Development of DNA aptamers universally bound to single-chain fragment variables and their applications in bioprocess monitoring.

Single-chain fragment variables (scFvs), composed of variable heavy and light chains joined together by a peptide linker, can be produced using a cost-effective bacterial expression system, making them promising candidates for pharmaceutical applications. However, a versatile method for monitoring recombinant-protein production has not yet been developed. Herein, we report a novel anti-scFv aptamer-based biosensing system with high specificity and versatility. First, anti-scFv aptamers were screened using the competitive systematic evolution of ligands by exponential enrichment, focusing on a unique scFv-specific peptide linker. We selected two aptamers, P1-12 and P2-63, with KD = 2.1 µM or KD = 1.6 µM toward anti-human epidermal growth factor receptor (EGFR) scFv, respectively. These two aptamers can selectively bind to scFv but not to anti-EGFR Fv. Furthermore, the selected aptamers recognized various scFvs with different CDRs, such as anti-4-1BB and anti-hemoglobin scFv, indicating that they recognized a unique peptide linker region. An electrochemical sensor for anti-EGFR scFv was developed using anti-scFv aptamers based on square wave voltammetry. Thus, the constructed sensor could monitor anti-EGFR scFv concentrations in the range of 10-500 nM in a diluted medium for bacterial cultivation, which covered the expected concentration range for the recombinant production of scFvs. These achievements promise the realization of continuous monitoring sensors for pharmaceutical scFv, which will enable the real-time and versatile monitoring of large-scale scFv production.

Exploration and Application of DNA-Binding Proteins to Make a Versatile DNA-Protein Covalent-Linking Patch (D-Pclip): The Case of a Biosensing Element.

DNA-protein complexes are attractive components with broad applications in various research fields, such as DNA aptamer-enzyme complexes as biosensing elements. However, noncovalent DNA-protein complexes often decrease detection sensitivity because they are highly susceptible to environmental conditions. In this study, we developed a versatile DNA-protein covalent-linking patch (D-Pclip) for fabricating covalent and stoichiometric DNA-protein complexes. We comprehensively explored the database to determine the DNA-binding ability of the candidates and selected UdgX as the only uracil-DNA glycosylase known to form covalent bonds with DNA via uracil, with a binding efficiency >90%. We integrated a SpyTag/SpyCatcher protein-coupling system into UdgX to create a universal and convenient D-Pclip. The usability of D-Pclip was shown by preparing a stoichiometric model complex of a hemoglobin (Hb)-binding aptamer and glucose oxidase (GOx) by mixing at 4 °C. The prepared aptamer-GOx complexes detected Hb in a dose-dependent manner within the clinically required detection range in buffer and human serum without any washing procedures. D-Pclip covalently connects any uracil-inserted DNA sequence and any SpyCatcher-fused protein stoichiometrically; therefore, it has a high potential for various applications.

Transient potentiometry based d-serine sensor using engineered d-amino acid oxidase showing quasi-direct electron transfer property.

d-Serine biosensing has been extensively reported based on enzyme sensors using flavin adenine dinucleotide (FAD) -dependent d-amino acid oxidase (DAAOx), based on the monitoring of hydrogen peroxide generated by the enzymatic reaction, which is affected by dissolved oxygen concentration in the measurement environment in in vivo use. Here we report a novel sensing principle for d-serine, transient potentiometry based d-serine sensor using engineered DAAOx showing quasi-direct electron transfer (DET) property. DAAOx Gly52Val mutant, revealed to possess dye-mediated dehydrogenase activity using artificial synthetic electron acceptors, while its oxidase activity was negligible. The enzyme was immobilized on electrode and was modified with amine-reactive phenazine ethosulfate, resulted an enzyme electrode showing quasi-DET type response. Although OCP based monitoring took more than several minutes to obtain steady state OCP value, the time dependent OCP change monitoring, transient potentiometry, provided rapid and sensitive sensor signals. While dOCP/dt based monitoring was suitable for sensing with longer than 5 s time resolution with d-serine concentration range between 0.5 mM and 5 mM, dOCP/d t based monitoring is suitable for d-serine monitoring with much shorter time resolution (less than 1 s) with high sensitivity with wider dynamic range (20 µM-30 mM). The maximum dOCP/d t was -39.2 ± 2.0 mV/s1/2, the Km(app) was 1.9 mM, and the lower limit of detection was 20 µM. In addition, d-serine monitoring was also possible in the artificial cerebrospinal fluid. The transient potentiometry based sensing reported in this study will be further utilized to realize miniaturized, continuous, real-time, in vivo sensor for d-serine monitoring.

Glycogen Production in Marine Cyanobacterial Strain Synechococcus sp. NKBG 15041c.

An important feature offered by marine cyanobacterial strains over freshwater strains is the capacity to grow in seawater, replacing the need for often-limited freshwater. However, there are only limited numbers of marine cyanobacteria that are available for genetic manipulation and bioprocess applications. The marine unicellular cyanobacteria Synechococcus sp. strain NKBG 15041c (NKBG15041c) has been extensively studied. Recombinant DNA technologies are available for this strain, and its genomic information has been elucidated. However, an investigation of carbohydrate production, such as glycogen production, would provide information for inevitable biofuel-related compound production, but it has not been conducted. In this study, glycogen production by marine cyanobacterium NKBG15041c was investigated under different cultivation conditions. NKBG15041c yielded up to 399 µg/ml/OD730 when cells were cultivated for 168 h in nitrogen-depleted medium (marine BG11ΔN) after medium replacement (336 h after inoculation). Cultivation under nitrogen-limited conditions also yielded an accumulation of glycogen in NKBG15041c cells (1 mM NaNO3, 301 µg/ml/OD730; 3 mM NaNO3, 393 µg/ml/OD730; and 5 mM NaNO3, 328 µg/ml/OD730) under ambient conditions. Transcriptional analyses were carried out for 13 putative genes responsible for glycogen synthesis and catabolism that were predicted based on homology analyses with Synechocystis sp. PCC 6803 (PCC6803) and Synechococcus sp. PCC7002 (PCC7002). The transcriptional analyses revealed that glycogen production in NKBG15041c under nitrogen-depleted conditions can be explained by the contribution of both increased carbon flux towards glycogen synthesis, similar to PCC6803 and PCC7002, and increased transcriptional levels of genes responsible for glycogen synthesis, which is different from the conventionally reported phenomenon, resulting in a relatively high amount of glycogen under ambient conditions compared to PCC6803 and PCC7002.