Discovery of Aptamers Against Cell Surface Markers Using Ligand-Guided Selection.

Oligonucleotide ligands (DNA, RNA, or XNA), also known as aptamers, are selected against various target molecules using an iterative, evolutionary process called systematic evolution of ligands by exponential enrichment (SELEX). To select aptamers against complex cell surface proteins in their native state, a variant of SELEX termed ligand-guided selection (LIGS) was recently introduced. The significance of LIGS is rooted in its strategy of exploiting the selection step in SELEX to identify highly specific aptamers against known cell surface markers. Thus, in LIGS, a higher-affinity secondary ligand, such as a monoclonal antibody (mAb) to a whole-cell bound to an evolved SELEX library, is introduced to outcompete sequences against the mAb targeting cell surface protein or induce a conformational switch to destabilize the aptamer-surface cell surface protein resulting in elution of the sequences. Here, we describe the detailed method of LIGS utilized in identifying aptamers against T-cell receptor cluster of differentiation three complex (TCR-CD3) expressed in human T-cells and T-cell leukemia.

An In Vitro Selection Platform to Identify Multiple Aptamers against Multiple Cell-Surface Markers Using Ligand-Guided Selection.

Aptamer ligand discovery against multiple molecules expressed on whole cells is an essential component in molecular tool development. However, owing to their intrinsic structural characteristics, cell-surface receptors have proven to be challenging targets in ligand discovery. Several variants to systematic evolution of ligands by exponential enrichment (SELEX) have been introduced to address the ″target problem″ for aptamer screening. To this end, we introduced a variant of SELEX, termed ligand-guided selection (LIGS), to identify highly specific aptamers against complex cell-surface markers in their native state. So far, the application of LIGS has been aimed at identifying aptamers against the most dominant receptors on the cell surface. Here, we report that LIGS can be expanded to identify two receptors on the same cell surface, paving the way to generate a multiplexed ligand discovery platform based on SELEX-targeting membrane receptors in their native functional state. Using CD19 and CD20 expressed on Toledo cells as a model system, multiple aptamer families were evolved against Toledo cells. We then utilized two monoclonal antibodies (mAbs) against CD20 and CD19 to selectively partition specific aptamers against CD19 and CD20. Following biochemical characterization, we introduce two specific aptamers against CD19 and two specific aptamers against CD20 with high affinity. Multi-target LIGS, as reported here, demonstrates a successful combinatorial approach for nucleic acid library screening to generate multiple artificial nucleic acid ligands against multiple receptors expressed on a single cell.

Bispecific Aptamer Sensor toward T-Cell Leukemia Detection in the Tumor Microenvironment.

The current detection methods of malignant cells are mainly based on the high expression levels of certain surface proteins on these cells. However, many of the same surface marker proteins are also expressed in normal cells. Growing evidence suggests that the molecular signatures of the tumor microenvironment (TME) are related to the biological state of a diseased cell. Exploiting the unique molecular signature of the TME, we have designed a molecular sensing agent consisting of a molecular switch that can sense the elevated concentration of a small molecule in the TME and promote precise recognition of a malignant cell. We accomplished this by designing and developing a bispecific aptamer that takes advantage of a high concentration of adenosine 5'-triphosphate in the TME. Thus, we report a prototype of a bispecific aptamer molecule, which serves as a dual detection platform and recognizes tumor cells only when a given metabolite concentration is elevated in the TME. This system overcomes hurdles in detecting tumor cells solely based on the elevated expression of cell surface markers, providing a universal platform for tumor targeting and sensing.

Utility of Multivalent Aptamers to Develop Nanoscale DNA Devices against Surface Receptors.

DNA nanotechnology is undergoing rapid progress in the assembly of functional devices with biological relevance. In particular, currently, the research attention is more focused on the application of nanodevices at the interface of chemistry and biology, on the cell membrane where protein receptors communicate with the extracellular environment. This review explores the use of multivalent nucleic acid ligands termed aptamers in the design of DNA-based nanodevices to probe cellular interactions followed by a perspective on the untapped utility of XNA and UBP nanotechnology in designing functional nanomaterials with broader structural space.

