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1.
Biosens Bioelectron ; 195: 113645, 2022 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-34571483

RESUMO

Various sensing platforms based on molecular or nanosystems are widely exploited through molecular diversity and specific recognition. However, it is extremely challenging to develop systems with tunable sensing ability and utilize the systems as information carriers/covers for communication and safety. Herein, DNA nanosensing systems based on cobalt oxyhydroxide (CoOOH) nanosheets were constructed for tunable detection and valence distinction of metal ions, molecular crypto-steganography, and information coding. CoOOH nanosheets absorb fluorescence-labeled single-stranded DNA with different bases and lengths, resulting in fluorescence quenching. The binding priority of bases with CoOOH nanosheets was guanine (G) > cytosine (C) > adenine (A) ≈ thymine (T) and the short chain excelled long chain. Due to the differences in the interaction among CoOOH, DNA, metal ions and variability of DNA bases, various DNA-CoOOH nanosystems have significantly different selective response patterns (that is selectivity) to metal ions and tunable linear ranges to Fe3+, Hg2+, Cr3+. Interestingly, by utilizing their molecular diversity, recognition, selective patterns, DNA-CoOOH sensing systems can be served as doubly cryptographic and steganographic systems to implement information encoding, encryption, and hiding and to reversely improve the selectivity of metal ions. This study provides an idea and platform for adjustable detection and valence distinction of metal ions, and gives a set of "molecular programming languages" for designing intelligent programmable sensing and molecular information communication and safety systems.


Assuntos
Técnicas Biossensoriais , DNA/genética , DNA de Cadeia Simples , Íons
2.
Chemosphere ; 287(Pt 2): 132163, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34509014

RESUMO

A structural switching in Single-stranded DNA (ssDNA) fluorescence biosensor for quick turn-on/off detection of Pb2+ ions and pesticide was reported. The design strategy of Hex-labelled ssDNA consists of two types of aptamer probe, G-rich base pair sequence forms G-quadruplex confirmation with Pb2+ ions. While other part of base pair sequence exhibits affinity to fold isocarbophos pesticide. MoS2 nanosheets were identified as quick quencher of Hex fluorescence intensity via Vander-Waals interaction and its significance was compared with other nanomaterials. This sensing mechanism proposes a specific affinity of GA-rich ssDNA with Pb2+ to form G-quadruplex via G-Pb2+-G sequences. Consequently, ssDNA relived from MoS2 nanosheets to restore the fluorescence intensity (turn-on). Subsequent addition of pesticide shows stronger affinity towards unfolded aptamer probe to form a random coil like structure. This causes Hex-labelled 5' end closer to the G-quadruplex connected at the 3' end of ssDNA resulting in a remarkable fluorescence quenching (turn-off) owing to PET process. Moreover, the sensing probe (Hex-labelled GA-rich ssDNA) was recycled by introducing acetylcholinesterase enzyme and thiocoline into the reaction mixture. The detection limits of Pb2+ and isocarbophos pesticide was estimated to be 0.6 nM and 0.018 µg/L respectively. Moreover, this study reveals a high sensitivity and selectivity towards target molecules in environmental samples.


Assuntos
Técnicas Biossensoriais , Praguicidas , Acetilcolinesterase , DNA de Cadeia Simples , Chumbo , Limite de Detecção , Compostos Organofosforados
3.
Anal Chem ; 93(45): 15216-15223, 2021 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-34736322

RESUMO

The development of a sensitive, facile, and cost-effective colorimetric method is of great significance for the point-of-care testing of viral nucleic acid. Herein, we reported a strand displacement amplification assisted CRISPR-Cas12a (SDACC) method for the colorimetric analysis of viral nucleic acid. The hepatitis B virus (HBV) DNA was chosen as the target to trigger strand displacement amplification (SDA) and generate abundant single-strand DNA (ssDNA) products. The ssDNA amplicon hybridized with template DNA to activate the trans-cleavage activity of CRISPR-Cas12a, leading to the nonspecific cleavage of ssDNA on GOx-ssDNA-modified magnetic beads and the release of GOx. The released GOx was capable of catalyzing the substrate solution to generate a color change, which could be directly observed by naked eyes. The SDACC strategy could identify a single-base mismatch located in the DNA sequence and achieve a sensitive detection for HBV DNA with the limit of detection as low as 41.8 fM. Notably, the sophisticated primer design for target amplification and complicated detection process could be circumvented. The current approach realizes a simple, low-cost, and sensitive colorimetric detection for viral nucleic acid and holds great promise for the practical application of virus infection diagnosis.


