ABSTRACT
Correction for 'Towards control of excitonic coupling in DNA-templated Cy5 aggregates: the principal role of chemical substituent hydrophobicity and steric interactions' by Sebastián A. Díaz et al., Nanoscale, 2023, 15, 3284-3299. https://doi.org/10.1039/D2NR05544A.
ABSTRACT
Peptide-based liquid-liquid phase separated domains, or coacervates, are a biomaterial gaining new interest due to their exciting potential in fields ranging from biosensing to drug delivery. In this study, we demonstrate that coacervates provide a simple and biocompatible medium to improve nucleic acid biosensors through the sequestration of both the biosensor and target strands within the coacervate, thereby increasing their local concentration. Using the well-established polyarginine (R9) - ATP coacervate system and an energy transfer-based DNA molecular beacon we observed three key improvements: i) a greater than 20-fold reduction of the limit of detection within coacervates when compared to control buffer solutions; ii) an increase in the kinetics, equilibrium was reached more than 4-times faster in coacervates; and iii) enhancement in the dye fluorescent quantum yields within the coacervates, resulting in greater signal-to-noise. The observed benefits translate into coacervates greatly improving bioassay functionality.
ABSTRACT
Scaffolded molecular networks are important building blocks in biological pigment-protein complexes, and DNA nanotechnology allows analogous systems to be designed and synthesized. System-environment interactions in these systems are responsible for important processes, such as the dissipation of heat and quantum information. This study investigates the role of nanoscale molecular parameters in tuning these vibronic system-environment dynamics. Here, genetic algorithm methods are used to obtain nanoscale parameters for a DNA-scaffolded chromophore network based on comparisons between its calculated and measured optical spectra. These parameters include the positions, orientations, and energy level characteristics within the network. This information is then used to compute the dynamics, including the vibronic population dynamics and system-environment heat currents, using the hierarchical equations of motion. The dissipation of quantum information is identified by the system's transient change in entropy, which is proportional to the heat currents according to the second law of thermodynamics. These results indicate that the dissipation of quantum information is highly dependent on the particular nanoscale characteristics of the molecular network, which is a necessary first step before gleaning the systematic optimization rules. Subsequently, the I-concurrence dynamics are calculated to understand the evolution of the vibronic system's quantum entanglement, which are found to be long-lived compared to these system-bath dissipation processes.
ABSTRACT
This work investigates the effect of plasmonic gold nanoparticle (AuNP) size on the rate of thermal release of single-stranded oligonucleotides under femtosecond (fs)-pulsed laser irradiation sources. Contrary to the theoretical predictions that larger AuNPs (50-60 nm diameter) would produce the most solution heating and fastest DNA release, it is found that smaller AuNP diameters (25 nm) lead to faster dsDNA denaturation rates. Controlling for the pulse energy fluence, AuNP concentration, DNA loading density, and the distance from the AuNP surface finds the same result. These results imply that the solution temperature increases around the AuNP during fs laser pulse optical heating may not be the only significant influence on dsDNA denaturation, suggesting that direct energy transfer from the AuNP to the DNA (phonon-phonon coupling), which is increased as AuNPs decrease in size, may play a significant role.
