Kinetic analysis of site-specific photoaptamer-protein cross-linking

ssDNA oligonucleotides containing bromodeoxyuridine, BrdU-photoaptamers, are rapidly emerging as specific protein capture reagents in protein microarray technologies. A mathematical model for the kinetic analysis of photoaptamer-protein photocross-linking reactions is presented. The model is based on specific aptamer/protein binding followed by laser excitation that can lead to either covalent cross-linking of the photoaptamer and protein in the complex or irreversible photodamage to the aptamer. Two distinct kinetic regimes, (1) frozen and (2) rapid equilibrium, are developed analytically to model binding kinetics between laser pulses. The models are used to characterize the photocross-linking between three photoaptamers and their cognate protein targets; photoaptamers 0650 and 0615 cross-link human basic fibroblast growth factor and 0518 cross-links HIV MN envelope glycoprotein. Data for cross-linking reaction yields as a function of both laser energy dose and target protein concentration are analyzed for affinity constants and cross-link reaction rates. The binding dissociation constants derived from the cross-linking data are in good accord with independent measurements; the rapid equilibrium model appears to produce results more consistent with the experimental observations, although there is significant overlap between the two models for most conditions explored here. The rate of photodamage for 0615 and 0518 is 3.5 and 2.5 times that of the specific cross-link, giving low maximum reaction yields of approximately 20% and approximately 30%, whereas 0650 cross-links with a rate over five times higher than its photodamage rate and has a maximum reaction yield exceeding 80%. Quantum yields for the three systems are estimated from the data; photoaptamer 0650 has a reasonably high quantum yield of approximately 0.2 for protein cross-linking, while 0518 and 0615 have quantum yields of 0.07 and 0.02. The work presented here provides a useful set of metrics that allow for refinement of photoaptamer properties.     

Functional RNA microarrays for high-throughput screening of antiprotein aptamers

High-throughput methods for generating aptamer microarrays are described. As a proof-of-principle, the microarrays were used to screen the affinity and specificity of a pool of robotically selected antilysozyme RNA aptamers. Aptamers were transcribed in vitro in reactions supplemented with biotinyl-guanosine 5'-monophosphate, which led to the specific addition of a 5' biotin moiety, and then spotted on streptavidin-coated microarray slides. The aptamers captured target protein in a dose-dependent manner, with linear signal response ranges that covered seven orders of magnitude and a lower limit of detection of 1 pg/mL (70 fM). Aptamers on the microarray retained their specificity for target protein in the presence of a 10,000-fold (w/w) excess of T-4 cell lysate protein. The RNA aptamer microarrays performed comparably to current antibody microarrays and within the clinically relevant ranges of many disease biomarkers. These methods should also prove useful for generating other functional RNA microarrays, including arrays for genomic noncoding RNAs that bind proteins. Integrating RNA aptamer microarray production with the maturing technology for automated in vitro selection of antiprotein aptamers should result in the high-throughput production of proteome chips.     

Photoaptameric heterodimeric constructs as a new approach to enhance the efficiency of formation of photocrosslinks with a target protein

Using DNA aptamers selectively recognizing anion-binding exosites 1 and 2 of thrombin as a model, it has been demonstrated that their conjugation by a poly-(dT)-linker (ranging from 5 to 65 nucleotides (nt) in length) to produce aptamer heterodimeric constructs results into affinity enhancement. At the linker lengths ranged from 35 to 55 nt the apparent dissociation constants (K _D^app) measured using the optical biosensor Biacore-3000 for complexes of thrombin with the heterodimeric constructs reached minimum values (K _D^app) = 0.2–0.4 nM), which were approximately 30-fold less than for the complexes with the initial aptamers. A photoaptamer heterodimeric construct was designed connecting photoaptamer and aptamer sequences with the poly-(dT)-linker of 35 nt long. The photoaptamer used could form photo-induced cross-links with the exosite 2 of thrombin and the aptamer could bind to the exosite 1. The (K _D^app value for the photoaptamer construct was approximately 40-fold less than that for the primary photoaptamer (5.3 and 190 nM, respectively). Upon exposure of the equimolar mixtures of thrombin with the photoaptamer construct to the UV radiation at 308 nm the equal yield of the crosslinked complexes was observed at concentrations, which were lower by two orders of magnitude than in the case of the primary photoaptamer. It was found that concurrently with crosslinking to thrombin a photo-induced inactivation of the photoaptamer occurs presumably due to formation of the intermolecular crosslinking.     

