Multivalent Aptamers Research Articles

Facile supermolecular aptamer inhibitors of L-selectin.

Multivalent interactions occur frequently in nature, where they mediate high-affinity interactions between cells, proteins, or molecules. Here, we report on a method to generate multivalent aptamers (Multi-Aptamers) that target L-selectin function using rolling circle amplification (RCA). We find that the L-selectin Multi-Aptamers have increased affinity compared to the monovalent aptamer, are specific to L-selectin, and are capable of inhibiting interactions with endogenous ligands. In addition, the Multi-Aptamers efficiently inhibit L-selectin mediated dynamic adhesion in vitro and homing to secondary lymphoid tissues in vivo. Importantly, our method of generating multivalent materials using RCA avoids many of the challenges associated with current multivalent materials in that the Multi-Aptamers are high affinity, easily produced and modified, and biocompatible. We anticipate that the Multi-Aptamers can serve as a platform technology to modulate diverse cellular processes.

Nanostructural morphology master-regulated the cell capture efficiency of multivalent aptamers

The nanostructural features of stretched multivalent aptamers significantly improve the cell enrichment efficiency to about 16 fold higher than normal multivalent aptamers.

Multivalent aptamer-RNA based fluorescent probes for carrier-free detection of cellular microRNA-34a in mucin1-expressing cancer cells.

In this study, multivalent carrier-free aptamer-RNA based fluorescent probes (CF-probes) were designed as a simpler, more reliable, timesaving strategy for cellular miRNA detection. CF-probes spontaneously delivered into cells without the need for additional carriers and visualized target microRNA-34a specifically.

Research highlights: aptamers on a chip.

Aptamers, oligonucleic acid or peptide molecules with binding affinity to a specific molecule, have gained broad scientific attention due to their stability, ease of production and modification, and durability, and therefore have been employed in a wide range of applications both in basic research and clinical science. Recent advances in high-throughput sequencing are poised to revolutionize the selection of aptamers, while microfluidic and microarray approaches have helped automate these processes of selection. Here we highlight work addressing several challenges in using aptamers more widely. We discuss array-based discovery of multivalent aptamers which is used to develop high affinity or paired aptamers by cleverly selecting new aptamers that bind to previously aptamer-bound proteins. Other highlighted work is addressing problems in analyzing local cell secretions, as well as refreshable sensors that detect signals over hundreds of cycles and can be refreshed with DI water scavenged from the air, leading to less reagent storage towards wearable sensors.

Array-based discovery of aptamer pairs.

Affinity reagent pairs that recognize distinct epitopes on a target protein can greatly improve the sensitivity and specificity of molecular detection. Importantly, such pairs can be conjugated to generate reagents that achieve two-site “bidentate” target recognition, with affinities greatly exceeding either monovalent component. DNA aptamers are especially well-suited for such constructs, because they can be linked via standard synthesis techniques without requiring chemical conjugation. Unfortunately, aptamer pairs are difficult to generate, primarily because conventional selection methods preferentially yield aptamers that recognize a dominant “hot spot” epitope. Our array-based discovery platform for multivalent aptamers (AD-MAP) overcomes this problem to achieve efficient discovery of aptamer pairs. We use microfluidic selection and high-throughput sequencing to obtain an enriched pool of aptamer sequences. Next, we synthesize a custom array based on these sequences, and perform parallel affinity measurements to identify the highest-affinity aptamer for the target protein. We use this aptamer to form complexes that block the primary binding site on the target, and then screen the same array with these complexes to identify aptamers that bind secondary epitopes. We used AD-MAP to discover DNA aptamer pairs that bind distinct sites on human angiopoietin-2 with high affinities, even in undiluted serum. To the best of our knowledge, this is the first work to discover new aptamer pairs using arrays. We subsequently conjugated these aptamers with a flexible linker to construct ultra-high-affinity bidentate reagents, with equilibrium dissociation constants as low as 97 pM: >200-fold better than either component aptamer. Functional studies confirm that both aptamers critically contribute to this ultrahigh affinity, highlighting the promise of such reagents for research and clinical use.

Multivalent comb-type aptamer-siRNA conjugates for efficient and selective intracellular delivery.

