Contributing to the management of viral infections through simple immunosensing of the arachidonic acid serum level.

A trendsetting direct competitive-based biosensing tool has been developed and implemented for the determination of the polyunsaturated fatty acid arachidonic acid (ARA), a highly significant biological regulator with decisive roles in viral infections. The designed methodology involves a competitive reaction between the target endogenous ARA and a biotin-ARA competitor for the recognition sites of anti-ARA antibodies covalently attached to the surface of carboxylic acid-coated magnetic microbeads (HOOC-MµBs), followed by the enzymatic label of the biotin-ARA residues with streptavidin-horseradish peroxidase (Strep-HRP) conjugate. The resulting bioconjugates were magnetically trapped onto the sensing surface of disposable screen-printed carbon transducers (SPCEs) to monitor the extent of the biorecognition reaction through amperometry. The operational functioning of the exhaustively optimized and characterized immunosensing bioplatform was highly convenient for the quantitative determination of ARA in serum samples from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2-) and respiratory syncytial virus (RSV)-infected individuals in a rapid, affordable, trustful, and sensitive manner.

Self-assembly of protein-DNA hybrids dedicated to an accelerated and self-primed strand displacement amplification for reinforced serum microRNA probing.

BACKGROUND: High-efficiency and highly reliable analysis of microRNAs (miRNAs) in bodily fluids highlights its significance to be extensively utilized as candidates for non-invasive "liquid biopsy" approaches. DNA biosensors based on strand displacement amplification (SDA) methods have been successfully designed to detect miRNAs given the efficiently amplified and recycled of the target sequences. However, the unpredictable DNA framework and heavy reliance on free diffusion or random reactant collisions in existing approaches lead to delayed reaction kinetics and inadequate amplification. Thus, it is crucial to create a modular probe with a controlled structure, high local concentration, and ease of synthesis. RESULTS: Inspired by the natural spatial-confinement effect based on a well-known streptavidin-biotin interaction, we constructed a protein-DNA hybrid, named protein-scaffolded DNA tetrads (PDT), which consists of four biotinylated Y-shaped DNA (Y-DNA) surrounding a streptavidin protein center via a streptavidin-biotin bridge. The streptavidin-biotin recognition system significantly increased the local concentration and intermolecular distance of the probes to achieve enhanced reaction efficiency and kinetics. The PDT-based assay starts with the target miRNA binding to Y-DNA, which disassembles the Y-DNA structures into three types of hairpin-shaped structures via self-primed strand displacement amplification (SPSDA) and generates remarkable fluorescence signal that is proportional to the miRNA concentration. Results demonstrated that PDT enabled a more efficient detection of miRNA-21 with a sensitivity of 1 fM. Moreover, it was proven reliable for the detection of clinical serum samples, suggesting great potential for advancing the development of rapid and robust signal amplification technologies for early diagnosis. SIGNIFICANCE: This simple yet robust system contributes to the early diagnosis of miR-21 with satisfactory sensitivity and specificity, and display a significantly improved nuclease resistance owing to their unique structure. The results suggested that the strategy is expected to provide a promising potential platform for tumor diagnosis, prognosis and therapy.

Construction of a streptavidin-based dual-localized DNAzyme walker for disease biomarker detection.

A dual-localized DNAzyme walker (dlDW) was constructed by utilizing multiple split DNAzymes with probes, and their substrates are separately localized on streptavidin and AuNPs, serving as walking pedals and tracks, respectively. Based on dlDW, biosensing platform was successfully constructed and showed great potential application in clinical disease diagnosis.

Controlled In Situ Self-Assembly of Biotinylated Trans-Cyclooctene Nanoparticles for Orthogonal Dual-Pretargeted Near-Infrared Fluorescence and Magnetic Resonance Imaging.