Developing aptamer probes for acute myelogenous leukemia detection and surface protein biomarker discovery

Background

The majority of patients with acute myelogenous leukemia (AML) still die of their disease. In order to improve survival rates in AML patients, new strategies are necessary to discover biomarkers for the detection and targeted therapy of AML. One of the advantages of the aptamer-based technology is the unique cell-based selection process, which allows us to efficiently select for cell-specific aptamers without knowing which target molecules are present on the cell surface.

Methods

The NB4 AML cell line was used as the target cell population for selecting single stranded DNA aptamers. After determining the affinity of selected aptamers to leukocytes, the aptamers were used to phenotype human bone marrow leukocytes and AML cells in clinical specimens. Then a biotin-labelled aptamer was used to enrich and identify its target surface protein.

Results

Three new aptamers were characterized from the selected aptamer pools (JH6, JH19, and K19). All of them can selectively recognize myeloid cells with Kd in the low nanomole range (2.77 to 12.37 nM). The target of the biotin-labelled K19 aptamer probe was identified as Siglec-5, a surface membrane protein in low abundance whose expression can serve as a biomarker of granulocytic maturation and be used to phenotype AML. More importantly, Siglec-5 expression can be used to detect low concentrations of AML cells in human bone marrow specimens, and functions as a potential target for leukemic therapy.

Conclusions

We have demonstrated a pipeline approach for developing single stranded DNA aptamer probes, phenotyping AML cells in clinical specimens, and then identifying the aptamer-recognized target protein. The developed aptamer probes and identified Siglec-5 protein may potentially be used for leukemic cell detection and therapy in our future clinical practice.     

Diagnostic potential of PhotoSELEX-evolved ssDNA aptamers

High sensitivity and specificity of two modified ssDNA aptamers capable of photocross-linking recombinant human basic fibroblast growth factor (bFGF((155))) were demonstrated. The aptamers were identified through a novel, covalent, in vitro selection methodology called photochemical systematic evolution of ligands by exponential enrichment (PhotoSELEX). The aptamers exhibited high sensitivity for bFGF((155)) comparable with commercially available ELISA monoclonal antibodies with an absolute sensitivity of at least 0.058 ppt bFGF((155)) under prevailing test conditions. The aptamers exquisitely distinguished bFGF((155)) from consanguine proteins, vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF). A commercially viable diagnostic system incorporating PhotoSELEX-evolved aptamers capable of simultaneous quantification of a large number of analyte molecules is also described. Such a system benefits from covalent bonding of aptamer to target protein allowing vigorous washing with denaturants to improve signal to noise.     

Aptamers as tools for target validation

Synthetic nucleic acid ligands, called aptamers, bind to protein targets with high specificity and affinity. They are very potent inhibitors of protein function and their application can greatly enhance the process of target validation and drug development. An important benefit of this technology is the recent development of rapidly identifying these sophisticated ligands for almost any target molecule in multi-parallel, automated workstations. The aptamer technology is thus well-suited to addressing the growing demand for high-throughput analysis and functional validation of potential drug targets. Numerous examples have shown the potency of aptamers in inhibiting the function of proteins in cell culture and in vivo models. The technology is complementary to genetic knockout or siRNA approaches as it provides highly valuable information at the proteomic level. In addition, the aptamer technology has recently been extended to developing aptamer drugs and identifying functionally equivalent small molecule leads.     

