Plug-and-play protein biosensors using aptamer-regulated in vitro transcription.

Molecular biosensors that accurately measure protein concentrations without external equipment are critical for solving numerous problems in diagnostics and therapeutics. Modularly transducing the binding of protein antibodies, protein switches or aptamers into a useful output remains challenging. Here, we develop a biosensing platform based on aptamer-regulated transcription in which aptamers integrated into transcription templates serve as inputs to molecular circuits that can be programmed to a produce a variety of responses. We modularly design molecular biosensors using this platform by swapping aptamer domains for specific proteins and downstream domains that encode different RNA transcripts. By coupling aptamer-regulated transcription with diverse transduction circuits, we rapidly construct analog protein biosensors and digital protein biosensors with detection ranges that can be tuned over two orders of magnitude and can exceed the binding affinity of the aptamer. Aptamer-regulated transcription is a straightforward and inexpensive approach for constructing programmable protein biosensors that could have diverse applications in research and biotechnology.

In vitro selection of a trans aptamer complex for target-responsive fluorescence activation.

BACKGROUND: Most biological molecular complexes consist of multiple functional domains, yet rationally constructing such multifunctional complexes is challenging. Aptamers, the nucleic acid-based functional molecules, can perform multiple tasks including target recognition, conformational changes, and enzymatic activities, while being chemically synthesizable and tunable, and thus provide a basis for engineering enhanced functionalities through combination of multiple units. However, the conventional approach of simply combining aptamer units in a serial manner is susceptible to undesired crosstalk or interference between the aptamer units and to false interactions with non-target molecules; besides, the approach would require additional mechanisms to separate the units if they are desired to function independently. It is clearly a challenge to develop multi-aptamer complexes that preserve independent functions of each unit while avoiding undesired interference and non-specific interactions. RESULTS: By directly in vitro selecting a 'trans' aptamer complex, we demonstrate that one aptamer unit ('utility module') can remain hidden or 'inactive' until a target analyte triggers the other unit ('sensing module') and separates the two aptamers. Since the operation of the utility module occurs free from the sensing module, unnecessary crosstalk between the two units can be avoided. Because the utility module is kept inactive until separated from the complex, non-specific interactions of the hidden module with noncognate targets can be naturally prevented. In our demonstration, the sensing module was selected to detect serotonin, a clinically important neurotransmitter, and the target-binding-induced structure-switching of the sensing module reveals and activates the utility module that turns on a fluorescence signal. The aptamer complex exhibited a moderately high affinity and an excellent specificity for serotonin with ∼16-fold discrimination against common neurotransmitter molecules, and displayed strong robustness to perturbations in the design, disallowing nonspecific reactions against various challenges. SIGNIFICANCE: This work represents the first example of a trans aptamer complex that was in vitro selected de novo. The trans aptamer complex selected by our strategy does not require chemical modifications or immediate optimization processes to function, because the complex is directly selected to perform desired functions. This strategy should be applicable to a wide range of functional nucleic acid moieties, which will open up diverse applications in biosensing and molecular therapeutics.

Ionic liquid-caged nucleic acids enable active folding-based molecular recognition with hydrolysis resistance.

Beyond storage and transmission of genetic information in cellular life, nucleic acids can perform diverse interesting functions, including specific target recognition and biochemical reaction acceleration; the versatile biopolymers, however, are acutely vulnerable to hydrolysis-driven degradation. Here, we demonstrate that the cage effect of choline dihydrogen phosphate permits active folding of nucleic acids like water, but prevents their phosphodiester hydrolysis unlike water. The choline-based ionic liquid not only serves as a universal inhibitor of nucleases, exceptionally extending half-lives of nucleic acids up to 6 500 000 times, but highly useful tasks of nucleic acids (e.g. mRNA detection of molecular beacons, ligand recognition of aptamers, and transesterification reaction of ribozymes) can be also conducted with well-conserved affinities and specificities. As liberated from the function loss and degradation risk, the presence of undesired and unknown nucleases does not undermine desired molecular functions of nucleic acids without hydrolysis artifacts even in nuclease cocktails and human saliva.

Type-Independent 3D Writing and Nano-Patterning of Confined Biopolymers.

Biopolymers are essential building blocks that constitute cells and tissues with well-defined molecular structures and diverse biological functions. Their three-dimensional (3D) complex architectures are used to analyze, control, and mimic various cells and their ensembles. However, the free-form and high-resolution structuring of various biopolymers remain challenging because their structural and rheological control depend critically on their polymeric types at the submicron scale. Here, direct 3D writing of intact biopolymers is demonstrated using a systemic combination of nanoscale confinement, evaporation, and solidification of a biopolymer-containing solution. A femtoliter solution is confined in an ultra-shallow liquid interface between a fine-tuned nanopipette and a chosen substrate surface to achieve directional growth of biopolymer nanowires via solvent-exclusive evaporation and concurrent solution supply. The evaporation-dependent printing is biopolymer type-independent, therefore, the 3D motor-operated precise nanopipette positioning allows in situ printing of nucleic acids, polysaccharides, and proteins with submicron resolution. By controlling concentrations and molecular weights, several different biopolymers are reproducibly patterned with desired size and geometry, and their 3D architectures are biologically active in various solvents with no structural deformation. Notably, protein-based nanowire patterns exhibit pin-point localization of spatiotemporal biofunctions, including target recognition and catalytic peroxidation, indicating their application potential in organ-on-chips and micro-tissue engineering.