Subject(s)
Gold , Metal Nanoparticles , Heating , Lasers , DNAABSTRACT
Almost all pathogens, whether viral or bacterial, utilize key proteolytic steps in their pathogenesis. The ability to detect a pathogen's genomic material along with its proteolytic activity represents one approach to identifying the pathogen and providing initial evidence of its viability. Here, we report on a prototype biosensor design assembled around a single semiconductor quantum dot (QD) scaffold that is capable of detecting both nucleic acid sequences and proteolytic activity by using orthogonal energy transfer (ET) processes. The sensor consists of a central QD assembled via peptidyl-PNA linkers with multiple DNA sequences that encode complements to genomic sequences originating from the Ebola, Influenza, and COVID-19 viruses, which we use as surrogate targets. These are hybridized to complement strands labeled with a terbium (Tb) chelate, AlexaFluor647 (AF647), and Cy5.5 dyes, giving rise to two potential FRET cascades: the first includes Tb â QD â AF647 â Cy5.5 (â = ET step), which is detected in a time-gated modality, and QD â AF647 â Cy5.5, which is detected from direct excitation. The labeled DNA-displaying QD construct is then further assembled with a RuII-modified peptide, which quenches QD photoluminescence by charge transfer and is recognized by a protease to yield the full biosensor. Each of the labeled DNAs and peptides can be ratiometrically assembled to the QD in a controllable manner to tune each of the ET pathways. Addition of a given target DNA displaces its labeled complement on the QD, disrupting that FRET channel, while protease addition disrupts charge transfer quenching of the central QD scaffold and boosts its photoluminescence and FRET relay capabilities. Along with characterizing the ET pathways and verifying biosensing in both individual and multiplexed formats, we also demonstrate the ability of this construct to function in molecular logic and perform Boolean operations; this highlights the construct's ability to discriminate and transduce signals between different inputs or pathogens. The potential application space for such a sensor device is discussed.
Subject(s)
Biosensing Techniques , Carbocyanines , Quantum Dots , Quantum Dots/chemistry , Peptide Hydrolases/metabolism , Fluorescence Resonance Energy Transfer , Peptides/chemistry , DNA/chemistry , Endopeptidases/metabolismABSTRACT
Introducción: El uso excesivo de las redes sociales ha generado diversas secuelas en la salud mental y visual-ocular, dando lugar al Síndrome Visual Informático (SVI) por sobreexposición a las pantallas. Objetivo: Analizar la influencia del uso de las redes sociales en el síndrome visual informático en adolescentes en una realidad peruana. Métodos: Se realizó una investigación de enfoque cuantitativo, hipotético-deductivo, transaccional, correlacional-causal, con una muestra de 126 adolescentes. Para recabar información acerca del uso de las redes sociales, se utilizó el ARS de Escurra y Salas. Para verificar la prevalencia del SVI, se empleó el Cuestionario de Síndrome Visual Informático de Seguí y colaboradores; ambos instrumentos fueron adaptados a formato Google Forms para su aplicación en línea. Resultados: Se encontró una asociación significativa entre el tiempo de permanencia en las redes sociales (RS) y el SVI (p=0,027<0,05); además, se evidenció el impacto significativo de la Adicción a las Redes Sociales (ARS) sobre el SVI (p=0,000<0.01), los síntomas visuales (p=0,000<0.01), oculares (p=0,000<0.01) y astenópicos (p=0,003<0.01). Conclusiones: El estudio demuestra una clara conexión entre el uso excesivo de redes sociales y el SVI en adolescentes peruanos, subrayando la necesidad de una acción conjunta de padres y educadores para mitigar los riesgos y promover un uso digital saludable(AU)
Introduction: The excessive use of social media has generated various sequelae on mental and visual-ocular health, leading to Computer Vision Syndrome (CVS) due to screen overexposure. Objective: To analyze the influence of social media use on computer vision syndrome in adolescents in a Peruvian context. Methods: A quantitative, hypothetical-deductive, transactional, correlational-causal study was conducted with a sample of 126 adolescents. To gather information about social media use, the ARS by Escurra and Salas was used. To verify the prevalence of CVS, the Computer Vision Syndrome Questionnaire by Seguí and collaborators was employed; both instruments were adapted to Google Forms format for online application. Results: A significant association was found between the time spent on social media (SM) and CVS (p=0.027<0.05); additionally, a significant impact of Social Media Addiction (SMA) on CVS (p=0.000<0.01), visual symptoms (p=0.000<0.01), ocular symptoms (p=0.000<0.01), and asthenopic symptoms (p=0.003<0.01) was evidenced. Conclusions: The study demonstrates a clear connection between excessive use of social media and CVS in Peruvian adolescents, highlighting the need for joint action by parents and educators to mitigate risks and promote healthy digital use(AU)
Subject(s)
Humans , Adolescent , Syndrome , Asthenopia/epidemiology , Social Networking , Internet Addiction Disorder , Coping Skills , Peru , Mental Health , Internet Addiction Disorder/etiologyABSTRACT
Lizards, except geckos, are generally considered voiceless organisms, although some species emit oral sounds. For most of these "vocal lizards", however, there is almost no information on the characteristics of the sounds, precluding exploration of the functionality and evolution of the sounds. Pristidactylus are known as "grunter lizards" since individuals emit oral sounds under predation risk. We explored the characteristics of the sounds emitted by P. valeriae, recording 17 adults and 1 juvenile when they were threatened and captured by a predator. Only adults emitted sounds with open mouths and displayed aggressive postures, e.g., biting attempts. These sounds correspond to hisses, which lack amplitude or frequency modulation. The lizards emitted longer hisses when threatened than when captured by the predator, which may provide honest information on individuals' ability to escape. In addition, males may experience higher distress during threats since their hisses had higher aggregate entropy than those of the females. Finally, hissing has been documented in four of the five Leiosauridae genera, the family to which Pristidactylus belongs, suggesting that sound emission is ancestral to the family.