A Homodimeric Aptamer Variant Generated from Ligand-Guided Selection Activates the T Cell Receptor Cluster of Differentiation 3 Complex.

Recently, immunotherapeutic modalities with engineered cells and monoclonal antibodies have been effective in treating several malignancies. Nucleic acid aptamers can serve as alternative molecules to design immunotherapeutic agents with high functional diversity. Here we report a synthetic prototype consisting of DNA aptamers that can activate the T cell receptor cluster of differentiation 3 (TCR-CD3) complex in cultured T cells. We show that the activation potential is similar to that of a monoclonal antibody (mAb) against TCR-CD3, suggesting potential for aptamers in developing efficacious synthetic immunomodulators. The synthetic prototype of anti-TCR-CD3ε, as described here, was designed using aptamer ZUCH-1 against TCR-CD3ε, generated by ligand-guided selection (LIGS). Aptamer ZUCH-1 was truncated and modified with nuclease-resistant RNA analogs to enhance stability. Several dimeric analogs with truncated and modified variants were designed with variable linker lengths to investigate the activation potential of each construct. Among them, a dimeric aptamer with dimensions approximately similar to those of an antibody showed the highest T cell activation, suggesting the importance of optimizing linker lengths in engineering functional aptamers. The observed activation potential of dimeric aptamers shows the vast potential of aptamers in designing synthetically versatile immunomodulators with tunable pharmacokinetic properties, expanding immunotherapeutic designs by using nucleic acid-based ligands such as aptamers.

Ligand Guided Selection (LIGS) of Artificial Nucleic Acid Ligands against Cell Surface Targets.

With the success of RNA-based therapeutic drugs, the demand has increased for sophisticated nucleic-acid-based targeting agents. Nucleic acid aptamers (NAAs), in this regard, represent a suitable class of molecules with synthetic versatility. Aptamers are composed of single-stranded RNA/DNA/XNA molecules, which can be identified using a method called systematic evolution of ligands by exponential enrichment (SELEX) against any molecule. This Spotlight summarizes the recent introduction of ligand guided selection (LIGS), which will permit the identification of a wide range of functional aptamers against complex targets such as cell surface receptors while maintaining their native functional state. Aptamers identified from LIGS will allow researchers to develop aptamers in biomedicine as low-cost, stable therapeutic agents and diagnostic molecules or biochemical devices.

Ligand-Guided Selection with Artificially Expanded Genetic Information Systems against TCR-CD3ε.

Here we are reporting, for the first time, a ligand-guided selection (LIGS) experiment using an artificially expanded genetic information system (AEGIS) to successfully identify an AEGIS-DNA aptamer against T cell receptor-CD3ε expressed on Jurkat.E6 cells. Thus, we have effectively combined the enhanced diversity of an AEGIS DNA library with LIGS to develop a superior screening platform to discover superior aptamers. Libraries of DNA molecules from highly diversified building blocks will provide better ligands due to more functional diversity and better-controlled folding. Thus, a DNA library with AEGIS components (dZ and dP) was used in LIGS experiments against TCR-CD3ε in its native state using two clinically relevant monoclonal antibodies to identify an aptamer termed JZPO-10, with nanomolar affinity. Multiple specificity assays using knockout cells, and competition experiments using monoclonal antibodies utilized in LIGS, show unprecedented specificity of JZPO-10, suggesting that the combination of LIGS with AEGIS-DNA libraries will provide a superior screening platform to discover artificial ligands against critical cellular targets.

Integrating Ligand-Receptor Interactions and In Vitro Evolution for Streamlined Discovery of Artificial Nucleic Acid Ligands.

