Sensitive Detection of Tumor Cells Using Protein Nanoparticles with Multiple Displays of DNA Aptamers and Bioluminescent Reporters.

Simple and effective detection methods for circulating tumor cells are essential for early detection and progression monitoring of tumors. The use of DNA aptamer and bioluminescence is expected to be a key tool for the simple, effective, and sensitive detection of tumor cells. Herein, we designed multifunctional protein nanoparticles for the detection of tumor cells using DNA aptamer and bioluminescence. Fusion proteins (ELP-poly(d)-POIs), composed of elastin-like polypeptide (ELP) fused with protein of interests (POIs) via poly(aspartic acid) (poly(d)), formed the protein nanoparticles based on the temperature responsivity of ELP sequences, leading to multiply displayed POIs on the protein nanoparticles. In the present study, we focused on porcine circovirus type 2 replication initiation protein (Rep), which covalently conjugated with DNA aptamers, and NanoLuc luciferase (Nluc), which emitted a strong bioluminescence, as POIs. ELP-poly(d)-Rep and ELP-poly(d)-Nluc were constructed and formed the protein nanoparticles with multiply displayed Nluc and Rep (DNA aptamer) that amplified the bioluminescence signal and tumor recognition ability. Mucin-1 (MUC1)-overexpressing human breast tumor MCF7 cells and MUC1-recognizing aptamer (MUC1 aptamer) were selected as models. The MUC1 aptamer-conjugated protein nanoparticles exhibited a 13.7-fold higher bioluminescence signal to MCF-7 cells than to human embryonic kidney 293 (HEK293) cells, which express low levels of MUC1. Furthermore, the protein nanoparticles could detect up to 70.7 cells/mL of MCF-7 cells from a cell suspension containing HEK-293. The protein nanoparticles with multiple Rep and Nluc show a great potential as a material for detecting CTCs.

Polysarcosine-Coated liposomes attenuating immune response induction and prolonging blood circulation.

HYPOTHESIS: Liposomes coated with long polysarcosine (PSar) chains at a high density might enable long blood circulation and attenuate accelerated blood clearance (ABC) phenomenon. EXPERIMENTS: In this study, we controlled the length (23, 45, 68 mers) and density (5, 10, 15 mol%) of PSar on liposomal coatings and, furthermore, investigated the effects of PSar length and density on the blood circulation time, biodistribution, immune response, and ABC phenomenon induction. Length-controlled PSar-bound lipids (PSar-PEs) were synthesized using a click reaction and inserted into bare liposomes at different combinations of chain lengths and proportions. FINDINGS: Although all PSar-coated liposomes (PSar-lipos) had similar morphological, physical, and chemical properties, they had different blood circulation times and biodistribution, and exerted varied effects on the immune system. All PSar-lipos with different PSar length and density showed a similar anti-PSar IgM response. Liposomes modified with the longest PSar chain (68 mers) at a high density (15 mol%) showed the longest blood circulation time and, additionally, attenuated ABC phenomenon compared with PEG-lipo. The ex vivo analysis of the biodistribution of liposomes revealed that a thick PSar layer enhanced the blood circulation time of liposomes due to the reduction of the accumulation of liposomes in the liver and spleen. These findings provide new insights into the relationship between IgM expression and ABC phenomenon inhibition.

Development of an enhanced immunoassay based on protein nanoparticles displaying an IgG-binding domain and luciferase.

Detection of small amounts of target molecules with high sensitivity is important for the diagnosis of many diseases, including cancers, and is particularly important to detect early stages of disease. Here, we report the development of a temperature-responsive fusion protein (ELP-DCN) comprised of an elastin-like polypeptide (ELP), poly-aspartic acid (D), antibody-binding domain C (C), and NanoLuc luciferase (N). ELP-DCN proteins form nanoparticles above a certain threshold temperature that display an antibody-binding domain and NanoLuc luciferase on their surface. ELP-DCN nanoparticles can be applied for enhancement of immunoassay systems because they provide more antibody-binding sites and an increased number of luciferase molecules, resulting in an increase in assay signal. Here, we report the detection of human serum albumin (HSA) as a model protein using anti-HSA and ELP-DCN proteins. Upon formation of ELP-DCN nanoparticles, the detection limit improved tenfold compared to the monomeric form of ELP-DCN.

