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.

Stimuli-Responsive DNA-Functionalized Metal-Organic Frameworks (MOFs).

The synthesis of nucleic acid-functionalized metal-organic frameworks (MOFs) is described. The metal-organic frameworks are loaded with a dye being locked in the structures by means of stimuli-responsive nucleic acid caps. The pH and K+ -ion-triggered release, and switchable release, are demonstrated.

Gossypol-Capped Mitoxantrone-Loaded Mesoporous SiO2 NPs for the Cooperative Controlled Release of Two Anti-Cancer Drugs.

Mesoporous SiO2 nanoparticles, MP-SiO2 NPs, are functionalized with the boronic acid ligand units. The pores of the MP-SiO2 NPs are loaded with the anticancer drug mitoxantrone, and the pores are capped with the anticancer drug gossypol. The resulting two-drug-functionalized MP-SiO2 NPs provide a potential stimuli-responsive anticancer drug carrier for cooperative chemotherapeutic treatment. In vitro experiments reveal that the MP-SiO2 NPs are unlocked under environmental conditions present in cancer cells, e.g., acidic pH and lactic acid overexpressed in cancer cells. The effective unlocking of the capping units under these conditions is attributed to the acidic hydrolysis of the boronate ester capping units and to the cooperative separation of the boronate ester bridges by the lactate ligand. The gossypol-capped mitoxantrone-loaded MP-SiO2 NPs reveals preferential cytotoxicity toward cancer cells and cooperative chemotherapeutic activities toward the cancer cells. The MCF-10A epithelial breast cells and the malignant MDA-MB-231 breast cancer cells treated with the gossypol-capped mitoxantrone-loaded MP-SiO2 NPs revealed after a time-interval of 5 days a cell death of ca. 8% and 60%, respectively. Also, the gossypol-capped mitoxantrone-loaded MP-SiO2 NPs revealed superior cancer-cell death (ca. 60%) as compared to control carriers consisting of ß-cyclodextrin-capped mitoxantrone-loaded (ca. 40%) under similar loading of the mitoxantrone drug. The drugs-loaded MP-SiO2 NPs reveal impressive long-term stabilities.

Addressing, amplifying and switching DNAzyme functions by electrochemically-triggered release of metal ions.

The design of artificial cells, which mimic the functions of native cells, is an ongoing scientific goal. The development of stimuli-responsive chemical systems that stimulate cascaded catalytic transformations, trigger chemical networks, and control vectorial branched transformations and dose-controlled processes, are the minimum requirements for mimicking cell functions. We have studied the electrochemical programmed release of ions from electrodes, which trigger selective DNAzyme-driven chemical reactions, cascaded reactions that self-assemble catalytic DNAzyme polymers, and the ON-OFF switching and dose-controlled operation of catalytic reactions. The addressable and potential-controlled release of Pb2+ or Ag+ ions into an electrolyte that includes a mixture of nucleic acids, results in the metal ion-guided selection of nucleic acids yielding the formation of specific DNAzymes, which stimulate orthogonal reactions or activate DNAzyme cascades.

Multiplexed analysis of genes and of metal ions using enzyme/DNAzyme amplification machineries.

The progressive development of amplified DNA sensors using nucleic acid-based machineries, involving the isothermal autonomous synthesis of the Mg(2+)-dependent DNAzyme, is used for the amplified, multiplexed analysis of genes (Smallpox, TP53) and metal ions (Ag(+), Hg(2+)). The DNA sensing machineries are based on the assembly of two sensing modules consisting of two nucleic acid scaffolds that include recognition sites for the two genes and replication tracks that yield the nicking domains for Nt.BbvCI and two different Mg(2+)-dependent DNAzyme sequences. In the presence of any of the genes or the genes together, their binding to the respective recognition sequences triggers the nicking/polymerization machineries, leading to the synthesis of two different Mg(2+)-dependent DNAzyme sequences. The cleavage of two different fluorophore/quencher-modified substrates by the respective DNAzymes leads to the fluorescence of F1 and/or F2 as readout signals for the detection of the genes. The detection limits for analyzing the Smallpox and TP53 genes correspond to 0.1 nM. Similarly, two different nucleic acid scaffolds that include Ag(+)-ions or Hg(2+)-ions recognition sequences and the replication tracks that yield the Nt.BbvCI nicking domains and the respective Mg(2+)-dependent DNAzyme sequences are implemented as nicking/replication machineries for the amplified, multiplexed analysis of the two ions, with detection limits corresponding to 1 nM. The ions sensing modules reveal selectivities dominated by the respective recognition sequences associated with the scaffolds.