ABSTRACT
DNA nanotechnology has now enabled the self-assembly of almost any prescribed 3-dimensional nanoscale structure in large numbers and with high fidelity. These structures are also amenable to site-specific modification with a variety of small molecules ranging from drugs to reporter dyes. Beyond obvious application in biotechnology, such DNA structures are being pursued as programmable nanoscale optical breadboards where multiple different/identical fluorophores can be positioned with sub-nanometer resolution in a manner designed to allow them to engage in multistep excitonic energy-transfer (ET) via Förster resonance energy transfer (FRET) or other related processes. Not only is the ability to create such complex optical structures unique, more importantly, the ability to rapidly redesign and prototype almost all structural and optical analogues in a massively parallel format allows for deep insight into the underlying photophysical processes. Dynamic DNA structures further provide the unparalleled capability to reconfigure a DNA scaffold on the fly in situ and thus switch between ET pathways within a given assembly, actively change its properties, and even repeatedly toggle between two states such as on/off. Here, we review progress in developing these composite materials for potential applications that include artificial light harvesting, smart sensors, nanoactuators, optical barcoding, bioprobes, cryptography, computing, charge conversion, and theranostics to even new forms of optical data storage. Along with an introduction into the DNA scaffolding itself, the diverse fluorophores utilized in these structures, their incorporation chemistry, and the photophysical processes they are designed to exploit, we highlight the evolution of DNA architectures implemented in the pursuit of increased transfer efficiency and the key lessons about ET learned from each iteration. We also focus on recent and growing efforts to exploit DNA as a scaffold for assembling molecular dye aggregates that host delocalized excitons as a test bed for creating excitonic circuits and accessing other quantum-like optical phenomena. We conclude with an outlook on what is still required to transition these materials from a research pursuit to application specific prototypes and beyond.
Subject(s)
Fluorescence Resonance Energy Transfer , Quantum Dots , Quantum Dots/chemistry , Biotechnology , Fluorescent Dyes/chemistry , DNA/chemistryABSTRACT
This article presents a 3D-printed cylindrical dielectric resonator antenna operating at 5.8 GHz that achieves circular polarization by integrating a fully dielectric parasitic helix with a higher permittivity than the cylindrical resonator. The antenna polarization can be right-handed or left-handed depending on the turning sense of the helix. An extensive parametric study was done for the helix design to evaluate the effects of the dimensions and dielectric constant of the helix over the matching and axial ratio of the antenna. The manufacturing is made using low-loss dielectric filaments and a low-cost 3D printer. Simulation and measurement results show that both antennas are well-matched and operate with the corresponding circular polarization, with an axial ratio bandwidth compatible with UAV applications.
