RESUMO
DNA-templated silver nanoclusters (AgNC@DNA) are a novel type of nanomaterial with advantageous optical properties. Only a few atoms in size, the fluorescence of nanoclusters can be tuned using DNA overhangs. In this study, we explored the properties of AgNCs manufactured on a short single-stranded (dC)12 when adjacent G-rich sequences (dGN , with N = 3-15) were added. The 'red' emission of AgNC@dC12 with λMAX = 660 nm dramatically changed upon the addition of a G-rich overhang with NG = 15. The pattern of the emission-excitation matrix (EEM) suggested the emergence of two new emissive states at λMAX = 575 nm and λMAX = 710 nm. The appearance of these peaks provides an effective way to design biosensors capable of detecting specific nucleic acid sequences with low fluorescence backgrounds. We used this property to construct an NA-based switch that brings AgNC and the G overhang near one another, turning 'ON' the new fluorescence peaks only when a specific miRNA sequence is present. Next, we tested this detection switch on miR-371, which is overexpressed in prostate cancer. The results presented provide evidence that this novel fluorescent switch is both sensitive and specific with a limit of detection close to 22 picomoles of the target miR-371 molecule.
Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , MicroRNAs , Neoplasias da Próstata , Humanos , Masculino , MicroRNAs/genética , Fluorescência , Guanina , Espectrometria de Fluorescência/métodos , DNARESUMO
Silver has a long history of antibacterial effectiveness. The combination of atomically precise metal nanoclusters with the field of nucleic acid nanotechnology has given rise to DNA-templated silver nanoclusters (DNA-AgNCs) which can be engineered with reproducible and unique fluorescent properties and antibacterial activity. Furthermore, cytosine-rich single-stranded DNA oligonucleotides designed to fold into hairpin structures improve the stability of AgNCs and additionally modulate their antibacterial properties and the quality of observed fluorescent signals. In this work, we characterize the sequence-specific fluorescence and composition of four representative DNA-AgNCs, compare their corresponding antibacterial effectiveness at different pH, and assess cytotoxicity to several mammalian cell lines.
Assuntos
Antibacterianos/química , DNA de Cadeia Simples/química , Nanopartículas Metálicas/química , Prata/química , Antibacterianos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Fluorescência , Humanos , Nanopartículas Metálicas/toxicidade , Células THP-1RESUMO
Micro RNA (miR) are regulatory non-coding RNA molecules, which contain a small number of nucleotides ~18-28 nt. There are many various miR sequences found in plants and animals that perform important functions in developmental, metabolic, and disease processes. miRs can bind to complementary sequences within mRNA molecules thus silencing mRNA. Other functions include cardiovascular and neural development, stem cell differentiation, apoptosis, and tumors. In tumors, some miRs can function as oncogenes, others as tumor suppressors. Levels of certain miR molecules reflect cellular events, both normal and pathological. Therefore, miR molecules can be used as biomarkers for disease diagnosis and prognosis. One of these promising molecules is miR-21, which can serve as a biomarker with high potential for early diagnosis of various types of cancer. Here, we present a novel design of miR detection and demonstrate its efficacy on miR-21. The design employs emissive properties of DNA-silver nanoclusters (DNA/AgNC). The detection probe is designed as a hairpin DNA structure with one side of the stem complimentary to miR molecule. The binding of target miR-21 opens the hairpin structure, dramatically modulating emissive properties of AgNC hosted by the C12 loop of the hairpin. "Red" fluorescence of the DNA/AgNC probe is diminished in the presence of the target miR. At the same time, "green" fluorescence is activated and its intensity increases several-fold. The increase in intensity of "green" fluorescence is strong enough to detect the presence of miR-21. The intensity change follows the concentration dependence of the target miR present in a sample, which provides the basis of developing a new, simple probe for miR detection. The detection strategy is specific, as demonstrated using the response of the DNA/AgNC probe towards the scrambled miR-21 sequence and miR-25 molecule. Additionally, the design reported here is very sensitive with an estimated detection limit at ~1 picomole of miR-21.