A pretargeted strategy that decouples targeting vectors from radionuclides has shown promise for nuclear imaging and/or therapy in vivo. However, the current pretargeted approach relies on the use of antibodies or nanoparticles as the targeting vectors, which may be compromised by poor tissue penetration and limited accumulation of targeting vectors in the tumor tissues. Herein, we present an orthogonal dual-pretargeted approach by combining stimuli-triggered in situ self-assembly strategy with fast inverse electron demand Diels-Alder (IEDDA) reaction and strong biotin-streptavidin (SA) interaction for near-infrared fluorescence (NIR FL) and magnetic resonance (MR) imaging of tumors. This approach uses a small-molecule probe (P-Cy-TCO&Bio) containing both biotin and trans-cyclooctene (TCO) as a tumor-targeting vector. P-Cy-TCO&Bio can efficiently penetrate subcutaneous HeLa tumors through biotin-assisted targeted delivery and undergo in situ self-assembly to form biotinylated TCO-bearing nanoparticles (Cy-TCO&Bio NPs) on tumor cell membranes. Cy-TCO&Bio NPs exhibited an "off-on" NIR FL and retained in the tumors, offering a high density of TCO and biotin groups for the concurrent capture of Gd-chelate-labeled tetrazine (Tz-Gd) and IR780-labeled SA (SA-780) via the orthogonal IEDDA reaction and SA-biotin interaction. Moreover, Cy-TCO&Bio NPs offered multiple-valent binding modes toward SA, which additionally regulated the cross-linking of Cy-Gd&Bio NPs into microparticles (Cy-Gd&Bio/SA MPs). This process could significantly (1) increase r1 relaxivity and (2) enhance the accumulation of Tz-Gd and SA-780 in the tumors, resulting in strong NIR FL, bright MR contrast, and an extended time window for the clear and precise imaging of HeLa tumors.

Interface-constrained catalytic hairpin assembly permits highly sensitive SERS signaling of miRNA.

The rapid and precise monitoring of peripheral blood miRNA levels holds paramount importance for disease diagnosis and treatment monitoring. In this study, we propose an innovative research strategy that combines the catalytic hairpin assembly reaction with SERS signal congregation and enhancement. This combination can significantly enhance the stability of SERS detection, enabling stable and efficient detection of miRNA. Specifically, our paper-based SERS detection platform incorporates a streptavidin-modified substrate, biotin-labeled catalytic hairpin assembly reaction probes, 4-ATP, and primer-co-modified gold nanoparticles. In the presence of miRNA, the 4-ATP and primer-co-modified gold nanoparticles can specifically recognize the miRNA and interact with the biotin-labeled CHA probes to initiate an interfacial catalytic hairpin assembly reaction. This enzyme-free high-efficiency catalytic process can accumulate a large amount of biotin on the gold nanoparticles, which then bind to the streptavidin on the substrate with the assistance of the driving liquid, forming red gold nanoparticle stripes. These provide a multitude of hotspots for SERS, enabling enhanced signal detection. This innovative design achieves a low detection limit of 3.47 fM while maintaining excellent stability and repeatability. This conceptually innovative detection platform offers new technological possibilities and solutions for clinical miRNA detection.

Combining the Benefits of Biotin-Streptavidin Aptamer Immobilization with the Versatility of Ni-NTA Regeneration Strategies for SPR.

The high affinity of the biotin-streptavidin interaction has made this non-covalent coupling an indispensable strategy for the immobilization and enrichment of biomolecular affinity reagents. However, the irreversible nature of the biotin-streptavidin bond renders surfaces functionalized using this strategy permanently modified and not amenable to regeneration strategies that could increase assay reusability and throughput. To increase the utility of biotinylated targets, we here introduce a method for reversibly immobilizing biotinylated thrombin-binding aptamers onto a Ni-nitrilotriacetic acid (Ni-NTA) sensor chip using 6xHis-tagged streptavidin as a regenerable capture ligand. This approach enabled the reproducible immobilization of aptamers and measurements of aptamer-protein interaction in a surface plasmon resonance assay. The immobilized aptamer surface was stable during five experiments over two days, despite the reversible attachment of 6xHis-streptavidin to the Ni-NTA surface. In addition, we demonstrate the reproducibility of this immobilization method and the affinity assays performed using it. Finally, we verify the specificity of the biotin tag-streptavidin interaction and assess the efficiency of a straightforward method to regenerate and reuse the surface. The method described here will allow researchers to leverage the versatility and stability of the biotin-streptavidin interaction while increasing throughput and improving assay efficiency.