ABSTRACT
Objetivo: En marzo del año 2020, se declaró pandemia la enfermedad producida por el coro-navirus SARS-CoV-2 (COVID 19). Se estimaba inicialmente que el 5% de la población afectada por COVID-19 requeriría ingreso a unidades de cuidados intensivos con soporte de ventilación mecánica invasiva, pudiendo desarrollar secue-las a partir de la hospitalización. El equipo de rehabilitación domiciliaria se propone el desafío de realizar una serie de evaluaciones con la fi-nalidad de poder valorar la rehabilitación en el ámbito domiciliario.Método: Ensayo clínico no controlado de pa-cientes de la unidad de hospitalización domici-liaria que hayan sufrido COVID-19 con uso de VMI, entre junio 2020 y junio 2021. Ingresaron 193 pacientes, a los cuales se le realizó eva-luaciones al inicio y al final del proceso de re-habilitación por un equipo multidisciplinar en el domicilio del paciente.Resultados: Prevalencia de comorbilidades de hipertensión arterial y obesidad. en la forma grave de dicha enfermedad. Diferencia significa en todas las evaluaciones P (Wilcoxon)<0,001 entre el estado inicial y posterior a la rehabilita-ción, presencia de mayor deterioro en extremi-dades superiores.Conclusión: Pacientes con múltiples secuelas que requieren de la evaluación e intervención precoz de un equipo multidisciplinario, siendo la hospitalización domiciliaria una alternativa segura, eficiente y eficaz. Se logró el restable-cimiento de la deambulación segura e indepen-diente, la prevención de caídas, alimentación segura, recuperación de las destrezas cogniti-vas-comunicativas, y el empoderamiento de la familia en un contexto domiciliario. (AU)
Objective: In March 2020, the disease caused by the coronavirus SARS-CoV-2 (COVID-19) was declared a pandemic. It was initially esti-mated that 5% of the population affected by COVID-19 required admission to intensive care units with invasive mechanical ventilation sup-port, and may develop sequelae from hospitali-zation. The home rehabilitation team proposes the challenge of carrying out a series of evalua-tions in order to be able to assess rehabilitation in the home environment.Method: Uncontrolled clinical trial of patients from the home hospitalization unit who have suf-fered from COVID-19 with the use of IMV, be-tween June 2020 and June 2021. 193 patients were admitted, who underwent surgery at the beginning and at the end of the rehabilitation process for a multidisciplinary team at the pa-tients home.Results: Prevalence of comorbidities of arterial hypertension and obesity. in the severe form of this disease. Mean difference in all P (Wilcoxon) scores <0.001 between baseline and post-reha-bilitation status, presence of greater impairment in upper extremities.Conclusions: Patients with multiple sequelae that require early evaluation and intervention by a multidisciplinary team, home hospitalization being a safe, efficient and effective alternative. The restoration of safe and independent ambu-lation, the prevention of falls, safe eating, recov-ery of cognitive-communicative skills, and the empowerment of the family in a home context were achieved. (AU)
Subject(s)
Humans , Pandemics , House Calls , Coronavirus Infections/epidemiology , Physical Therapy Specialty , Physical Therapy Department, Hospital , Occupational Therapists , RehabilitationABSTRACT
Access to efficient enzymatic channeling is desired for improving all manner of designer biocatalysis. We demonstrate that enzymes constituting a multistep cascade can self-assemble with nanoparticle scaffolds into nanoclusters that access substrate channeling and improve catalytic flux by orders of magnitude. Utilizing saccharification and glycolytic enzymes with quantum dots (QDs) as a model system, nanoclustered-cascades incorporating from 4 to 10 enzymatic steps are prototyped. Along with confirming channeling using classical experiments, its efficiency is enhanced several fold more by optimizing enzymatic stoichiometry with numerical simulations, switching from spherical QDs to 2-D planar nanoplatelets, and by ordering the enzyme assembly. Detailed analyses characterize assembly formation and clarify structure-function properties. For extended cascades with unfavorable kinetics, channeled activity is maintained by splitting at a critical step, purifying end-product from the upstream sub-cascade, and feeding it as a concentrated substrate to the downstream sub-cascade. Generalized applicability is verified by extending to assemblies incorporating other hard and soft nanoparticles. Such self-assembled biocatalytic nanoclusters offer many benefits towards enabling minimalist cell-free synthetic biology.