In this study, a simple and efficient strategy for selective intracellular delivery of RNA therapeutics into target cancer cells was designed using direct complementary base pairing between chemically conjugated multimeric antisense strands and aptamer-incorporating sense strands.

A cancer cell-activatable aptamer-reporter system for one-step assay of circulating tumor cells.

The current antibody-mediated numeration assays of circulating tumor cells (CTCs) require multiple steps and are time-consuming. To overcome these technical limitations, a cancer cell-activatable aptamer-reporter was formulated by conjugating a biomarker-specific aptamer sequence with paired fluorochrome-quencher molecules. In contrast to the antibody probes, the intact aptamer-reporter was optically silent in the absence of cells of interest. However, when used in an assay, the aptamer selectively targeted cancer cells through interaction with a specific surface biomarker, which triggered internalization of the aptamer-reporter and, subsequently, into cell lysosomes. Rapid lysosomal degradation of the aptamer-reporter resulted in separation of the paired fluorochrome-quencher molecules. The released fluorochrome emitted bright fluorescent signals exclusively within the targeted cancer cells, with no background noise in the assay. Thus, the assays could be completed in a single step within minutes. By using this one-step assay, CTCs in whole blood and marrow aspirate samples of patients with lymphoma tumors were selectively highlighted and rapidly detected with no off-target signals from background blood cells. The development of the cancer cell-activatable aptamer-reporter system allows for the possibility of a simple and robust point-of-care test for CTC detection, which is currently unavailable.

Immunotherapy of CD30-expressing lymphoma using a highly stable ssDNA aptamer

CD30 is highly expressed on Hodgkins lymphoma and anaplastic large cell lymphoma, making it an attractive target for therapy. We describe the generation of serum-stabilized ssDNA aptamers that bind CD30 via a hybrid SELEX methodology. The selected aptamer bound CD30 with high affinity and specificity. Further optimization of the aptamer led to a short, truncated variant with a 50-fold higher affinity than its longer counterpart. The multivalent aptamer was able to induce oligomerization of CD30 receptors and, in effect, activate downstream signaling, which led to apoptosis of ALCL cells. Immunotherapy using aptamer-based co-stimulation provides an alternative to antibodies, and has potential to transform cancer treatment.

Multivalent DNA nanospheres for enhanced capture of cancer cells in microfluidic devices.

Isolation of circulating tumor cells (CTCs) from peripheral blood or cancer cells from bone marrow has significant applications in cancer diagnosis, therapy monitoring, and drug development. CTCs are cancer cells shed from primary tumors; they circulate in the bloodstream, leading to metastasis. The extraordinary rarity of CTCs in the bloodstream makes their isolation a significant technological challenge. Herein, we report the development of a platform combining multivalent DNA aptamer nanospheres with microfluidic devices for efficient isolation of cancer cells from blood. Gold nanoparticles (AuNPs) were used as an efficient platform for assembling a number of aptamers for high-efficiency cell capture. Up to 95 aptamers were attached onto each AuNP, resulting in enhanced molecular recognition capability. An increase of 39-fold in binding affinity was confirmed by flow cytometry for AuNP-aptamer conjugates (AuNP-aptamer) when compared with aptamer alone. With a laminar flow flat channel microfluidic device, the capture efficiency of human acute leukemia cells from a cell mixture in buffer increased from 49% using aptamer alone to 92% using AuNP-aptamer. We also employed AuNP-aptamer in a microfluidic device with herringbone mixing microstructures for isolation of leukemia cells in whole blood. The cell capture efficiency was also significantly increased with the AuNP-aptamer over aptamer alone, especially at high flow rates. Our results show that the platform combining DNA nanostructures with microfluidics has a great potential for sensitive isolation of CTCs and is promising for cancer diagnosis and prognosis.

A Systematic Study of the Features Critical for Designing a High Avidity Multivalent Aptamer

Macromolecular interactions are central to the regulation and execution of many key biological processes, and therefore, they are attractive targets for drug discovery. Previously, we identified an RNA aptamer for the heat shock factor (HSF1), which is capable of interfering with the binding of HSF1 to its cognate DNA elements. Here we report the significant enhancement of avidity through dimerization of this aptamer. In particular, we describe the effect of 2 factors in designing a multivalent aptamer: the distance between active subunits and the flexibility of the linkage.