Aptamer affinity chromatography:: combinatorial chemistry applied to protein purification

The systematic evolution of ligands by exponential enrichment process is a combinatorial chemistry method that allows the identification of specific oligonucleotide sequences, known as aptamers, that bind to a desired target molecule with high affinity and specificity. Here, a DNA-aptamer specific for human -selectin was immobilized to a chromatography support to create an affinity column. This column was effectively applied as either the first or second step in the purification of a recombinant human -selectin–Ig fusion protein from Chinese hamster ovary cell-conditioned medium. The fusion protein was efficiently bound to the column and efficiently eluted by gentle elution schemes. Application of the aptamer column as the initial purification step resulted in a 1500-fold purification with an 83% single step recovery. These results demonstrate that oligonucleotide aptamers can be effective affinity purification reagents.     

Facile Discovery of Cell-Surface Protein Targets of Cancer Cell Aptamers

Cancer biomarker discovery constitutes a frontier in cancer research. In recent years, cell-binding aptamers have become useful molecular probes for biomarker discovery. However, there are few successful examples, and the critical barrier resides in the identification of the cell-surface protein targets for the aptamers, where only a limited number of aptamer targets have been identified so far. Herein, we developed a universal SILAC-based quantitative proteomic method for target discovery of cell-binding aptamers. The method allowed for distinguishing specific aptamer-binding proteins from nonspecific proteins based on abundance ratios of proteins bound to aptamer-carrying bait and control bait. In addition, we employed fluorescently labeled aptamers for monitoring and optimizing the binding conditions. We were able to identify and validate selectin L and integrin α4 as the protein targets for two previously reported aptamers, Sgc-3b and Sgc-4e, respectively. This strategy should be generally applicable for the discovery of protein targets for other cell-binding aptamers, which will promote the applications of these aptamers.Cancer is the leading cause of morbidity and mortality worldwide, with ∼14 million new cases and 8.2 million cancer-related deaths in 2012, and the number of new cases is expected to rise by ∼ 70% over the next two decades (1). Individual tumors may have distinct molecular profiles emanating from genetic and epigenetic alterations along with the activation of complex signaling networks (2). The use of reliable cancer biomarkers for early detection, staging, and individualized therapy may improve patient care. Along this line, Anderson et al. (3) predicted the need of biomarker panels for the detection of multiple proteins for a complex disease like cancer. Nevertheless, the elucidation of molecular alterations of cancer cells is limited by the lack of effective probes that can identify and recognize the protein biomarkers for cancer cells.Aptamers are single-stranded DNA or RNA molecules evolved from random oligonucleotide libraries by repetitive binding of the oligonucleotides to target molecules, a process known as systematic evolution of ligands by exponential enrichment (SELEX)¹ (4, 5). Similar to antibodies, aptamers can bind to their target molecules with high affinity and specificity (4, 5). Additionally, a large number of aptamers exhibiting specific binding toward a variety of cells has been identified by employing cell-based SELEX (6). These aptamers can recognize the molecular signatures of certain types of cancer cells; thus, cell-surface protein targets of aptamers may serve as candidate biomarkers for these cells.Identification of the molecular targets of the cancer-cell-specific aptamers is a crucial step toward the revelation of the molecular signatures of cancer cells and the applications of the aptamers. Although recent studies have led to the selection of more than 100 cell aptamers, protein targets for only a very limited number of these aptamers have been identified (7), which greatly hampered their applications. In this vein, aptamer-target protein binding requires a native conformation of the aptamer. On the other hand, membrane proteins are hydrophobic, poorly soluble in water, and of relatively low abundance. Thus, the identification of target protein(s) for aptamers is a challenging task. Through extraction and affinity purification of proteins of cancer cells with the use of cell-recognition aptamers, protein tyrosine kinase 7 and Siglec-5 were identified as protein targets for aptamers that can bind to T-lineage acute lymphoblastic leukemia cells (8) and acute myelogenous leukemia cells (9), respectively. In addition, an aptamer-facilitated biomarker discovery method was developed for the identification of biomarkers of immature and mature dendritic cells (10). However, it remains difficult to identify biomarkers of low abundance. By employing cross-linking with the use of an aptamer harboring a photochemically activatable nucleoside, Mallikaratchy et al. (11) identified membrane-bound immunoglobin heavy mu chain as the cell-surface protein target for aptamer TD05. However, chemical modification of an aptamer may alter its binding property, and the method is labor-intensive, rendering it impractical for large-scale discovery of aptamer targets. Recently, the same group employed a formaldehyde-induced cross-linking method and identified stress-induced phosphoprotein 1 as a potential ovarian cancer biomarker (12); many proteins were identified by mass spectrometry, rendering it very difficult to ascertain which protein is the true aptamer target.Recently, rapid advances have been made for the identification and quantifications of proteins by mass spectrometry. Among the many quantitative proteomic methods, stable-isotope labeling by amino acids in cell culture (SILAC) is simple, efficient, and accurate, and it is also suitable for the quantitative analysis of membrane proteins (13, 14). In the present study, we set out to develop a SILAC-based quantitative proteomic approach to identify cell-surface target proteins of two previously reported cell aptamers, Sgc-3b and Sgc-4e (6, 15), and we were able to identify unique cell-surface proteins that can bind to the two aptamers.     