Subject(s)
Nanoparticles , Quantum Dots , Nanoparticles/chemistry , Quantum Dots/chemistry , Biocatalysis , Catalysis , KineticsABSTRACT
Understanding and controlling exciton coupling in dye aggregates has become a greater focus as potential applications such as coherent exciton devices, nanophotonics, and biosensing have been proposed. DNA nanostructure templates allow for a powerful modular approach. Using DNA Holliday junction (HJ) templates variations of dye combinations and precision dye positions can be rapidly assayed, as well as creating aggregates of dyes that could not be prepared (either due to excess or lack of solubility) through alternative means. Indodicarbocyanines (Cy5) have been studied in coupled systems due to their large transition dipole moment, which contributes to strong coupling. Cy5-R dyes were recently prepared by chemically modifying the 5,5'-substituents of indole rings, resulting in varying dye hydrophobicity/hydrophilicity, steric considerations, and electron-donating/withdrawing character. We utilized Cy5-R dyes to examine the formation and properties of 30 unique DNA templated homodimers. We find that in our system the sterics of Cy5-R dyes play the determining factor in orientation and coupling strength of dimers, with coupling strengths ranging from 50-138 meV. The hydrophobic properties of the Cy5-R modify the percentage of dimers formed, and have a secondary role in determining the packing characteristics of the dimers when sterics are equivalent. Similar to other reports, we find that positioning of the Cy5-R within the HJ template can favor particular dimer interactions, specifically oblique or H-type dimers.
Subject(s)
Coloring Agents , DNA , DNA/chemistry , Carbocyanines/chemistry , DNA, CruciformABSTRACT
BACKGROUND: Despite representing the largest fraction of animal life, the number of insect species whose genome has been sequenced is barely in the hundreds. The order Dermaptera (the earwigs) suffers from a lack of genomic information despite its unique position as one of the basally derived insect groups and its importance in agroecosystems. As part of a national educational and outreach program in genomics, a plan was formulated to engage the participation of high school students in a genome sequencing project. Students from twelve schools across Chile were instructed to capture earwig specimens in their geographical area, to identify them and to provide material for genome sequencing to be carried out by themselves in their schools. RESULTS: The school students collected specimens from two cosmopolitan earwig species: Euborellia annulipes (Fam. Anisolabididae) and Forficula auricularia (Fam. Forficulidae). Genomic DNA was extracted and, with the help of scientific teams that traveled to the schools, was sequenced using nanopore sequencers. The sequence data obtained for both species was assembled and annotated. We obtained genome sizes of 1.18 Gb (F. auricularia) and 0.94 Gb (E. annulipes) with the number of predicted protein coding genes being 31,800 and 40,000, respectively. Our analysis showed that we were able to capture a high percentage (≥ 93%) of conserved proteins indicating genomes that are useful for comparative and functional analysis. We were also able to characterize structural elements such as repetitive sequences and non-coding RNA genes. Finally, functional categories of genes that are overrepresented in each species suggest important differences in the process underlying the formation of germ cells, and modes of reproduction between them, features that are one of the distinguishing biological properties that characterize these two distant families of Dermaptera. CONCLUSIONS: This work represents an unprecedented instance where the scientific and lay community have come together to collaborate in a genome sequencing project. The versatility and accessibility of nanopore sequencers was key to the success of the initiative. We were able to obtain full genome sequences of two important and widely distributed species of insects which had not been analyzed at this level previously. The data made available by the project should illuminate future studies on the Dermaptera.