De novo selected hACE2 mimics that integrate hotspot peptides with aptameric scaffolds for binding tolerance of SARS-CoV-2 variants.

The frequent occurrence of viral variants is a critical problem in developing antiviral prophylaxis and therapy; along with stronger recognition of host cell receptors, the variants evade the immune system-based vaccines and neutralizing agents more easily. In this work, we focus on enhanced receptor binding of viral variants and demonstrate generation of receptor-mimicking synthetic reagents, capable of strongly interacting with viruses and their variants. The hotspot interaction of viruses with receptor-derived short peptides is maximized by aptamer-like scaffolds, the compact and stable architectures of which can be in vitro selected from a myriad of the hotspot peptide-coupled random nucleic acids. We successfully created the human angiotensin-converting enzyme 2 (hACE2) receptor-mimicking hybrid ligand that recruits the hACE2-derived receptor binding domain-interacting peptide to directly interact with a binding hotspot of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Experiencing affinity boosting by ~500% to Omicron, the de novo selected hACE2 mimic exhibited a great binding tolerance to all SARS-CoV-2 variants of concern.

Noncovalent minimal assembly of exogenous histamine with hemin cofactor as a peroxidase-mimicking cooperative catalyst.

By mimicking the synergistic interplay of primary and secondary coordination spheres within native peroxidases, we demonstrate a scaffold-free, yet highly effective molecular-level cooperation between an iron(III)-containing hemin cofactor and exogenous histamine in accelerating a peroxidase-like reaction. Density functional theory computations predict that, among structurally similar molecules, the histamine is the most interactive partner of hemin to elicit a spontaneous peroxidation by electrostatically attracting the proton of hydrogen peroxide to its own imidazole and thermodynamically stabilizing a transition-state intermediate. Although the molecular weight of hemin-histamine pair is 763, 1.7% of the horseradish peroxidase, cooperative catalysis of two natural molecules exhibits 17.3 times greater catalytic efficiency (17.93 M-1s-1) and 57.8 times larger specific activity (36.45 µmol/min·mg) than the hemin alone (1.04 M-1s-1 and 0.63 µmol/min·mg). Despite no scaffold or covalent linkage, the self-assembly with hemin is highly histamine-specific in complex environments, leading rapid color changes by substrate oxidation within 10 s.

Live Streaming of a Single Cell's Life over a Local pH-Monitoring Nanowire Waveguide.

Spatiotemporal pH monitoring of single living cells across rigid cell and organelle membranes has been challenging, despite its significance in understanding cellular heterogeneity. Here, we developed a mechanically robust yet tolerably thin nanowire waveguide that enables in situ monitoring of pH dynamics at desired cellular compartments via direct optical communication. By chemically labeling fluorescein at one end of a poly(vinylbenzyl azide) nanowire, we continuously monitored pH variations of different compartments inside a living cell, successfully observing organelle-exclusive pH homeostasis and stimuli-selective pH regulations. Importantly, it was demonstrated for the first time that, during the mammalian cell cycle, the nucleus displays pH homeostasis in interphase but a tidal pH curve in the mitotic phase, implying the existence of independent pH-regulating activities by the nuclear envelope. The rapid and accurate local pH-reporting capability of our nanowire waveguide would be highly valuable for investigating cellular behaviors under diverse biological situations in living cells.

A unified computational view of DNA duplex, triplex, quadruplex and their donor-acceptor interactions.

DNA can assume various structures as a result of interactions at atomic and molecular levels (e.g., hydrogen bonds, π-π stacking interactions, and electrostatic potentials), so understanding of the consequences of these interactions could guide development of ways to produce elaborate programmable DNA for applications in bio- and nanotechnology. We conducted advanced ab initio calculations to investigate nucleobase model structures by componentizing their donor-acceptor interactions. By unifying computational conditions, we compared the independent interactions of DNA duplexes, triplexes, and quadruplexes, which led us to evaluate a stability trend among Watson-Crick and Hoogsteen base pairing, stacking, and even ion binding. For a realistic solution-like environment, the influence of water molecules was carefully considered, and the potassium-ion preference of G-quadruplex was first analyzed at an ab initio level by considering both base-base and ion-water interactions. We devised new structure factors including hydrogen bond length, glycosidic vector angle, and twist angle, which were highly effective for comparison between computationally-predicted and experimentally-determined structures; we clarified the function of phosphate backbone during nucleobase ordering. The simulated tendency of net interaction energies agreed well with that of real world, and this agreement validates the potential of ab initio study to guide programming of complicated DNA constructs.

Catalytic RNA, ribozyme, and its applications in synthetic biology.