CD28 Aptamers as Powerful Immune Response Modulators

CD28 is one of the main costimulatory receptors responsible for the proper activation of T lymphocytes. We have isolated two aptamers that bind to the CD28 receptor. As a monomer, one of them interfered with the binding of CD28 to its ligand (B7), precluding the costimulatory signal, whereas the other one was inactive. However, dimerization of any of the anti-CD28 aptamers was sufficient to provide an artificial costimulatory signal. No antibody has featured a dual function (i.e., the ability to work as agonist and antagonist) to date. Two different agonistic structures were engineered for each anti-CD28 aptamer. One showed remarkably improved costimulatory properties, surpassing the agonistic effect of an anti-CD28 antibody. Moreover, we showed in vivo that the CD28 agonistic aptamer is capable of enhancing the cellular immune response against a lymphoma idiotype and of prolonging survival of mice which receive the aptamer together with an idiotype vaccine. The CD28 aptamers described in this work could be used to modulate the immune response either blocking the interaction with B7 or enhancing vaccine-induced immune responses in cancer immunotherapy.

Selection is more intelligent than design: improving the affinity of a bivalent ligand through directed evolution

Multivalent molecular interactions can be exploited to dramatically enhance the performance of an affinity reagent. The enhancement in affinity and specificity achieved with a multivalent construct depends critically on the effectiveness of the scaffold that joins the ligands, as this determines their positions and orientations with respect to the target molecule. Currently, no generalizable design rules exist for construction of an optimal multivalent ligand for targets with known structures, and the design challenge remains an insurmountable obstacle for the large number of proteins whose structures are not known. As an alternative to such design-based strategies, we report here a directed evolution-based method for generating optimal bivalent aptamers. To demonstrate this approach, we fused two thrombin aptamers with a randomized DNA sequence and used a microfluidic in vitro selection strategy to isolate scaffolds with exceptionally high affinities. Within five rounds of selection, we generated a bivalent aptamer that binds thrombin with an apparent dissociation constant (Kd) <10 pM, representing a ∼200-fold improvement in binding affinity over the monomeric aptamers and a ∼15-fold improvement over the best designed bivalent construct. The process described here can be used to produce high-affinity multivalent aptamers and could potentially be adapted to other classes of biomolecules.

Selection strategy to generate aptamer pairs that bind to distinct sites on protein targets.

Many analytical techniques benefit greatly from the use of affinity reagent pairs, wherein each reagent recognizes a discrete binding site on a target. For example, antibody pairs have been widely used to dramatically increase the specificity of enzyme linked immunosorbent assays (ELISA). Nucleic acid-based aptamers offer many advantageous features relative to protein-based affinity reagents, including well-established chemical synthesis, thermostability, and low production cost. However, the generation of suitable aptamer pairs has posed a significant challenge, and few such pairs have been reported to date. To address this important challenge, we present multivalent aptamer isolation systematic evolution of ligands by exponential enrichment (MAI-SELEX), a technique designed for the efficient selection of aptamer pairs. In contrast to conventional selection methods, our method utilizes two selection modules to generate separate aptamer pools that recognize distinct binding sites on a single target. Using MAI-SELEX, we have isolated two groups of 2'-fluoro-modified RNA aptamers that specifically recognize the αV or β3 subunits of integrin αVβ3. These aptamers exhibit low nanomolar affinities for their targets, with minimal cross-reactivity to other closely related integrin homologues. Moreover, we show that these aptamer pairs do not interfere with each other's binding and effectively detect the target even in complex mixtures such as undiluted serum.