Subject(s)
Insecta , Animals , Insecta/genetics , Sequence Analysis, DNA , ChileABSTRACT
Nature uses chromophore networks, with highly optimized structural and energetic characteristics, to perform important chemical functions. Due to its modularity, predictable aggregation characteristics, and established synthetic protocols, structural DNA nanotechnology is a promising medium for arranging chromophore networks with analogous structural and energetic controls. However, this high level of control creates a greater need to know how to optimize the systems precisely. This study uses the system's modularity to produce variations of a coupled 14-Site chromophore network. It uses machine-learning algorithms and spectroscopy measurements to reveal the energy-transport roles of these Sites, paying particular attention to the cooperative and inhibitive effects they impose on each other for transport across the network. The physical significance of these patterns is contextualized, using molecular dynamics simulations and energy-transport modeling. This analysis yields insights about how energy transfers across the Donor-Relay and Relay-Acceptor interfaces, as well as the energy-transport pathways through the homogeneous Relay segment. Overall, this report establishes an approach that uses machine-learning methods to understand, in fine detail, the role that each Site plays in an optoelectronic molecular network.
ABSTRACT
Coherently coupled pseudoisocyanine (PIC) dye aggregates have demonstrated the ability to delocalize electronic excitations and ultimately migrate excitons with much higher efficiency than similar designs where excitations are isolated to individual chromophores. Here, we report initial evidence of a new type of PIC aggregate, formed through heterogeneous nucleation on DNA oligonucleotides, displaying photophysical properties that differ significantly from previously reported aggregates. This new aggregate, which we call the super aggregate (SA) due to the need for elevated dye excess to form it, is clearly differentiated from previously reported aggregates by spectroscopic and biophysical characterization. In emission spectra, the SA exhibits peak narrowing and, in some cases, significant quantum yield variation, indicative of stronger coupling in cyanine dyes. The SA was further characterized with circular dichroism and atomic force microscopy observing unique features depending on the DNA substrate. Then by integrating an AlexaFluorTM647 (AF) dye as an energy transfer acceptor into the system, we observed mixed energy transfer characteristics using the different DNA. For example, SA formed with a rigid DNA double crossover tile (DX-tile) substrate resulted in AF emission sensitization. While SA formed with more flexible non-DX-tile DNA (i.e. duplex and single strand DNA) resulted in AF emission quenching. These combined characterizations strongly imply that DNA-based PIC aggregate properties can be controlled through simple modifications to the DNA substrate's sequence and geometry. Ultimately, we aim to inform rational design principles for future device prototyping. For example, one key conclusion of the study is that the high absorbance cross-section and efficient energy transfer observed with rigid substrates made for better photonic antennae, compared to flexible DNA substrates.
Subject(s)
Quinolines , Quinolines/chemistry , DNA/chemistry , DNA, Single-Stranded , Circular DichroismABSTRACT
Background An overwhelming majority of matriculating medical students in the USA are keen to deliver quality health care to all people, including the socioeconomically disadvantaged populations in remote, resource-scarce regions nationally and worldwide. Here, we describe a protocol developed to evaluate the interest of our medical students in global health activities. We also examined the relationship between students' interest in global health and readiness to pursue a future career in the primary care specialty. Materials and methods We designed a survey in Qualtrics online software and reached all first-year and third-year medical students between 2019 and 2022 enrolled at the Alabama College of Osteopathic Medicine (ACOM). The survey utilized ordinal scale items to explore the medical students' interest in primary care residency programs, their interest in global health and international travel, and their perceptions of how a range of factors might motivate their desire to participate in global health activities. The study was approved by ACOM's Institutional Review Board (IRB). In order to compare findings from this study with data from other medical schools, we developed constructs using the national aggregate data, in percentages, from matriculants and graduates of Doctor of Osteopathic Medicine (DO) degree-granting medical schools according to gender, published by the American Association of Colleges of Osteopathic Medicine (AACOM). Statistical analysis of national aggregate data was performed using the unpaired t-test. Results Both female and male participants had lived or traveled abroad before starting medical school. Female (98%, n=249) and male (95%, n=140) participants in the first-year cohorts considered helping the underserved population as important or very important as it is related to a career in medicine. Females in the third-year cohorts (97%, n=71) also ranked this statement as important or very important compared to male cohorts (89%, n=31). A higher proportion of females (43%, n=108) compared to males (35%, n=52) in first-year cohorts agreed or strongly agreed that they would likely pursue a residency in primary care. More females (59%, n=43) than males (46%, n=16) in the third-year cohorts agreed or strongly agreed with the same statement. Analysis of the aggregate national data (2009-2022) revealed that the percentage (actual count not available) of female students who planned to practice in underserved/shortage area was higher both at the time of matriculation (M=51%, SD=4%) and before graduation (M=40%, SD=4%) compared to males (matriculation: M=40%, SD=5%; graduation: M=33%, SD=4%) presenting a significant difference (matriculation t(24)=6.7, p<0.0001; graduation t(24)=5.4, p<0.0001). Furthermore, a higher percentage of females at the time of matriculation (M=25%, SD=5%) and graduation (M=40%, SD=6%) planned to practice in the primary care specialties compared to males (matriculation: M=17%, SD=4%; graduation: M=29%, SD=6%) presenting a significant difference (matriculation: t(24)=4.6, p = 0.0001; graduation: t(24)=4.8, p<0.0001). Conclusions Interest in global health activities may be associated with interest in pursuing a future career in the primary care specialty. In this study, more female medical students expressed interest in participating in global health experiences, serving the underserved population domestically and abroad, and expressing interest in primary care than males.