Assuntos
Técnicas Biossensoriais/métodos , MicroRNAs/análise , Nanoestruturas/química , Prata/química , Técnicas Biossensoriais/instrumentação , Cor , DNA/química , Fluorescência , MicroRNAs/metabolismo , Técnicas de Sonda Molecular/instrumentação , Sondas Moleculares/química , Conformação de Ácido Nucleico , Moldes Genéticos , Termodinâmica , Raios UltravioletaRESUMO
The segmentation clock, a genetic oscillator in the presomitic mesoderm (PSM), is known to be influenced by biochemical signals, yet its potential regulation by mechanical cues remains unclear. The complex PSM microenvironment has made it challenging to isolate the effects of mechanical perturbations on clock behavior. Here we investigated how mechanical stimuli affect clock oscillations by culturing zebrafish PSM cells on PDMS micropost arrays with tunable rigidities (0.6-1200 kPa). We observed an inverse sigmoidal relationship between surface rigidity and both the percentage of oscillating cells and the number of oscillation cycles, with a switching threshold between 3-6 kPa. The periods of oscillating cells showed a consistently broad distribution across rigidity changes. Moreover, these cells exhibited distinct biophysical properties, such as reduced motility, contractility, and sustained circularity. These findings highlight the crucial role of cell-substrate interactions in regulating segmentation clock behavior, providing insights into the mechanobiology of somitogenesis.
RESUMO
Silver nanoclusters (AgNCs) are the next-generation nanomaterials representing supra-atomic structures where silver atoms are organized in a particular geometry. DNA can effectively template and stabilize these novel fluorescent AgNCs. Only a few atoms in size - the properties of nanoclusters can be tuned using only single nucleobase replacement of C-rich templating DNA sequences. A high degree of control over the structure of AgNC could greatly contribute to the ability to fine-tune the properties of silver nanoclusters. In this study, we explore the properties of AgNCs formed on a short DNA sequence with a C12 hairpin loop structure (AgNC@hpC12). We identify three types of cytosines based on their involvement in the stabilization of AgNCs. Computational and experimental results suggest an elongated cluster shape with 10 silver atoms. We found that the properties of the AgNCs depend on the overall structure and relative position of the silver atoms. The emission pattern of the AgNCs depends strongly on the charge distribution, while all silver atoms and some DNA bases are involved in optical transitions based on molecular orbital (MO) visualization. We also characterize the antibacterial properties of silver nanoclusters and propose a possible mechanism of action based on the interactions of AgNCs with molecular oxygen.
RESUMO
Combining atomically resolved DNA-templated silver nanoclusters (AgNCs) with nucleic acid nanotechnology opens new exciting possibilities for engineering bioinorganic nanomaterials with uniquely tunable properties. In this unforeseen cooperation, nucleic acids not only drive the formation of AgNCs but also promote their spatial organization in supra-assemblies. In this work, we confirm the feasibility of this approach using programmable RNA rings to control formation and optical properteis of six individual AgNCs. "Red" (λEXC/λEM = 565/623 nm) and "green" (λEXC/λEM = 440/523 nm) emitting AgNCs are templated on cytosine-rich DNA fragments embedded into the RNA rings. Optical properties of the AgNCs formed on the RNA rings are characterized in detail. While all "red" species passively transition to "green" emitters with time, the initial fluorescent properties and relative stabilities of "red" AgNCs can be regulated by altering the relative orientation of AgNCs within the RNA rings. As such, the oxidative stability increases dramatically for AgNC positioned towards the center of the RNA rings rather than facing outward. Overall, our findings expand the existing AgNC fluorescent toolkit while uncovering the complexity of the AgNC electronic structures with the abundance of possibilities for controlling de-excitation processes.
Assuntos
Nanopartículas Metálicas , Nanoestruturas , DNA , RNA , PrataRESUMO
Besides being a passive carrier of genetic information, DNA can also serve as an architecture template for the synthesis of novel fluorescent nanomaterials that are arranged in a highly organized network of functional entities such as fluorescent silver nanoclusters (AgNCs). Only a few atoms in size, the properties of AgNCs can be tuned using a variety of templating DNA sequences, overhangs, and neighboring duplex regions. In this study, we explore the properties of AgNCs manufactured on a short DNA sequence-an individual element designed for a construction of a larger DNA-based functional assembly. The effects of close proximity of the double-stranded DNA, the directionality of templating single-stranded sequence, and conformational heterogeneity of the template are presented. We observe differences between designs containing the same AgNC templating sequence-twelve consecutive cytosines, (dC)12. AgNCs synthesized on a single "basic" templating element, (dC)12, emit in "red". The addition of double-stranded DNA core, required for the larger assemblies, changes optical properties of the silver nanoclusters by adding a new population of clusters emitting in "green". A new population of "blue" emitting clusters forms only when ssDNA templating sequence is placed on the 5' end of the double-stranded core. We also compare properties of silver nanoclusters, which were incorporated into a dimeric structure-a first step towards a larger assembly.