Investigation of the Interaction between a Bivalent Aptamer and Thrombin by AFM

Aptamers are a new class of molecular probes for protein recognition, detection, and inhibition. Multivalent aptamer-protein binding through aptamer assembly has been currently developed as an effective way to achieve higher protein affinity and selectivity. In this study, the specific interaction between bivalent aptamer Bi-8S and thrombin has been measured directly and quantitatively by atomic force microscopy to investigate the unbinding dynamics and dissociation energy landscape of the multivalent interaction. Bivalent aptamer Bi-8S contains thrombin's two aptamers, 15apt and 27apt, which are linked by eight spacer phosphoramidites. The results revealed the sequential dissociation of the two aptamers. Moreover, the dynamic force spectroscopy data revealed that the 27apt's binding to the thrombin remains largely unaffected by the eight-spacer phosphoramidites within Bi-8S. In contrast, the eight-spacer phosphoramidites stabilized the 15apt-thrombin binding.

Targeted Cell–Cell Interactions by DNA Nanoscaffold‐Templated Multivalent Bispecific Aptamers

Cell-cell interactions are essential for multicellular organisms, playing important roles in their development, function, and immunity. Herein a bottom-up strategy to construct self-assembled DNA nanostructures is reported, consisting of multivalent, bispecific, cell-targeting aptamers to specifically induce cell-cell interactions. Various DNA nanoscaffolds are rationally designed to assemble aptamers with different valencies and flexibilities, and their cellular binding capabilities are tested. Multivalent aptamers, assembled on more rigid scaffolds, display higher binding activities. Further, multivalent bispecific aptamer fusion molecules are constructed based on this configuration, and successfully link two types of cells. Using cell-targeting aptamers, the presented strategy eliminates the need to chemically modify cell surfaces and offers excellent cell specificity, binding efficiency, and stability. This proof-of-concept study establishes that multivalent bispecific aptamers linked on DNA-nanoscaffolds can mediate cellular engagement, which could lead to their use in directing or guiding cell-cell interactions in many biological events.

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.

Multivalent DNA Aptamer-Based Therapeutic Agents for Lymphoma and Leukemia.

Abstract 2711Poster Board II-687Non-Hodgkins lymphomas affect 450,000 patients in the United States and even with recent advances in antibody-based therapies; more than 20,000 people will die of their disease annually. The goal of this work was to develop a high affinity, stable aptamer selective for B-cell leukemias and lymphomas. Aptamers are small DNA molecules that have the ability to bind to proteins with high affinity and specificity. They are also ideal candidates as therapeutic carriers. Aptamer binding is based on the ability of small oligonucleotide polymers (typically 20–80mers) to fold into unique three-dimensional structures that can interact with a specific target. Based on nature of this interaction, aptamers could be considered to be antibody analogs. Compared to antibodies however, one of the inherent properties of aptamers is that their small size (typically 10–20,000 daltons for aptamers vs 150,000 daltons for antibodies,) might address some difficult pharmacologic issues of antibodies, which penetrate slowly into tumors and clear the blood slowly. Recently, the TDO5 aptamer was identified and it was found to bind to membrane bound human IgM, a component of the BCR complex in B-Cell neoplasms. In contrast to currently available monoclonal antibodies, TD05 binds to membrane bound human IgM only and not with soluble IgM, eliminating the possibility of competitive inhibition by soluble IgM in the serum. The specificity of the aptamer was confirmed by screening with 24 cell lines and fresh clinical leukemia samples. Out of 24 cell lines, the IgM-negative cells, including T-cell leukemias and solid tumor lines such as breast, kidney, and colon and ovarian, showed no binding with TD05 indicating there is no non-specific adhesion with cell lines. One of the challenges of using the current form of TD05 as a drug carrier is that it is not yet suitable for use in vivo because of low avidity (>10uM) and stability (t ½= 1min) at physiological conditions. In order to increase the affinity of this aptamer, a new truncated multivalent and nuclease stable aptamer was designed. First, truncation of the original version of TD05 was considered because reduced size may lead to more efficient chemical syntheses and better pharmacologic properties. The resulting TD05.1 has a 5-fold increased affinity compared to original version of TD05. Second, bivalent (BV) aptamers utilizing TD05.1 with various polyethylene glycol (PEG) linker lengths were designed. Linker length is critical in designing multivalent aptamers to avoid loss of binding due to steric hindrance and to optimize the binding geometry, both of which would affect binding affinity. An optimal linker length of ∼16nm was chosen after empiric binding studies. Nuclease stability was also addressed by the introduction of chemical functionalities into TD05.1. The introduction of non-natural bases such as LNA bases (Locked Nucleic Acids), have been demonstrated to be effective in this regard. The stability of LNA stems from the bicyclic furanose unit locked in a sugar conformation. In order to retain the specific recognition and 3-dimensional nature of the aptamer's folding, LNA analogues were only introduced at the regions that are not involved in binding. Additionally, to further improve nuclease resistance, increase circulation time in vivo and to prevent non-specific adhesion to serum proteins and cells, the LNA modified BV aptamer TD05.1 was modified with polyethylene glycol at the 3' and 5' ends. The introduction of LNA and polyethylene glycol further stabilized the secondary structure, increasing the affinity and nuclease resistance in serum from one minute to seven hours(t ½> 420 min). We then constructed a trivalent analog of the aptamer with multiple functionalities, including chelators and fluorophores, which showed ∼25-fold higher affinity compared to monomeric aptamer at physiological conditions. In conclusion, this study demonstrates the development of a trivalent, high affinity aptamer selective for the membrane bound human IgM found in B-cell leukemias and lymphoma. Studies to assess the biological activity and the use of this construct as a drug carrier to treat B-NHL and B-CLL are in progress. Disclosures:No relevant conflicts of interest to declare.