Use of DNA-Aptamers for Enrichment of Low Abundant Proteins in Cellular Extracts for Quantitative Detection by Selected Reaction Monitoring

The relationship between the amount of a target protein in a complex biological sample and its amount measured by selected reaction monitoring (SRM) mass spectrometry upon the affinity enrichment of the target protein with aptamers immobilized on a solid phase has been investigated. Human thrombin added in known concentrations to cellular extracts derived from bacterial cells was used as a model target protein. The affinity enrichment of thrombin in cellular extracts by means of the thrombin-binding aptamer immobilized on the surface of magnetic microbeads resulted in an approximately 10-fold increase of the concentration of the target protein and a 100-fold decrease of the low limit of a target protein concentration range where its quantitative detection by SRM was possible without interference from other peptides present in the tryptic digest.     

Probing high affinity sequences of DNA aptamer against VEGF165

Vascular endothelial growth factor (VEGF(165)) is a potent angiogenic mitogen commonly overexpressed in cancerous cells. It contains two main binding domains, the receptor-binding domain (RBD) and the heparin-binding domain (HBD). This study attempted to identify the specific sequences of the VEa5 DNA aptamer that exhibit high binding affinity towards the VEGF(165) protein by truncating the original VEa5 aptamer into different segments. Using surface plasmon resonance (SPR) spectroscopy for binding affinity analysis, one of the truncated aptamers showed a >200-fold increase in the binding affinity for HBD. This truncated aptamer also exhibited high specificity to HBD with negligible binding affinity for VEGF(121), an isoform of VEGF lacking HBD. Exposing colorectal cancer cells to the truncated aptamer sequence further confirmed the binding affinity and specificity of the aptamer to the target VEGF(165) protein. Hence, our approach of aptamer truncation can potentially be useful in identifying high affinity aptamer sequences for the biological molecules and targeting them as antagonist for cancer cell detection.     

Aptamer directly evolved from live cells recognizes membrane bound immunoglobin heavy mu chain in Burkitt's lymphoma cells

The identification of tumor related cell membrane protein targets is important in understanding tumor progression, the development of new diagnostic tools, and potentially for identifying new therapeutic targets. Here we present a novel strategy for identifying proteins that are altered in their expression levels in a diseased cell using cell specific aptamers. Using an intact viable B-cell Burkitt's lymphoma cell line (Ramos cells) as the target, we have selected aptamers that recognize cell membrane proteins with high affinity. Among the selected aptamers that showed different recognition patterns with different cell lines of leukemia, the aptamer TD05 showed binding with Ramos cells. By chemically modifying TD05 to covalently cross-link with its target on Ramos cells to capture and to enrich the target receptors using streptavidin coated magnetic beads followed by mass spectrometry, we were able to identify membrane bound immunoglobin heavy mu chain as the target for TD05 aptamer. Immunoglobin heavy mu chain is a major component of the B-cell antigen receptor, which is expressed in Burkitt's lymphoma cells. This study demonstrates that this two step strategy, the development of high quality aptamer probes and then the identification of their target proteins, can be used to discover new disease related potential markers and thus enhance tumor diagnosis and therapy. The aptamer based strategy will enable effective molecular elucidation of disease related biomarkers and other interesting molecules.     