To discover DNA ligands against a predetermined receptor protein complex, we introduce a comprehensive version of ligand-guided selection (LIGS). LIGS is, itself, a variant of systematic evolution of ligands by exponential enrichment (SELEX). Herein, we have optimized LIGS to identify higher affinity aptamers with high specificity. In addition, we demonstrate the expandability of LIGS by performing specific aptamer elution at 25°C, utilizing multiple monoclonal antibodies (mAbs) against cultured cells and primary cells obtained from human donors expressing the same receptor. Eluted LIGS libraries obtained through Illumina high-throughput (HT) DNA sequencing were analyzed by bioinformatics tools to discover five DNA aptamers with apparent affinities ranging from 3.06 ± 0.485 nM to 325 ± 62.7 nM against the target, T cell receptor-cluster of differentiation epsilon (TCR-CD3ε) expressed on human T cells. The specificity of the aptamers was validated utilizing multiple strategies, including competitive binding analysis and a double-knockout Jurkat cell line generated by CRISPR technology. The cross-competition experiments using labeled and unlabeled aptamers revealed that all five aptamers compete for the same binding site. Collectively, the data in this report introduce a modified LIGS strategy as a universal platform to identify highly specific multiple aptamers toward multi-component receptor proteins in their native state without changing the cell-surface landscape.

The role of G-quadruplex structures of LIGS-generated aptamers R1.2 and R1.3 in IgM specific recognition.

Exploiting a variant of SELEX called "Ligand-Guided Selection" (LI-GS), we recently identified two novel truncated G-rich aptamers, called R1.2 and R1.3, specific for membrane-bound IgM (mIgM), the hallmark of B cells. Herein, the conformational behaviour of these aptamers has been analysed by multiple biophysical methods. In order to investigate their functional secondary structures, these studies have been carried out in pseudo-physiological buffers mimicking different cellular environments. Both aptamers proved to be highly polymorphic, folding into stable, unimolecular G-quadruplex structures in K+-rich buffers. In turn, in buffered solutions containing Na+/Mg2+ ions, R1.2 and R1.3 formed mainly duplex structures. Remarkably, these aptamers were able to effectively bind mIgM on B-cell lymphoma exclusively in the presence of potassium ions. These findings demonstrate the key role of G-quadruplex folding in the molecular recognition and efficient binding of R1.2 and R1.3 to mIgM expressed in lymphoma and leukemia cells, providing a precious rational basis for the design of effective aptamer-based biosensors potentially useful for the detection of cancer-relevant biomarkers.

Synthesis of stable azide and alkyne functionalized phosphoramidite nucleosides.

The use of CuAAC chemistry to crosslink and stabilize oligonucleotides has been limited by the incompatibility of azides with the phosphoramidites used in automated oligonucleotide synthesis. Herein we report optimized reaction conditions to synthesize azide derivatives of thymidine and cytidine phosphoramidites. Investigation of the stability of the novel phosphoramidites using 31P NMR at room temperature showed less than 10% degradation after 6 hours. The azide modified thymidine was successfully utilized as an internal modifier in the standard phosphoramidite synthesis of a DNA sequence. The synthesized azide and alkyne derivatives of pyrimidines will allow efficient incorporation of azide and alkyne click pairs into nucleic acids, thus widening the applicability of click chemistry in investigating the chemistry of nucleic acids.

Dimerization of an aptamer generated from Ligand-guided selection (LIGS) yields a high affinity scaffold against B-cells.

Nucleic Acid Aptamers (NAAs) are a class of synthetic DNA or RNA molecules that bind specifically to their target. We recently introduced an aptamer termed R1.2 against membrane Immunoglobulin M (mIgM) expressing B-cell neoplasms using Ligand Guided Selection (LIGS). While LIGS-generated aptamers are highly specific, their lower affinity prevents aptamers from being used for translational applications. Highly specific aptamers with higher affinity can increase targetability, boosting the application of aptamers as diagnostic and therapeutic molecules. Herein, we report that dimerization of R1.2, an aptamer generated from LIGS, leads to high affinity variants without compromising the specificity. Three dimeric aptamer analogues with variable linker lengths were designed to evaluate the effect of linker length in affinity. The optimized dimeric R1.2 against cultured B-cell neoplasms, four donor B-cell samples and mIgM-positive Waldenström's Macroglobulinemia (WM) showed specificity. Furthermore, confocal imaging of dimeric aptamer and anti-IgM antibody in purified B-cells suggests co-localization. Binding assays against IgM knockout Burkitt's Lymphoma cells utilizing CRISPR/Cas9 further validated specificity of dimeric R1.2. Collectively, our findings show that LIGS-generated aptamers can be re-engineered into dimeric aptamers with high specificity and affinity, demonstrating wide-range of applicability of LIGS in developing clinically practical diagnostic and therapeutic aptamers.