Advancing MicroRNA Detection: Enhanced Biotin-Streptavidin Dual-Mode Phase Imaging Surface Plasmon Resonance Aptasensor.

MicroRNAs (miRNAs) are novel tumor biomarkers owing to their important physiological functions in cell communication and the progression of multiple diseases. Due to the small molecular weight, short sequence length, and low concentration levels of miRNA, miRNA detection presents substantial challenges, requiring the advancement of more refined and sensitive techniques. There is an urgent demand for the development of a rapid, user-friendly, and sensitive miRNA analysis method. Here, we developed an enhanced biotin-streptavidin dual-mode phase imaging surface plasmon resonance (PI-SPR) aptasensor for sensitive and rapid detection of miRNA. Initially, we evaluated the linear sensing range for miRNA detection across two distinct sensing modalities and investigated the physical factors that influence the sensing signal in the aptamer-miRNA interaction within the PI-SPR aptasensor. Then, an enhanced biotin-streptavidin amplification strategy was introduced in the PI-SPR aptasensor, which effectively reduced the nonspecific adsorption by 20% and improved the limit of detection by 548 times. Furthermore, we have produced three types of tumor marker chips, which utilize the rapid sensing mode (less than 2 min) of PI-SPR aptasensor to achieve simultaneous detection of multiple miRNA markers in the serum from clinical cancer patients. This work not only developed a new approach to detect miRNA in different application scenarios but also provided a new reference for the application of the biotin-streptavidin amplification system in the detection of other small biomolecules.

In vivo-stable bis-iminobiotin for targeted radionuclide delivery with the mutant streptavidin.

Targeted delivery of radionuclides to tumors is significant in theranostics applications for precision medicine. Pre-targeting, in which a tumor-targeting vehicle and a radionuclide-loaded effector small molecule are administered separately, holds promise since it can reduce unnecessary internal radiation exposure of healthy cells and can minimize radiation decay. The success of the pre-targeting delivery requires an in vivo-stable tumor-targeting vehicle selectively binding to tumor antigens and an in vivo-stable small molecule effector selectively binding to the vehicle accumulated on the tumor. We previously reported a drug delivery system composed of a low-immunogenic streptavidin with weakened affinity to endogenous biotin and a bis-iminobiotin with high affinity to the engineered streptavidin. It was, however, unknown whether the bis-iminobiotin is stable in vivo when administered alone for the pre-targeting applications. Here we report a new in vivo-stable bis-iminobiotin derivative. The keys to success were the identification of the degradation site of the original bis-iminobiotin treated with mouse plasma and the structural modification of the degradation site. We disclosed the successful pre-targeting delivery of astatine-211 (211At), α-particle emitter, to the CEACAM5-positive tumor in xenograft mouse models.

A strategy to enhance the water solubility of luminescent ß-diketonate-Europium(III) complexes for time-gated luminescence bioassays.