Detection of SARS-CoV-2 by antigen ELISA test is highly swayed by viral load and sample storage condition.

BACKGROUND: Rapid increase in COVID-19 suspected cases has rendered disease diagnosis challenging, mainly depending upon RT-qPCR. Reliable, rapid, and cost-effective diagnostic assays that complement RT-qPCR should be introduced after thoroughly evaluating their performance upon various disease phases, viral load, and sample storage conditions. OBJECTIVE: We investigated the correlation of cycle threshold (Ct) value, which implies the viral load and infection phase, and the storage condition of the clinical specimen with the diagnosis of SARS-CoV-2 through our newly developed in-house rapid enzyme-linked immunosorbent assay (ELISA) system. METHOD: Naso-oropharyngeal samples of 339 COVID-19 suspected cases were collected and evaluated through RT-qPCR that were stored up to 30 days in different conditions (i.e. -80°C, -20°C and initially at 4°C followed by -80°C). The clinical specimens were evaluated with our in-house ELISA system after finalizing the assay method through checkerboard assay and minimizing the signal/noise ratio. RESULT: The ELISA system showed the highest sensitivity (92.9%) for samples with Ct ≤30 and preserving at -80°C temperature. The sensitivity reduced proportionally with increasing Ct value and preserving temperature. However, the specificity ranged between 98.3% and 100%. CONCLUSION: The results indicate the necessity of early infection phase diagnosis and lower temperature preservation of samples to perform rapid antigen ELISA tests.

Antibody response to the first dose of AZD1222 vaccine in COVID-19 convalescent and uninfected individuals in Bangladesh.

BACKGROUND: Vaccination with the Oxford-AstraZeneca COVID-19 vaccine (AZD1222) initially started in the UK and quickly implemented around the Globe, including Bangladesh. Up to date, more than nine million doses administrated to the Bangladeshi public. METHOD: Herein, we studied the antibody response to the first dose of AZD1222 in 86 Bangladeshi individuals using in-house ELISA kits. Study subjects were categorized into two groups, convalescent and uninfected, based on prior infection history and SARS-CoV-2 nucleocapsid-IgG profiles. RESULTS: All the convalescent individuals presented elevated spike-1-IgG compared to 90% of uninfected ones after the first dose. Day >28 post-vaccination, the convalescent group showed six times higher antibody titer than the uninfected ones. The most elevated antibody titers for the former and later group were found at Day 14 and Days >28 post-vaccination, respectively. The spike-1-IgA titer showed a similar pattern as spike-1-IgG, although in a low-titer. In contrast, the IgM titer did not show any significant change in either group. CONCLUSION: High antibody titer in the convalescent group, signify the importance of the first dose among the uninfected group. This study advocates the integration of antibody tests in vaccination programs in the healthcare system for maximizing benefit.

AuNP Coupled Rapid Flow-Through Dot-Blot Immuno-Assay for Enhanced Detection of SARS-CoV-2 Specific Nucleocapsid and Receptor Binding Domain IgG.