Photoaptamer arrays applied to multiplexed proteomic analysis

Multiplexed photoaptamer-based arrays that allow for the simultaneous measurement of multiple proteins of interest in serum samples are described. Since photoaptamers covalently bind to their target analytes before fluorescent signal detection, the arrays can be vigorously washed to remove background proteins, providing the potential for superior signal-to-noise ratios and lower limits of quantification in biological matrices. Data are presented here for a 17-plex photoaptamer array exhibiting limits of detection below 10 fM for several analytes including interleukin-16, vascular endothelial growth factor, and endostatin and able to measure proteins in 10% serum samples. The assays are simple, scalable, and reproducible. Affinity of the capture reagent is shown to be directly correlated to the limit of detection for the analyte on the array.     

Using aptamers as capture reagents in bead-based assay systems for diagnostics and hit identification

Most applications of xMAP (Luminex) bead-based assay technology in diagnostics and drug discovery use immobilized antigens or antibodies. Here the authors describe the development of novel assay systems in which synthetic oligonucleotides that specifically bind and inhibit other biomolecules--so-called aptamers--are directly immobilized on beads. The robustness, specificity, and sensitivity of aptamer-based assays were demonstrated in a test system that detected human alpha-thrombin in serum samples. xMAP technology was also adapted to competitive screening formats where an aptamer/protein complex was disrupted by a functionally analogous competitor. The results indicate that such assays are excellently suited for diagnostic applications or drug screening, where aptamers serve as competitive binding probes for the identification of small-molecule hits. These methods should be transferable to a large number of applications because specific aptamers can be rapidly generated for almost any protein target.     

Rational design of modular allosteric aptamer sensor for label-free protein detection

An aptamer can be redesigned to new functional molecules by conjugating with other oligonucleotides. However, it requires experimental trials to optimize the conjugating module with the sensitivity and selectivity toward a target. To reduce these efforts, we report rationally-designed modular allosteric aptamer sensor (MAAS), which is composed of coupled two aptamers and the regulator. For label-free protein detection, the protein-aptamer was conjugated with the malachite green (MG) aptamer for signaling. The MAAS additionally has the regulator domain which is designed to hybridize to a protein binding domain. The regulator makes MAAS to be inactive by destructing the original structure of the two aptamers. However, its conformation becomes active by dissociating the hybridization from the protein recognition signal, thereby inducing the binding of MG emitting the enhanced fluorescence. The design of regulator is based on the thermodynamic energy difference by the RNA conformational change and protein-aptamer affinity. Here we first demonstrated the MAAS for hepatitis C helicase and replicase. The target proteins were detected up to 250nM with minimized blank signals and displayed high specificities 10-fold greater than in non-specific proteins. The MAAS provides valuable tools that can be adapted to a wide range of configurations in bioanalytical applications.     

Targeted delivery of chemotherapy agents using a liver cancer-specific aptamer

Background

Using antibody/aptamer-drug conjugates can be a promising method for decreasing toxicity, while increasing the efficiency of chemotherapy.

Methodology/Principal Findings

In this study, the antitumor agent Doxorubicin (Dox) was incorporated into the modified DNA aptamer TLS11a-GC, which specifically targets LH86, a human hepatocellular carcinoma cell line. Cell viability tests demonstrated that the TLS11a-GC-Dox conjugates exhibited both potency and target specificity. Importantly, intercalating Dox into the modified aptamer inhibited nonspecific uptake of membrane-permeable Dox to the non-target cell line. Since the conjugates are selective for cells that express higher amounts of target proteins, both criteria noted above are met, making TLS11a-GC-Dox conjugates potential candidates for targeted delivery to liver cancer cells.

Conclusions/Significance

Considering the large number of available aptamers that have specific targets for a wide variety of cancer cells, this novel aptamer-drug intercalation method will have promising implications for chemotherapeutics in general.     