ABSTRACT
BACKGROUND: Despite representing the largest fraction of animal life, the number of insect species whose genome has been sequenced is barely in the hundreds. The order Dermaptera (the earwigs) suffers from a lack of genomic information despite its unique position as one of the basally derived insect groups and its importance in agroecosystems. As part of a national educational and outreach program in genomics, a plan was formulated to engage the participation of high school students in a genome sequencing project. Students from twelve schools across Chile were instructed to capture earwig specimens in their geographical area, to identify them and to provide material for genome sequencing to be carried out by themselves in their schools. RESULTS: The school students collected specimens from two cosmopolitan earwig species: Euborellia annulipes (Fam. Anisolabididae) and Forficula auricularia (Fam. Forficulidae). Genomic DNA was extracted and, with the help of scientific teams that traveled to the schools, was sequenced using nanopore sequencers. The sequence data obtained for both species was assembled and annotated. We obtained genome sizes of 1.18 Gb (F. auricularia) and 0.94 Gb (E. annulipes) with the number of predicted protein coding genes being 31,800 and 40,000, respectively. Our analysis showed that we were able to capture a high percentage (≥ 93%) of conserved proteins indicating genomes that are useful for comparative and functional analysis. We were also able to characterize structural elements such as repetitive sequences and non-coding RNA genes. Finally, functional categories of genes that are overrepresented in each species suggest important differences in the process underlying the formation of germ cells, and modes of reproduction between them, features that are one of the distinguishing biological properties that characterize these two distant families of Dermaptera. CONCLUSIONS: This work represents an unprecedented instance where the scientific and lay community have come together to collaborate in a genome sequencing project. The versatility and accessibility of nanopore sequencers was key to the success of the initiative. We were able to obtain full genome sequences of two important and widely distributed species of insects which had not been analyzed at this level previously. The data made available by the project should illuminate future studies on the Dermaptera.
Subject(s)
Animals , Insecta/genetics , Chile , Sequence Analysis, DNAABSTRACT
Cell-free synthetic biology has emerged as a valuable tool for the development of rapid, portable biosensors that can be readily transported in the freeze-dried form to the point of need eliminating cold chain requirements. One of the challenges associated with cell-free sensors is the ability to simultaneously detect multiple analytes within a single reaction due to the availability of a limited set of fluorescent and colorimetric reporters. To potentially provide multiplexing capabilities to cell-free biosensors, we designed a modular semiconductor quantum dot (QD)-based reporter platform that is plugged in downstream of the transcription-translation functionality in the cell-free reaction and which converts enzymatic activity in the reaction into distinct optical signals. We demonstrate proof of concept by converting restriction enzyme activity, utilized as our prototypical sensing output, into optical changes across several distinct spectral output channels that all use a common excitation wavelength. These hybrid Förster resonance energy transfer (FRET)-based QD peptide PNA-DNA-Dye reporters (QD-PDDs) are completely self-assembled and consist of differentially emissive QD donors paired to a dye-acceptor displayed on a unique DNA encoding a given enzyme's cleavage site. Three QD-based PDDs, independently activated by the enzymes BamHI, EcoRI, and NcoI, were prototyped in mixed enzyme assays where all three demonstrated the ability to convert enzymatic activity into fluorescent output. Simultaneous monitoring of each of the three paired QD-donor dye-acceptor spectral channels in cell-free biosensing reactions supplemented with added linear genes encoding each enzyme confirmed robust multiplexing capabilities for at least two enzymes when co-expressed. The modular QD-PDDs are easily adapted to respond to other restriction enzymes or even proteases if desired.