Therapeutic Applications of DNA and RNA Aptamers

Structured single-stranded nucleic acids, or aptamers, bind target molecules with high affinity and specificity, which translates into unique therapeutic possibilities. Currently, aptamers can be identified to most proteins, including blood-clotting factors, cell-surface receptors, and transcription factors. Chemical modifications to the oligonucleotides enhance their pharmacokinetics and pharmacodynamics, thus extending their therapeutic potential. Several aptamers have entered the clinical pipeline for applications and diseases such as macular degeneration, coronary artery bypass graft surgery, and various types of cancer. Furthermore, the functional repertoire of aptamers has expanded with the descriptions of multivalent agonistic aptamers and aptamers-siRNA chimeras. This review highlights those aptamers and aptamer-based approaches with particular likelihood of achieving therapeutic application.

Composite RNA aptamers as functional mimics of proteins

Individual RNA aptamers are often used to modulate the function of their target proteins, and multi-valent aptamers have been constructed to enhance their activity. To expand the utility of aptamers in manipulating and controlling biological processes, here we advance a general method for the design and construction of composite aptamers. The resulting molecular constructs resemble proteins in that they can form specific interactions with three or more different partners and be readily integrated into existing protein regulatory networks. As the first embodiment of this method, we created a tetra-valent aptamer that simultaneously binds to two molecules of the Drosophila protein B52 and two copies of streptavidin, thus mimicking the function of an antibody in immunochemical assays. We demonstrated that the performance of this ‘aptabody’ rivals that of a monoclonal antibody against B52 in these assays. While this study was performed in vitro and the composite aptamer we made was intended to mimic an existing protein, the same method can be used to accommodate arbitrary combinations of individual aptamers in composite molecular contexts, and these constructs can be delivered into living cells, where they are able to utilize existing cellular infrastructure for their production and processing.

Multivalent 4-1BB binding aptamers costimulate CD8+ T cells and inhibit tumor growth in mice

4-1BB is a major costimulatory receptor that promotes the survival and expansion of activated T cells. Administration of agonistic anti-4-1BB Abs has been previously shown to enhance tumor immunity in mice. Abs are cell-based products posing significant cost, manufacturing, and regulatory challenges. Aptamers are oligonucleotide-based ligands that exhibit specificity and avidity comparable to, or exceeding, that of Abs. To date, various aptamers have been shown to inhibit the function of their cognate target. Here, we have described the development of an aptamer that binds 4-1BB expressed on the surface of activated mouse T cells and shown that multivalent configurations of the aptamer costimulated T cell activation in vitro and mediated tumor rejection in mice. Because aptamers can be chemically synthesized, manufacturing and the regulatory approval process should be substantially simpler and less costly than for Abs. Agonistic aptamers could therefore represent a superior alternative to Abs for the therapeutic manipulation of the immune system.