Systematic optimization and modification of a DNA aptamer with 2'-O-methyl RNA analogues.

Nucleic acid aptamers (NAAs) are short synthetic DNA or RNA molecules that specifically fold into distinct three-dimensional structures able to specifically recognize a target. While NAAs show unprecedented promise in a variety of applications, including sensing, therapeutics and diagnostics, one major limitation involves the lack of stability towards omnipresent nucleases. Therefore, we herein report a systematic truncation and incorporation of 2'-O-methyl bases to a DNA aptamer, which results in increased stability without affecting affinity. One of the newly designed analogues is stable up to 24 hours, demonstrating that 2'-O-methyl RNA is an attractive modification to DNA aptamers, especially when therapeutic applications are intended.

Engineered Aptamers to Probe Molecular Interactions on the Cell Surface.

Significant progress has been made in understanding the nature of molecular interactions on the cell membrane. To decipher such interactions, molecular scaffolds can be engineered as a tool to modulate these events as they occur on the cell membrane. To guarantee reliability, scaffolds that function as modulators of cell membrane events must be coupled to a targeting moiety with superior chemical versatility. In this regard, nucleic acid aptamers are a suitable class of targeting moieties. Aptamers are inherently chemical in nature, allowing extensive site-specific chemical modification to engineer sensing molecules. Aptamers can be easily selected using a simple laboratory-based in vitro evolution method enabling the design and development of aptamer-based functional molecular scaffolds against wide range of cell surface molecules. This article reviews the application of aptamers as monitors and modulators of molecular interactions on the mammalian cell surface with the aim of increasing our understanding of cell-surface receptor response to external stimuli. The information gained from these types of studies could eventually prove useful in engineering improved medical diagnostics and therapeutics.

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.

Evolution of Complex Target SELEX to Identify Aptamers against Mammalian Cell-Surface Antigens.

The demand has increased for sophisticated molecular tools with improved detection limits. Such molecules should be simple in structure, yet stable enough for clinical applications. Nucleic acid aptamers (NAAs) represent a class of molecules able to meet this demand. In particular, aptamers, a class of small nucleic acid ligands that are composed of single-stranded modified/unmodified RNA/DNA molecules, can be evolved from a complex library using Systematic Evolution of Ligands by EXponential enrichment (SELEX) against almost any molecule. Since its introduction in 1990, in stages, SELEX technology has itself undergone several modifications, improving selection and broadening the repertoire of targets. This review summarizes these milestones that have pushed the field forward, allowing researchers to generate aptamers that can potentially be applied as therapeutic and diagnostic agents.

Ligand-guided selection of aptamers against T-cell Receptor-cluster of differentiation 3 (TCR-CD3) expressed on Jurkat.E6 cells.

We recently introduced a screening technology termed ligand-guided selection, (LIGS), to selectively identify target-specific aptamers from an evolved cell-SELEX library. Cell-SELEX utilizes a large combinatorial single-stranded oligonucleotide library and progressively selects DNA ligands against whole cells with variable DNA-binding affinities and specificities by repeated rounds of partition and amplification. LIGS exploits the partition step and introduces a secondary, pre-existing high-affinity monoclonal antibody (mAb) ligand to outcompete and elute specific aptamers towards the binding target of the antibody, not the cell. Here, using anti-CD3ε mAb against the cluster of differentiation 3 (CD3ε), as the guiding ligand against one of the domains of the T-cell Receptor (TCR) complex expressed on Jurkat.E6 cells, we discovered three specific aptamers against TCR complex expressed on an immortalized line of human T lymphocyte cells. In sum, we demonstrate that specific aptamers can be identified utilizing an antibody against a single domain of a multidomain protein complex in their endogenous state with neither post- nor pre-SELEX protein manipulation.