Ribozymes are functional RNA molecules that can catalyze biochemical reactions. Since the discovery of the first catalytic RNA, various functional ribozymes (e.g., self-cleaving ribozymes, splicing ribozymes, RNase P, etc.) have been uncovered, and their structures and mechanisms have been identified. Ribozymes have the advantage of possessing features of "RNA" molecules; hence, they are highly applicable for manipulating various biological systems. To fully employ ribozymes in a broad range of biological applications in synthetic biology, a variety of ribozymes have been developed and engineered. Here, we summarize the main features of ribozymes and the methods used for engineering their functions. We also describe the past and recent efforts towards exploiting ribozymes for effective and novel applications in synthetic biology. Based on studies on their significance in biological applications till date, ribozymes are expected to advance technologies in artificial biological systems.

A Dual-Sensing DNA Nanostructure with an Ultrabroad Detection Range.

Despite considerable interest in the development of biosensors that can measure analyte concentrations with a dynamic range spanning many orders of magnitude, this goal has proven difficult to achieve. We describe here a modular biosensor architecture that integrates two different readout mechanisms into a single-molecule construct that can achieve target detection across an extraordinarily broad dynamic range. Our dual-mode readout DNA biosensor combines an aptamer and a DNAzyme to quantify adenosine triphosphate (ATP) with two different mechanisms, which respond to low (micromolar) and high (millimolar) concentrations by generating distinct readouts based on changes in fluorescence and absorbance, respectively. Importantly, we have also devised regulatory strategies to fine-tune the target detection range of each sensor module by controlling the target-sensitivity of each readout mechanism. Using this strategy, we report the detection of ATP at a dynamic range spanning 1-500 000 µM, more than 5 orders of magnitude, representing the largest dynamic range reported to date with a single biosensor construct.

Adsorption of Cd, Cu and Zn from aqueous solutions onto ferronickel slag under different potentially toxic metal combination.

Adsorption characteristics of potentially toxic metals in single- and multi-metal forms onto ferronickel slag were evaluated. Competitive sorption of metals by ferronickel slag has never been reported previously. The maximum adsorption capacities of toxic metals on ferronickel were in the order of Cd (10.2 mg g(-1)) > Cu (8.4 mg g(-1)) > Zn (4.4 mg g(-1)) in the single-metal adsorption isotherm and Cu (6.1 mg g(-1)) >> Cd (2.3 mg g(-1)) > Zn (0.3 mg g(-1)) in the multi-metal adsorption isotherm. In comparison with single-metal adsorption isotherm, the reduction rates of maximum toxic metal adsorption capacity in the multi-metal adsorption isotherm were in the following order of Zn (93%) > Cd (78%) >> Cu (27%). The Freundlich isotherm provides a slightly better fit than the Langmuir isotherm equation using ferronickel slag for potentially toxic metal adsorption. Multi-metal adsorption behaviors differed from single-metal adsorption due to competition, based on data obtained from Freundlich and Langmuir adsorption models and three-dimensional simulation. Especially, Cd and Zn were easily exchanged and substituted by Cu during multi-metal adsorption. Further competitive adsorption studies are necessary in order to accurately estimate adsorption capacity of ferronickel slag for potentially toxic metals in natural environments.

In vitro virucidal effect of mouthrinse containing C31G on seasonal influenza viruses.

C31G is a potent antimicrobial agent and can disrupt the microbial membrane by the alkyl portion of the molecule. The objective of this study was to evaluate the virucidal effectiveness of C31G and mouthrinse containing C31G (Sense-Time) on seasonal influenza viruses. Evaluation of the virucidal activity against influenza viruses was performed with end-point titration in 10-day-old chicken embryos and Madin-Darby canine kidney cells. In vitro studies demonstrated that C31G and Sense-Time inhibited the growth of seasonal influenza viruses even in the presence of 5% organic material. Gargling with C31G or Sense-Time would enhance oropharyngeal hygiene, which would be helpful for reducing influenza transmission.

Rapid identification of potentially probiotic Bifidobacterium species by multiplex PCR using species-specific primers based on the region extending from 16S rRNA through 23S rRNA.

This study aimed at developing a novel multiplex polymerase chain reaction (PCR) primer set for identification of the potentially probiotic Bifidobacterium species B. adolescentis, B. animalis subsp. animalis (B. animalis), B. bifidum, B. breve, B. longum biovar infantis (B. infantis), B. animalis subsp. lactis B. lactis, B. longum biovar longum (B. longum) and B. pseudolongum. The primer set comprised specific and conserved primers and was derived from the integrated sequences of 16S and 23S rRNA genes and the rRNA intergenic spacer region (ISR) of each species. It could detect and identify type strains and isolates from pharmaceuticals or dairy products corresponding to the eight Bifidobacterium species with high specificity. It was also useful for screening of the related strains from natural sources such as the gastro-intestinal tract and feces. We suggest that the assay system from this study is an efficient tool for simple, rapid and reliable identification of Bifidobacterium species for which probiotic strains are known.