Luminescent ß-diketonate-europium(III) complexes have been found a wide range of applications in time-gated luminescence (TGL) bioassays, but their poor water solubility is a main problem that limits their effective uses. In this work we propose a simple and general strategy to enhance the water solubility of luminescent ß-diketonate-europium(III) complexes that permits facile synthesis and purification. By introducing the fluorinated carboxylic acid group into the structures of ß-diketone ligands, two highly water-soluble and luminescent Eu3+ complexes, PBBHD-Eu3+ and CPBBHD-Eu3+, were designed and synthesized. An excellent solubility exceeding 20 mg/mL for PBBHD-Eu3+ was found in a pure aqueous buffer, while it also displayed strong and long-lived luminescence (quantum yield φ = 26%, lifetime τ = 0.49 ms). After the carboxyl groups of PBBHD-Eu3+ were activated, the PBBHD-Eu3+-labeled streptavidin-bovine serum albumin (SA-BSA) conjugate was prepared, and successfully used for the immunoassay of human α-fetoprotein (AFP) and the imaging of an environmental pathogen Giardia lamblia under TGL mode, which demonstrated the practicability of PBBHD-Eu3+ for highly sensitive TGL bioassays. The carboxyl groups of PBBHD can also be easily derivatized with other reactive chemical groups, which enables PBBHD-Eu3+ to meet diverse requirements of biolabeling technique, to provide new opportunities for developing functional europium(III) complex biolabels serving for TGL bioassays.

Redox-Responsive "Catch and Release" Cryogels: A Versatile Platform for Capture and Release of Proteins and Cells.

Macroporous cryogels are attractive scaffolds for biomedical applications, such as biomolecular immobilization, diagnostic sensing, and tissue engineering. In this study, thiol-reactive redox-responsive cryogels with a porous structure are prepared using photopolymerization of a pyridyl disulfide poly(ethylene glycol) methacrylate (PDS-PEG-MA) monomer. Reactive cryogels are produced using PDS-PEG-MA and hydrophilic poly(ethylene glycol) methyl ether methacrylate (PEGMEMA) monomers, along with a PEG-based cross-linker and photoinitiator. Functionalization of cryogels using a fluorescent dye via the disulfide-thiol exchange reactions is demonstrated, followed by release under reducing conditions. For ligand-mediated protein immobilization, first, thiol-containing biotin or mannose is conjugated onto the cryogels. Subsequently, fluorescent dye-labeled proteins streptavidin and concanavalin A (ConA) are immobilized via ligand-mediated conjugation. Furthermore, we demonstrate that the mannose-decorated cryogel could capture ConA selectively from a mixture of lectins. The efficiency of protein immobilization could be easily tuned by changing the ratio of the thiol-sensitive moiety in the scaffold. Finally, an integrin-binding cell adhesive peptide is attached to cryogels to achieve successful attachment, and the on-demand detachment of integrin-receptor-rich fibroblast cells is demonstrated. Redox-responsive cryogels can serve as potential scaffolds for a variety of biomedical applications because of their facile synthesis and modification.

Quantifying platinum binding on protein-functionalized magnetic microparticles using single particle-ICP-TOF-MS.

This work describes an analytical procedure, single particle-inductively coupled plasma-time-of-flight-mass spectrometry (SP-ICP-TOF-MS), that was developed to determine the platinum binding efficiency of protein-coated magnetic microparticles. SP-ICP-TOF-MS is advantageous due to its ability to quasi-simultaneously detect all nuclides (7Li-242Pu), allowing for both platinum and iron (composition of magnetic microparticles) to be measured concurrently. This method subsequently allows for the differentiation between bound and unbound platinum. The 1 µm magnetic microparticles were fully characterized for their iron concentration, particle concentration, and trace element composition by bulk digestion-ICP-MS and SP-ICP-TOF-MS. The results of both approaches agreed with the certificate values. Using the single particle methodology the platinum loading was quantified to be to 0.18 ± 0.02 fg per particle and 0.32 ± 0.02 fg per particle, for the streptavidin-coated and azurin-coated microparticles, respectively. Both streptavidin-coated and the azurin-coated microparticles had a particle-platinum association of >65%. Platinum bound samples were also analyzed via bulk digestion-based ICP-MS. The bulk ICP-MS results overestimated platinum loading due to free platinum in the samples. This highlights the importance of single particle analysis for a closer inspection of platinum binding performance. The SP-ICP-TOF-MS approach offers advantages over typical bulk digestion methods by eliminating laborious sample preparation, enabling differentiation between bound/unbound platinum in a solution, and quantification of platinum on a particle-by-particle basis. The procedure presented here enables quantification of metal content per particle, which could be broadly implemented for other single particle applications.