BACKGROUND: Serological tests detecting severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are widely used in seroprevalence studies and evaluating the efficacy of the vaccination program. Some of the widely used serological testing techniques are enzyme-linked immune-sorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and lateral flow immunoassay (LFIA). However, these tests are plagued with low sensitivity or specificity, time-consuming, labor-intensive, and expensive. We developed a serological test implementing flow-through dot-blot assay (FT-DBA) for SARS-CoV-2 specific IgG detection, which provides enhanced sensitivity and specificity while being quick to perform and easy to use. METHODS: SARS-CoV-2 antigens were immobilized on nitrocellulose membrane to capture human IgG, which was then detected with anti-human IgG conjugated gold nanoparticle (hIgG-AuNP). A total of 181 samples were analyzed in-house. Within which 35 were further evaluated in US FDA-approved CLIA Elecsys SARS-CoV-2 assay. The positive panel consisted of RT-qPCR positive samples from patients with both <14 days and >14 days from the onset of clinical symptoms. The negative panel contained samples collected from the pre-pandemic era dengue patients and healthy donors during the pandemic. Moreover, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FT-DBA were evaluated against RT-qPCR positive sera. However, the overall efficacies were assessed with sera that seroconverted against either nucleocapsid (NCP) or receptor-binding domain (RBD). RESULTS: In-house ELISA selected a total of 81 true seropositive and 100 seronegative samples. The sensitivity of samples with <14 days using FT-DBA was 94.7%, increasing to 100% for samples >14 days. The overall detection sensitivity and specificity were 98.8% and 98%, respectively, whereas the overall PPV and NPV were 99.6% and 99%. Moreover, comparative analysis between in-house ELISA assays and FT-DBA revealed clinical agreement of Cohen's Kappa value of 0.944. The FT-DBA showed sensitivity and specificity of 100% when compared with commercial CLIA kits. CONCLUSION: The assay can confirm past SARS-CoV-2 infection with high accuracy within 2 minutes compared to commercial CLIA or in-house ELISA. It can help track SARS-CoV-2 disease progression, population screening, and vaccination response. The ease of use of the assay without requiring any instruments while being semi-quantitative provides the avenue of its implementation in remote areas around the globe, where conventional serodiagnosis is not feasible.

Longitudinal Antibody Dynamics Against Structural Proteins of SARS-CoV-2 in Three COVID-19 Patients Shows Concurrent Development of IgA, IgM, and IgG.

BACKGROUND: Dynamics and persistence of neutralizing and non-neutralizing antibodies can give us the knowledge required for serodiagnosis, disease management, and successful vaccine design and development. The disappearance of antibodies, absence of humoral immunity activation, and sporadic reinfection cases emphasize the importance of longitudinal antibody dynamics against variable structural antigens. METHODS: In this study, twenty-five healthy subjects working in a SARS-COV-2 serodiagnostic assay development project were enrolled, and their sign and symptoms were followed up to six months. Three subjects showed COVID-19-like symptoms, and three subjects' antibody dynamics were followed over 120 days by analyzing 516 samples. We have developed 12 different types of in-house ELISAs to observe the kinetics of IgG, IgM, and IgA against four SARS-CoV-2 proteins, namely nucleocapsid, RBD, S1, and whole spike (S1+S2). For the development of these assays, 30-104 pre-pandemic samples were taken as negative controls and 83 RT-qPCR positive samples as positive ones. RESULTS: All three subjects presented COVID-19-like symptoms twice, with mild symptoms in the first episode were severe in the second, and RT-qPCR confirmed the latter. The initial episode did not culminate with any significant antibody development, while a multifold increase in IgG antibodies characterized the second episode. Interestingly, IgG antibody development concurrent with IgM and IgA and persisted, whereas the latter two weans off rather quickly if appeared. CONCLUSION: Antibody kinetics observed in this study can provide a pathway to the successful development of sero-diagnostics and epidemiologists to predict the fate of vaccination currently in place.

Versatile Mitogenic and Differentiation-Inducible Layer Formation by Underwater Adhesive Polypeptides.