Subject(s)
Biosensing Techniques , Nucleic Acids , Quantum Dots , Fluorescence Resonance Energy Transfer , DNAABSTRACT
Strategies utilizing the CRISPR/Cas nucleases Cas13 and Cas12 have shown great promise in the development of highly sensitive and rapid diagnostic assays for the detection of pathogenic nucleic acids. The most common approaches utilizing fluorophore-quencher molecular beacons require strand amplification strategies or highly sensitive optical setups to overcome the limitations of the readout. Here, we demonstrate a flexible strategy for assembling highly luminescent and colorimetric quantum dot-nucleic acid hairpin (QD-HP) molecular beacons for use in CRISPR/Cas diagnostics. This strategy utilizes a chimeric peptide-peptide nucleic acid (peptide-PNA) to conjugate fluorescently labeled DNA or RNA hairpins to ZnS-coated QDs. QDs are particularly promising alternatives for molecular beacons due to their greater brightness, strong UV absorbance with large emission offset, exceptional photostability, and potential for multiplexing due to their sharp emission peaks. Using Förster resonance energy transfer (FRET), we have developed ratiometric reporters capable of pM target detection (without nucleotide amplification) for both target DNA and RNA, and we further demonstrated their capabilities for multiplexing and camera-phone detection. The flexibility of this system is imparted by the dual functionality of the QD as both a FRET donor and a central nanoscaffold for arranging nucleic acids and fluorescent acceptors on its surface. This method also provides a generalized approach that could be applied for use in other CRISPR/Cas nuclease systems.
Subject(s)
Nucleic Acids , Quantum Dots , Quantum Dots/chemistry , CRISPR-Cas Systems , DNA/chemistry , RNA , Peptides/chemistry , Fluorescence Resonance Energy Transfer/methodsABSTRACT
Molecular excitons are useful for applications in light harvesting, organic optoelectronics, and nanoscale computing. Electronic energy transfer (EET) is a process central to the function of devices based on molecular excitons. Achieving EET with a high quantum efficiency is a common obstacle to excitonic devices, often owing to the lack of donor and acceptor molecules that exhibit favorable spectral overlap. EET quantum efficiencies may be substantially improved through the use of heteroaggregates-aggregates of chemically distinct dyes-rather than individual dyes as energy relay units. However, controlling the assembly of heteroaggregates remains a significant challenge. Here, we use DNA Holliday junctions to assemble homo- and heterotetramer aggregates of the prototypical cyanine dyes Cy5 and Cy5.5. In addition to permitting control over the number of dyes within an aggregate, DNA-templated assembly confers control over aggregate composition, i.e., the ratio of constituent Cy5 and Cy5.5 dyes. By varying the ratio of Cy5 and Cy5.5, we show that the most intense absorption feature of the resulting tetramer can be shifted in energy over a range of almost 200 meV (1600 cm-1). All tetramers pack in the form of H-aggregates and exhibit quenched emission and drastically reduced excited-state lifetimes compared to the monomeric dyes. We apply a purely electronic exciton theory model to describe the observed progression of the absorption spectra. This model agrees with both the measured data and a more sophisticated vibronic model of the absorption and circular dichroism spectra, indicating that Cy5 and Cy5.5 heteroaggregates are largely described by molecular exciton theory. Finally, we extend the purely electronic exciton model to describe an idealized J-aggregate based on Förster resonance energy transfer (FRET) and discuss the potential advantages of such a device over traditional FRET relays.