A sensitive fluoroimmunoassay for quantitative detection of imidacloprid based on quantum dot-streptavidin conjugate.

Imidacloprid (IMI), the most commonly used neonicotinoid, is widely present in both the environment and agro-products due to extensive and prolonged application, posing potential risks to ecological security and human health. This study introduced a sensitive and rapid fluorescence-linked immunosorbent assay, employing Quantum Dot-Streptavidin conjugate (QDs-SA-FLISA), for efficient monitoring of IMI residues in agro-products. Under optimized conditions, the QDs-SA-FLISA exhibited a half-maximal inhibition concentration (IC50) of 1.70 ng/mL and a limit of detection (LOD, IC20) of 0.5 ng/mL. Investigation into the sensitivity enhancement effect of the QDs-SA revealed that the sensitivity (IC50) of the QDs-SA-FLISA was 7.3 times higher than that of ELISA. The recoveries and relative standard deviation (RSD) ranged from 81.7 to 118.1 % and 0.5-9.4 %, respectively, for IMI in brown rice, tomato and pear. There was no significant difference in IMI residues obtained between QDs-SA-FLISA and UHPLC-MS/MS. Thus, the QDs-SA-FLISA represents a reliable approach for the quantitative determination of IMI in agro-products.

Exploring Options for Proximity-Dependent Biotinylation Experiments: Comparative Analysis of Labeling Enzymes and Affinity Purification Resins.

Proximity-dependent biotinylation (PDB) techniques provide information about the molecular neighborhood of a protein of interest, yielding insights into its function and localization. Here, we assessed how different labeling enzymes and streptavidin resins influence PDB results. We compared the high-confidence interactors of the DNA/RNA-binding protein transactive response DNA-binding protein 43 kDa (TDP-43) identified using either miniTurbo (biotin ligase) or APEX2 (peroxidase) enzymes. We also evaluated two commercial affinity resins for purification of biotinylated proteins: conventional streptavidin sepharose versus a new trypsin-resistant streptavidin conjugated to magnetic resin, which significantly reduces the level of contamination by streptavidin peptides following on-bead trypsin digestion. Downstream analyses involved liquid chromatography coupled to mass spectrometry in data-dependent acquisition mode, database searching, and statistical analysis of high-confidence interactors using SAINTexpress. The APEX2-TDP-43 experiment identified more interactors than miniTurbo-TDP-43, although miniTurbo provided greater overlap with previously documented TDP-43 interactors. Purifications on sepharose resin yielded more interactors than magnetic resin in small-scale experiments using a range of magnetic resin volumes. We suggest that resin-specific background protein binding profiles and different lysate-to-resin ratios cumulatively affect the distributions of prey protein abundance in experimental and control samples, which impact statistical confidence scores. Overall, we highlight key experimental variables to consider for the empirical optimization of PDB experiments.

Metabolic labeling-mediated visualization, capture, and inactivation of Gram-positive bacteria via biotin-streptavidin interactions.

We introduce a biotinylated D-amino acid probe capable of metabolically incorporating into bacterial PG. Leveraging the robust affinity between biotin and streptavidin, the probe has demonstrated efficacy in imaging, capture, and targeted inactivation of Gram-positive bacteria through synergistic pairings with commercially available streptavidin-modified fluorescent dyes and nanomaterials. The versatility of the probe is underscored by its compatibility with a variety of commercially available streptavidin-modified reagents. This adaptability allows the probe to be applied across diverse scenarios by integrating with these commercial reagents.

Improving the Performance of Selective Solid-State Nanopore Sensing Using a Polyhistidine-Tagged Monovalent Streptavidin.