Artificial materials have no biological functions, but they are important for medical devices such as artificial organs and matrices for regenerative medicine. In this study, mitogenic and differentiation-inducible materials are devised via the simple coating of polypeptides, which contain the sequence of epidermal growth factor or insulin-like growth factor with a key amino acid (3,4-dihydroxyphenylalanine) of underwater adhesive proteins. The adhesive polypeptides prepared via solid-phase synthesis form layers on various substrates involving organic and inorganic materials to provide biological surfaces. Through the direct activation of cognate receptors on interactive surfaces, the materials enable increased cell growth and differentiation compared to that achieved by soluble growth factors. This superior growth and differentiation are attributed to the long-lasting signal transduction (triggered by the bound growth factors), which do not cause receptor internalization and subsequent downregulation.

Development and performance evaluation of a rapid in-house ELISA for retrospective serosurveillance of SARS-CoV-2.

BACKGROUND: In the ongoing pandemic situation of COVID-19, serological tests can complement the molecular diagnostic methods, and can be one of the important tools of sero-surveillance and vaccine evaluation. AIM: To develop and evaluate a rapid SARS-CoV-2 specific ELISA for detection of anti-SARS-CoV2 IgG from patients' biological samples. METHODS: In order to develop this ELISA, three panels of samples (n = 184) have been used: panel 1 (n = 19) and panel 2 (n = 60) were collected from RT-PCR positive patients within 14 and after 14 days of onset of clinical symptoms, respectively; whereas panel 3 consisted of negative samples (n = 105) collected either from healthy donors or pre-pandemic dengue patients. As a capturing agent full-length SARS-CoV2 specific recombinant nucleocapsid was immobilized. Commercial SARS-CoV2 IgG kit based on chemiluminescent assay was used for the selection of samples and optimization of the assay. The threshold cut-off point, inter-assay and intra-assay variations were determined. RESULTS: The incubation/reaction time was set at a total of 30 minutes with the sensitivity of 84% (95% confidence interval, CI, 60.4%, 96.6%) and 98% (95% CI, 91.1%, 100.0%), for panel 1 and 2, respectively; with overall 94.9% sensitivity (95% CI 87.5%, 98.6%). Moreover, the clinical specificity was 97.1% (95% CI, 91.9%, 99.4%) with no cross reaction with dengue samples. The overall positive and negative predictive values are 96.2% (95% CI 89.2%, 99.2%) and 96.2% (95% CI, 90.6% 99.0%), respectively. In-house ELISA demonstrated 100% positive and negative percent agreement with Elecsys Anti-SARS-CoV-2, with Cohen's kappa value of 1.00 (very strong agreement), while comparing 13 positive and 17 negative confirmed cases. CONCLUSION: The assay is rapid and can be applied as one of the early and retrospective sero-monitoring tools in all over the affected areas.

Conjugation of biphenyl groups with poly(ethylene glycol) to enhance inhibitory effects on the PD-1/PD-L1 immune checkpoint interaction.

Monoclonal antibodies have been developed as anticancer agents to block immune checkpoint pathways associated with programmed cell death 1 (PD-1) and its ligand PD-L1. However, the high cost of antibodies has encouraged researchers to develop other inhibitor types. Here, biphenyl compounds were conjugated with poly(ethylene glycol) (PEG) to enhance the activity of small molecular inhibitors. Immunoassay results revealed the decrease in the inhibition activity following conjugation with linear PEG, suggesting that the PEG moiety reduced the interaction between the biphenyl structure and PD-L1. However, the inhibitory effect on PD-1/PD-L1 interaction was further enhanced by using branched PEG conjugates. The increase in the number of conjugated biphenyl compounds resulted in increased inhibitory activity. The highest IC50 value was 0.33 µM, which was about 5 times higher than that observed for a non-conjugated monovalent compound. The inhibitory activity was more than 20 times the activity reported for the starting compound. Considering the increase in the inhibition activity, this multivalent strategy can be useful in the design of new immune checkpoint inhibitors.

Direct visualization of the extracellular binding structure of E-cadherins in liquid.