Ligand-Guided Selection of Target-Specific Aptamers: A Screening Technology for Identifying Specific Aptamers Against Cell-Surface Proteins.

We report on a new strategy for identifying highly specific aptamers against a predetermined epitope of a target. Termed "ligand-guided selection" (LIGS), this method uniquely exploits the selection step, the core of SELEX (Systematic Evolution Exponential enrichment). LIGS uses a naturally occurring stronger and highly specific bivalent binder, an antibody (Ab) interacting with its cognate antigen to outcompete specific aptamers from a partially enriched SELEX pool, as a strategy. We demonstrate the hypothesis of LIGS by utilizing an Ab binding to membrane-bound Immunoglobulin M (mIgM) to selectively elute aptamers that are specific for mIgM from a SELEX pool that is partially enriched toward mIgM expressing Ramos cells. The selected aptamers show specificity toward Ramos cells. We identified three aptamer candidates utilizing LIGS that could be outcompeted by mIgM Ab, demonstrating that LIGS can be successfully applied to select aptamers from a partially evolved cell-SELEX library, against predetermined receptor proteins using a cognate ligand. This proof-of-concept study introduces a new biochemical-screening platform that exploits the binding of a secondary stronger molecular entity to its target as a partition step, to identify highly specific artificial nucleic acid ligands.

A self-assembling short oligonucleotide duplex suitable for pretargeting.

Monoclonal antibodies (mAbs) have naturally evolved as suitable, high affinity and specificity targeting molecules. However, the large size of full-length mAbs yields poor pharmacokinetic properties. A solution to this issue is the use of a multistep administration approach, in which the slower clearing mAb is administered first and allowed to reach the target site selectively, followed by administration of a rapidly clearing small molecule carrier of the cytotoxic or imaging ligand, which bears a cognate receptor for the mAb. Here, we introduce a novel pretargetable RNA based system comprised of locked nucleic acids (LNA) and 2'O-Methyloligoribonucleotides (2'OMe-RNA). The duplex shows fast hybridization, high melting temperatures, excellent affinity, and high nuclease stability in plasma. Using a prototype model system with rituximab conjugated to 2'OMe-RNA (oligo), we demonstrate that LNA-based complementary strand (c-oligo) effectively hybridizes with rituximab-oligo, which is slowly circulating in vivo, despite the high clearance rates of c-oligo.

A multivalent DNA aptamer specific for the B-cell receptor on human lymphoma and leukemia.

Long-term survival still eludes most patients with leukemia and non-Hodgkin's lymphoma. No approved therapies target the hallmark of the B cell, its mIgM, also known as the B-cell receptor (BCR). Aptamers are small oligonucleotides that can specifically bind to a wide range of target molecules and offer some advantages over antibodies as therapeutic agents. Here, we report the rational engineering of aptamer TD05 into multimeric forms reactive with the BCR that may be useful in biomedical applications. Systematic truncation of TD05 coupled with modification with locked nucleic acids (LNA) increased conformational stability and nuclease resistance. Trimeric and tetrameric versions with optimized polyethyleneglycol (PEG) linker lengths exhibited high avidity at physiological temperatures both in vitro and in vivo. Competition and protease studies showed that the multimeric, optimized aptamer bound to membrane-associated human mIgM, but not with soluble IgM in plasma, allowing the possibility of targeting leukemias and lymphomas in vivo. The B-cell specificity of the multivalent aptamer was confirmed on lymphoma cell lines and fresh clinical leukemia samples. The chemically engineered aptamers, with significantly improved kinetic and biochemical features, unique specificity and desirable pharmacological properties, may be useful in biomedical applications.