Solid-state (SS-) nanopore sensing has gained tremendous attention in recent years, but it has been constrained by its intrinsic lack of selectivity. To address this, we previously established a novel SS-nanopore assay that produces translocation signals only when a target biotinylated nucleic acid fragment binds to monovalent streptavidin (MS), a protein variant with a single high-affinity biotin-binding domain. While this approach has enabled selective quantification of diverse nucleic acid biomarkers, sensitivity enhancements are needed to improve the detection of low-abundance translational targets. Because the translocation dynamics that determine assay efficacy are largely governed by constituent charge characteristics, we here incorporate a polyhistidine-tagged MS (hMS) to alter the component detectability. We investigate the effects of buffer pH, salt concentration, and SS-nanopore diameter on the performance with the alternate reagent, achieve significant improvements in measurement sensitivity and selectivity, and expand the range of device dimensions viable for the assay. We used this improvement to detect as little as 1 nM miRNA spiked into human plasma. Overall, our findings improve the potential for broader applications of SS-nanopores in the quantitative analyses of molecular biomarkers.

Steric trapping strategy for studying the folding of helical membrane proteins.

Elucidating the folding energy landscape of membrane proteins is essential to the understanding of the proteins' stabilizing forces, folding mechanisms, biogenesis, and quality control. This is not a trivial task because the reversible control of folding is inherently difficult in a lipid bilayer environment. Recently, novel methods have been developed, each of which has a unique strength in investigating specific aspects of membrane protein folding. Among such methods, steric trapping is a versatile strategy allowing a reversible control of membrane protein folding with minimal perturbation of native protein-water and protein-lipid interactions. In a nutshell, steric trapping exploits the coupling of spontaneous denaturation of a doubly biotinylated protein to the simultaneous binding of bulky monovalent streptavidin molecules. This strategy has been evolved to investigate key elements of membrane protein folding such as thermodynamic stability, spontaneous denaturation rates, conformational features of the denatured states, and cooperativity of stabilizing interactions. In this review, we describe the critical methodological advancement, limitation, and outlook of the steric trapping strategy.

An array of femtoliter wells for sensitive detection of copper using click chemistry.

Sensitive detection of copper ion (Cu2+), which is of great importance for environmental pollution and human health, is crucial. In this study, we present a highly sensitive method for measuring Cu2+ in an array of femtoliter wells. In brief, magnetic beads (MBs) modified with alkyne groups were bound to the azide groups of biotin-PEG3-azide (bio-PEG-N3) via Cu+-catalyzed click chemistry. Cu+ in the click chemistry reaction was generated by reducing Cu2+ with sodium ascorbate. Following the ligation, the surface of the MBs was modified with biotin, which could be labeled with streptavidin-ß-galactosidase (SßG). The MBs complex was then suspended in ß-galactosidase substrate fluorescein-di-ß-d-galactopyranoside (FDG), and loaded into the array of femtoliter wells. The MBs sank into the wells due to gravity, and the resulting fluorescent product, generated from the reaction between SßG on the surface of the MBs and FDG, was confined within the wells. The number of fluorescent wells increased with higher Cu2+ concentrations. The bright-field and fluorescent images of the wells were acquired using an inverted fluorescent microscope. The detection limit of this assay for Cu2+ was 1 nM without signal amplification, which was 103 times lower than that of traditional fluorescence detection assays.

Drug-Loaded Bacillus Calmette-Guérin Bacteria for Immuno-Chemo Combo Therapy in Bladder Cancer.

Intravesical Bacillus Calmette-Guérin (BCG) is a well-established strategy for managing high-risk nonmuscle-invasive bladder cancer (NMIBC); however, over half of patients still experience disease recurrence or progression. Although the combined intravesical instillation of various chemotherapeutic drugs is implemented in clinical trials to enhance the BCG therapy, the outcome is far from satisfying due to severe irritative effects and treatment intolerance at high doses. Therefore, it is adopted the "biotin-streptavidin strategy" to doxorubicin (DOX)-encapsulated nanoparticles within live BCG bacteria (DOX@BCG) to improve treatment outcomes. Adherence of BCG to the bladder epithelium helps precisely target DOX@BCG to the local tumor cells and simultaneously increases intratumoral transport of therapeutic drugs. DOX@BCG effectively inhibits cancer progression and prolongs the survival of rats/mice with orthotopic bladder cancer owing to synergism between BCG-immunotherapy, DOX-chemotherapy, and DOX-induced immunogenic tumor cell death; furthermore, it exhibits improved tolerance and biosafety, and establishes antitumor immunity in the tumor microenvironment. Therefore, the drug-loaded live BCG bacterial delivery system holds considerable potential for clinical translation in the intravesical treatment of bladder cancer.