E-cadherin is a key Ca-dependent cell adhesion molecule, which is expressed on many cell surfaces and involved in cell morphogenesis, embryonic development, EMT, etc. The fusion protein E-cad-Fc consists of the extracellular domain of E-cadherin and the IgG Fc domain. On plates coated with this chimeric protein, ES/iPS cells are cultivated particularly well and induced to differentiate. The cells adhere to the plate via E-cad-Fc in the presence of Ca2+ and detach by a chelating agent. For the purpose of clarifying the structures of E-cad-Fc in the presence and absence of Ca2+, we analyzed the molecular structure of E-cad-Fc by AFM in liquid. Our AFM observations revealed a rod-like structure of the entire extracellular domain of E-cad-Fc in the presence of Ca2+ as well as trans-binding of E-cad-Fc with adjacent molecules, which may be the first, direct confirmation of trans-dimerization of E-cadherin. The observed structures were in good agreement with an X-ray crystallographic model. Furthermore, we succeeded in visualizing the changes in the rod-like structure of the EC domains with and without calcium. The biomatrix surface plays an important role in cell culture, so the analysis of its structure and function may help promote cell engineering based on cell recognition.

Thermally Induced Switch of Coupling Reaction Using the Morphological Change of a Thermoresponsive Polymer on a Reactive Heteroarmed Nanoparticle.

Control of the cross-linking reaction is imperative when developing a sophisticated in situ forming hydrogel in the body. In this study, a heteroarmed thermoresponsive (TR) nanoparticle was designed to investigate the mechanism of controlling reactivity of the functional groups introduced into the nanoparticles. The coupling reaction was suppressed/proceeded by utilizing temperature-induced morphological changes of the TR polymer. The heteroarmed TR nanoparticle was prepared by the coassembly of amphiphilic block copolymers possessing both a TR segment and hydrophilic segment with reactive functional groups of succinimide. The longer TR chain on the nanoparticle covered the succinimide group and suppressed the reaction with the primary amine on the external nanoparticle. In contrast, the coupling reaction was promoted at a high temperature to create the chemical cross-linking structure between the nanoparticles because of the exposure of the succinimide group on the surface of the particle as a consequence of the morphological change of the TR polymer. In addition, the thermally controlled chemical reaction modulated initiation of the gelation using a highly concentrated nanoparticle solution. The heteroarmed TR nanoparticle offers great practical advantages for clinical uses, such as embolization agents, through precise control of the reaction.

Preparation of Biphenyl-Conjugated Bromotyrosine for Inhibition of PD-1/PD-L1 Immune Checkpoint Interactions.

Cancer immunotherapy has been revolutionized by the development of monoclonal antibodies (mAbs) that inhibit interactions between immune checkpoint molecules, such as programmed cell-death 1 (PD-1), and its ligand PD-L1. However, mAb-based drugs have some drawbacks, including poor tumor penetration and high production costs, which could potentially be overcome by small molecule drugs. BMS-8, one of the potent small molecule drugs, induces homodimerization of PD-L1, thereby inhibiting its binding to PD-1. Our assay system revealed that BMS-8 inhibited the PD-1/PD-L1 interaction with IC50 of 7.2 µM. To improve the IC50 value, we designed and synthesized a small molecule based on the molecular structure of BMS-8 by in silico simulation. As a result, we successfully prepared a biphenyl-conjugated bromotyrosine (X) with IC50 of 1.5 µM, which was about five times improved from BMS-8. We further prepared amino acid conjugates of X (amino-X), to elucidate a correlation between the docking modes of the amino-Xs and IC50 values. The results suggested that the displacement of amino-Xs from the BMS-8 in the pocket of PD-L1 homodimer correlated with IC50 values. This observation provides us a further insight how to derivatize X for better inhibitory effect.

Construction of DNA-displaying nanoparticles by enzymatic conjugation of DNA and elastin-like polypeptides using a replication initiation protein.