Reversible Ag(+)-crosslinked DNA hydrogels.

DNA hydrogels, consisting of Y-shaped nucleic acid subunits or of nucleic acid-functionalized acrylamide chains, undergo switchable gel-to-solution transitions. The Ag(+)-stimulated formation of cytosine-Ag(+)-cytosine complexes results in the crosslinking of the units to yield the hydrogels, while the cysteamine-induced elimination of the Ag(+) ions dissociates the hydrogels into a solution phase.

Amplified and multiplexed detection of DNA using the dendritic rolling circle amplified synthesis of DNAzyme reporter units.

The amplified, highly sensitive detection of DNA using the dendritic rolling circle amplification (RCA) is introduced. The analytical platform includes a circular DNA and a structurally tailored hairpin structure. The circular nucleic acid template includes a recognition sequence for the analyte DNA (the Tay-Sachs mutant gene), a complementary sequence to the Mg(2+)-dependent DNAzyme, and a sequence identical to the loop region of the coadded hairpin structure. The functional hairpin in the system consists of the analyte-sequence that is caged in the stem region and a single-stranded loop domain that communicates with the RCA product. The analyte activates the RCA process, leading to DNA chains consisting of the Mg(2+)-dependent DNAzyme and sequences that are complementary to the loop of the functional hairpin structure. Opening of the coadded hairpin releases the caged analyte sequence, resulting in the dendritic RCA-induced synthesis of the Mg(2+)-dependent DNAzyme units. The DNAzyme-catalyzed cleavage of a fluorophore/quencher-modified substrate leads to a fluorescence readout signal. The method enabled the analysis of the target DNA with a detection limit corresponding to 1 aM. By the design of two different circular DNAs that include recognition sites for two different target genes, complementary sequences for two different Mg(2+)-dependent DNAzyme sequences and two different functional hairpin structures, the dendritic RCA-stimulated multiplexed analysis of two different genes is demonstrated. The amplified dendritic RCA detection of DNA is further implemented to yield the hemin/G-quadruplex horseradish peroxidase (HRP)-mimicking DNAzyme as catalytic labels that provide colorimetric or chemiluminescent readout signals.

Autonomous replication of nucleic acids by polymerization/nicking enzyme/DNAzyme cascades for the amplified detection of DNA and the aptamer-cocaine complex.

The progressive development of amplified DNA sensors and aptasensors using replication/nicking enzymes/DNAzyme machineries is described. The sensing platforms are based on the tailoring of a DNA template on which the recognition of the target DNA or the formation of the aptamer-substrate complex trigger on the autonomous isothermal replication/nicking processes and the displacement of a Mg(2+)-dependent DNAzyme that catalyzes the generation of a fluorophore-labeled nucleic acid acting as readout signal for the analyses. Three different DNA sensing configurations are described, where in the ultimate configuration the target sequence is incorporated into a nucleic acid blocker structure associated with the sensing template. The target-triggered isothermal autonomous replication/nicking process on the modified template results in the formation of the Mg(2+)-dependent DNAzyme tethered to a free strand consisting of the target sequence. This activates additional template units for the nucleic acid self-replication process, resulting in the ultrasensitive detection of the target DNA (detection limit 1 aM). Similarly, amplified aptamer-based sensing platforms for cocaine are developed along these concepts. The modification of the cocaine-detection template by the addition of a nucleic acid sequence that enables the autonomous secondary coupled activation of a polymerization/nicking machinery and DNAzyme generation path leads to an improved analysis of cocaine (detection limit 10 nM).