H1 RNA polymerase III promoter-driven expression of an RNA aptamer leads to high-level inhibition of intracellular protein activity

Aptamers offer advantages over other oligonucleotide-based approaches that artificially interfere with target gene function due to their ability to bind protein products of these genes with high affinity and specificity. However, RNA aptamers are limited in their ability to target intracellular proteins since even nuclease-resistant aptamers do not efficiently enter the intracellular compartments. Moreover, attempts at expressing RNA aptamers within mammalian cells through vector-based approaches have been hampered by the presence of additional flanking sequences in expressed RNA aptamers, which may alter their functional conformation. In this report, we successfully expressed a ‘pure’ RNA aptamer specific for NF-κB p50 protein (A-p50) utilizing an adenoviral vector employing the H1 RNA polymerase III promoter. Binding of the expressed aptamer to its target and subsequent inhibition of NF-κB mediated intracellular events were demonstrated in human lung adenocarcinoma cells (A549), murine mammary carcinoma cells (4T1) as well as a human tumor xenograft model. This success highlights the promise of RNA aptamers to effectively target intracellular proteins for in vitro discovery and in vivo applications.     

Structural optimization of an aptamer generated from Ligand-Guided Selection (LIGS) resulted in high affinity variant toward mIgM expressed on Burkitt's lymphoma cell lines

Aptamers are synthetic, short nucleic acid molecules capable of specific target recognition. Aptamers are selected using a screening method termed Systematic Evolution of Ligands by Exponential enrichment (SELEX). We recently have introduced a variant of SELEX called “Ligand-Guided-Selection” (LIGS) that allows the identification of specific aptamers against known cell-surface proteins. Utilizing LIGS, we introduced three specific aptamers against membrane-bound IgM (mIgM), which is the hallmark of B cells. Out of the three aptamers selected against mIgM, an aptamer termed R1, in particular, was found to be interesting due to its ability to recognize mIgM on target cells and then block anti-IgM antibodies binding their antigen. We systematically truncated parent aptamer R1 to design shorter variants with enhanced affinity. Importantly, herein we show that the specificity of the most optimized variant of R1 aptamer is similar to that of anti-IgM antibody, indicating that the specificity of the ligand utilized in selective elution of the aptamer determines the specificity of the LIGS-generated aptamer. Furthermore, we report that truncated variants of R1 are able to recognize mIgM-positive human B lymphoma BJAB cells at physiological temperature, demonstrating that LIGS-generated aptamers could be re-optimized into higher affinity variants. Collectively, these findings show the significance of LIGS in generating highly specific aptamers with potential applications in biomedicine.     

High-affinity five/six-letter DNA aptamers with superior specificity enabling the detection of dengue NS1 protein variants beyond the serotype identification

Genetic alphabet expansion of DNA by introducing unnatural bases (UBs), as a fifth letter, dramatically augments the affinities of DNA aptamers that bind to target proteins. To determine whether UB-containing DNA (UB-DNA) aptamers obtained by affinity selection could spontaneously achieve high specificity, we have generated a series of UB-DNA aptamers (K_D: 27−182 pM) targeting each of four dengue non-structural protein 1 (DEN-NS1) serotypes. The specificity of each aptamer is remarkably high, and the aptamers can recognize the subtle variants of DEN-NS1 with at least 96.9% amino acid sequence identity, beyond the capability of serotype identification (69−80% sequence identities). Our UB-DNA aptamers specifically identified two major variants of dengue serotype 1 with 10-amino acid differences in the DEN-NS1 protein (352 aa) in Singaporeans’ clinical samples. These results suggest that the high-affinity UB-DNA aptamers generated by affinity selection also acquire high target specificity. Intriguingly, one of the aptamers contained two different UBs as fifth and sixth letters, which are essential for the tight binding to the target. These two types of unnatural bases with distinct physicochemical properties profoundly expand the potential of DNA aptamers. Detection methods incorporating the UB-DNA aptamers will facilitate precise diagnoses of viral infections and other diseases.