DNA-displaying nanoparticles comprised of conjugates of single-stranded DNA (ssDNA) and elastin-like polypeptide (ELP) were developed. ssDNA was enzymatically conjugated to ELPs via a catalytic domain of Porcine Circovirus type 2 replication initiation protein (pRep) fused to ELPs. Nanoparticles were formed upon heating to temperatures above the phase transition temperature due to the hydrophobicity of ELPs and the hydrophilicity of conjugated ssDNA. We demonstrated the applicability of the resultant nanoparticles as drug carriers with tumor-targeting properties by conjugating a DNA aptamer, which is known to bind to Mucin 1 (MUC1), to ELPs. DNA aptamer-displaying nanoparticles encapsulating the anti-cancer drug paclitaxel were able to bind to cells overexpressing MUC1 and induce cell death.

Construction of multifunctional fusion proteins with a laminin-derived short peptide to promote neural differentiation of mouse induced pluripotent stem cells.

There is growing interest in the functional roles of the extracellular matrix (ECM) in regulating the fate of pluripotent stem cells (PSCs). An artificially bioengineered ECM provides an excellent model for studying the molecular mechanisms underlying self-renewal and differentiation of PSCs, without multiple unknown and variable factors associated with natural substrates. Here, we have engineered multifunctional fusion proteins that are based on peptides from laminin, including p20, RGD, and elastin-like polypeptide (ELP), where laminin peptides work as cell adhesion molecules (CAMs) and ELP to promote anchorage. The functionality of these chimeric proteins, referred to as ERE-p20 and E-p20, was assessed by determining their ability to immobilize cells on a hydrophobic polystyrene surface, improve mouse induced pluripotent stem cells (miPSCs) attachment, and promote miPSC differentiation to neural progenitors. ERE-p20 and E-p20 proteins showed hydrophobic binding saturation to the polystyrene plates around 500 nM (2.39 µg/cm2 ) and 750 nM (2.27 µg/cm2 ) protein concentrations, respectively. The apparent maximum cell binding to ERE-p20 and E-p20 was approximately 81% and 73%, respectively, relative to gelatin. For neural precursors, neurite outgrowth was enhanced by the presence of RGD and p20 peptides. The expression levels of neuronal marker protein MAP2 were upregulated approximately 2.5-fold and threefold by ERE-p20 and E-p20, respectively, relative to laminin. Overall, we have shown that elastin-mimetic fusion proteins consisting of p20 with and without RGD peptides are able to induce neuronal differentiation. In conclusion, our newly designed bioengineered fusion proteins allow preparation of specific bioactive matrices or coating/scaffold for miPSCs differentiation.

Temperature-Responsive Multifunctional Protein Hydrogels with Elastin-like Polypeptides for 3-D Angiogenesis.

Supramolecular protein hydrogels with tunable properties represent promising candidates for advanced designer extracellular matrices (ECMs). To control cellular functions, ECMs should be able to spatiotemporally regulate synergistic signaling between transmembrane receptors and growth factor (GF) receptors. In this study, we developed genetically engineered temperature-responsive multifunctional protein hydrogels. The designed hydrogel was fabricated by combining the following four peptide blocks: thermosensitive elastin-like polypeptides (ELPs), a polyaspartic acid (polyD) chain to control aggregation and delivery of GFs, a de novo-designed helix peptide that forms antiparallel homotetrameric coiled-coils, and a biofunctional peptide. The resultant coiled-coil unit bound ELPs (CUBEs) exhibit a controllable sol-gel transition with tunable mechanical properties. CUBEs were functionalized with bone sialoprotein-derived RGD (bRGD), and human umbilical vein endothelial cells (HUVECs) were three-dimensionally cultured in bRGD-modified CUBE (bRGD-CUBE) hydrogels. Proangiogenic activity of HUVECs was promoted by bRGD. Moreover, heparin-binding angiogenic GFs were immobilized to bRGD-CUBEs via electrostatic interactions. HUVECs cultured in GF-tethered bRGD-CUBE hydrogels formed three-dimensional (3-D) tubulelike structures. The designed CUBE hydrogels may demonstrate utility as advanced smart biomaterials for biomedical applications. Further, the protein hydrogel design strategy may provide a novel platform for constructing designer 3-D microenvironments for specific cell types.

