers and Their Use as Diagnostic and Therapeutic Agents.

Gold nanoparticles for nucleic acid delivery.

Metal requirements for nucleic acid binding proteins.

Molecular modeling of nucleic Acid structure: setup and analysis.

Peptide-nucleic acid nanostructures for transfection.

Digital Microfluidics for Nucleic Acid Amplification.

Nucleic acid detection systems for enteroviruses.

The search for scrapie agent nucleic acid.