Streptavidin-biotin system-mediated immobilization of a bivalent nanobody onto magnetosomes for separation and analysis of 3-phenoxybenzoic acid in urine.

The compound 3-phenoxybenzoic acid (3-PBA) is frequently utilized as a biomarker to detect exposure to various pyrethroids. In this study, a bivalent nanobody (Nb2) specifically targeting 3-PBA was biotinylated and immobilized onto streptavidin (SA)-modified bacterial magnetic nanoparticles (BMPs), resulting in the formation of BMP-SA-Biotin-Nb2 complexes. These complexes demonstrated remarkable stability when exposed to strongly acidic solutions (4 M HCl), methanol (80%), and high ionic strength (1.37 M NaCl). An immunoassay was subsequently developed utilizing BMP-SA-Biotin-Nb2 as the capture agent and 3-PBA-horseradish peroxidase as the detection probe. The immunoassay exhibited an IC50 value (half-maximum signal inhibition concentration) of 1.11 ng mL-1 for 3-PBA. To evaluate the accuracy of the assay, spiked sheep and cow urine samples (ranging from 3.0 to 240 ng mL-1) were analyzed. The quantitative recoveries ranged from 82.5% to 113.1%, which agreed well with the findings obtained using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Overall, the BMP-SA-Biotin-Nb2-based immunoassay holds great promise for rapid monitoring of 3-PBA following acid dissociation.

Optimisation of denaturing ion pair reversed phase HPLC for the purification of ssDNA in SELEX.

Aptamers have shown great promise as oligonucleotide-based affinity ligands for various medicinal and industrial applications. A critical step in the production of DNA aptamers via selective enhancement of ligands by exponential enrichment (SELEX) is the generation of ssDNA from dsDNA. There are a number of caveats associated with current methods for ssDNA generation, which can lower success rates of SELEX experiments. They often result in low yields thereby decreasing diversity or fail to eliminate parasitic PCR by-products leading to accumulation of by-products from round to round. Both contribute to the failure of SELEX protocols and therefore potentially limit the impact of aptamers compared to their peptide-based antibody counterparts. We have developed a novel method using ion pair reversed phase HPLC (IP RP HPLC) employed under denaturing conditions for the ssDNA re-generation stage of SELEX following PCR. We have utilised a range of 5' chemical modifications on PCR primers to amplify PCR fragments prior to separation and purification of the DNA strands using denaturing IP RP HPLC. We have optimised mobile phases to enable complete denaturation of the dsDNA at moderate temperatures that circumvents the requirement of high temperatures and results in separation of the ssDNA based on differences in their hydrophobicity. Validation of the ssDNA isolation and purity assessment was performed by interfacing the IP RP HPLC with mass spectrometry and fluorescence-based detection. The results show that using a 5' Texas Red modification on the reverse primer in the PCR stage enabled purification of the ssDNA from its complimentary strand via IP RP HPLC under denaturing conditions. Additionally, we have confirmed the purity of the ssDNA generated as well as the complete denaturation of the PCR product via the use of mass-spectrometry and fluorescence analysis therefore proving the selective elimination of PCR by-products and the unwanted complementary strand. Following lyophilisation, ssDNA yields of up to 80% were obtained. In comparison the streptavidin biotin affinity chromatography also generates pure ssDNA with a yield of 55%. The application of this method to rapidly generate and purify ssDNA of the correct size, offers the opportunity to improve the development of new aptamers via SELEX.