Synthesis of Photoreactive Poly(ethylene oxide)s for Surface Modification.

Photoreactive polymers that generate active species upon irradiation with light are very useful for modifying the surfaces of substrates. However, water solubility decreases as the number of photoreactive functional groups on the polymer increases because most photoreactive functional groups are hydrophobic. In order to improve the hydrophilicity of the photoreactive polymer, we synthesized polyethylene glycol-based photoreactive polymers bearing hydrophobic azidophenyl groups on their side chains. Because of the hydrophilicity of the ethylene glycol main chain, polymers with large numbers of azidophenyl groups were solubilized in protic solvents compared to hydrophobic alkylene chain-based polymers prepared by radical polymerization of methacrylate monomers. Polymers were immobilized on various substrates by irradiation with ultraviolet light and were shown to suppress nonspecific interactions between proteins and cells on the substrate. We conclude that such polymers are useful, highly water soluble antifouling agents.

Direct Labeling of Protein Nanoparticles with Fluorescent Compounds for Immunoassay Applications.

A fusion protein, designated ELP-D-C, comprised of a hydrophobic elastin-like polypeptide unit, a hydrophilic aspartic acid-rich peptide unit, and an antibody-binding domain as a functional unit, was constructed. Upon heat induction, ELP-D-C forms micellar nanoparticles displaying antibody-binding domains on their surfaces. The protein nanoparticles were able to incorporate hydrophobic fluorescent compounds and subsequently detect target molecules via antibody binding by the resulting fluorescence intensity, which was proportional to the log of the concentration of the target molecule.

Construction of DNA-NanoLuc luciferase conjugates for DNA aptamer-based sandwich assay using Rep protein.

OBJECTIVE: We developed a DNA-NanoLuc luciferase (NnaoLuc) conjugates for DNA aptamer-based sandwich assay using the catalytic domain of the replication initiator protein derived from porcine circovirus type 2 (pRep). RESULTS: For construction of DNA aptamer and NanoLuc conjugate using the catalytic domain of Rep from PCV2. pRep fused to NanoLuc was genetically constructed and expressed in E. coli. After purification, the activities of fused pRep and NanoLuc were evaluated, and DNA-NanoLuc conjugates were constructed via the fused pRep. Finally, constructed DNA-NanoLuc conjugates were applied for use in a DNA aptamer-based sandwich assay. Here, pRep was used not only for conjugation of the NanoLuc to the detection aptamer, but also for immobilization of the capture aptamer on the plate surface. CONCLUSION: We have demonstrated that DNA-NanoLuc conjugates via the catalytic domain of PCV2 Rep could be applied for DNA aptamer-based sandwich assay system.

Development of drug-loaded protein nanoparticles displaying enzymatically-conjugated DNA aptamers for cancer cell targeting.

Modification of protein-based drug carriers with tumor-targeting properties is an important area of research in the field of anticancer drug delivery. To this end, we developed nanoparticles comprised of elastin-like polypeptides (ELPs) with fused poly-aspartic acid chains (ELP-D) displaying DNA aptamers. DNA aptamers were enzymatically conjugated to the surface of the nanoparticles via genetic incorporation of Gene A* protein into the sequence of the ELP-D fusion protein. Gene A* protein, derived from bacteriophage ϕX174, can form covalent complexes with single-stranded DNA via the latter's recognition sequence. Gene A* protein-displaying nanoparticles exhibited the ability to deliver the anticancer drug paclitaxel (PTX), whilst retaining activity of the conjugated Gene A* protein. PTX-loaded protein nanoparticles displaying DNA aptamers known to bind to the MUC1 tumor marker resulted in increased cytotoxicity with MCF-7 breast cancer cells compared to PTX-loaded protein nanoparticles without the DNA aptamer modification.