Assuntos
Técnicas Biossensoriais , Ácidos Nucleicos , Sistemas CRISPR-Cas/genética , Colorimetria , DNA , DNA de Cadeia Simples/genética , Técnicas de Amplificação de Ácido Nucleico
4.
Phys Rev Lett ; 127(13): 138103, 2021 Sep 24.
Artigo em Inglês | MEDLINE | ID: mdl-34623840

RESUMO

Nanopores in 2D materials are highly desirable for DNA sequencing, yet achieving single-stranded DNA (ssDNA) transport through them is challenging. Using density functional theory calculations and molecular dynamics simulations we show that ssDNA transport through a pore in monolayer hexagonal boron nitride (h-BN) is marked by a basic nanomechanical conflict. It arises from the notably inhomogeneous flexural rigidity of ssDNA and causes high friction via transient DNA desorption costs exacerbated by solvation effects. For a similarly sized pore in bilayer h-BN, its self-passivated atomically smooth edge enables continuous ssDNA transport. Our findings shed light on the fundamental physics of biopolymer transport through pores in 2D materials.


Assuntos
Compostos de Boro/química , DNA de Cadeia Simples/química , Nanoestruturas/química , Fenômenos Biofísicos , Modelos Químicos , Simulação de Dinâmica Molecular , Nanoporos
5.
Anal Chem ; 93(43): 14568-14576, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34672523

RESUMO

Single-nucleotide polymorphisms (SNPs) are important hallmarks of human diseases. Herein, we develop a single quantum dot (QD)-mediated fluorescence resonance energy transfer (FRET) nanosensor with the integration of multiple primer generation rolling circle amplification (MPG-RCA) for sensitive detection of SNPs in cancer cells. This assay involves only a linear padlock probe for MPG-RCA. The presence of a mutant target facilitates the circularization of linear padlock probes to initiate RCA, producing three short single-stranded DNAs (ssDNAs) with the assistance of nicking endonuclease. The resulting ssDNAs can function as primers to induce cyclic MPG-RCA, resulting in the exponential amplification and generation of large numbers of linker probes. The linker probes can subsequently hybridize with the Cy5-labeled reporter probes and the biotinylated capture probes to obtain the sandwich hybrids. The assembly of these sandwich hybrids on the 605 nm-emission quantum dot (605QD) generates the 605QD-oligonucleotide-Cy5 nanostructures, resulting in efficient FRET from the 605QD to Cy5. This nanosensor is free from both the complicated probe design and the exogenous primers and has distinct advantages of high amplification efficiency, zero background signal, good specificity, and high sensitivity. It can detect SNPs with a large dynamic range of 8 orders of magnitude and a detection limit of 5.41 × 10-20 M. Moreover, this nanosensor can accurately distinguish as low as 0.001% mutation level from the mixtures, which cannot be achieved by previously reported methods. Furthermore, it can discriminate cancer cells from normal cells and even quantify SNP at the single-cell level.


Assuntos
Neoplasias , Pontos Quânticos , DNA de Cadeia Simples , Transferência Ressonante de Energia de Fluorescência , Humanos , Neoplasias/genética , Técnicas de Amplificação de Ácido Nucleico , Nucleotídeos , Polimorfismo de Nucleotídeo Único
6.
Int J Mol Sci ; 22(19)2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34638562

RESUMO

Genome editing is an indispensable tool for functional genomics. The caveat of the genome-editing pipeline is a prevalence of error-prone non-homologous end joining over homologous recombination, while only the latter is suitable to introduce particularly desired genetic variants. To overcome this problem, a toolbox of genome engineering was appended by a variety of improved instruments. In this work, we compared the efficiency of a number of recently suggested improved systems for genome editing applied to the same genome regions on a murine zygote model via microinjection. As a result, we observed that homologous recombination utilizing an ssDNA template following sgRNA directed Cas9 cleavage is still the method of choice for the creation of animals with precise genome alterations.


Assuntos
Edição de Genes/métodos , Zigoto/metabolismo , Animais , Sistemas CRISPR-Cas , Reparo do DNA por Junção de Extremidades , DNA de Cadeia Simples , Recombinação Homóloga , Camundongos , Microinjeções/métodos , Modelos Animais , RNA Guia
7.
Appl Microbiol Biotechnol ; 105(23): 8895-8906, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34714365

RESUMO

Canine parvovirus-2 (CPV-2) is ubiquitously distributed in dog population worldwide causing a severe and often fatal gastroenteritis. Owing to its highly contagious nature, rapid detection of CPV is crucial in effective control of the disease. Aptamers have emerged as potential alternative to antibodies as affinity reagents in diagnostic field. Present study was aimed to select and validate ssDNA aptamers specific to CPV. Systematic evolution of ligands through exponential enrichment (SELEX) method was employed for selection of CPV structural protein (VP2) specific DNA aptamers. SELEX was performed using a pool of ssDNA library comprising 30 random nucleotide region. A total of seven rounds of SELEX were performed using VP2 protein as target antigen which yielded ten aptamers (1A-10A) with distinct sequences. Target binding of all ten aptamers was assessed by dot blot and enzyme-linked oligonucleotide assay (ELONA) which revealed that 5A, 6A, 9A, and 10A were superior binders. In silico analysis of the aptamers revealed the existence of binding site on VP2 protein, and binding pattern was similar to in vitro findings. The affinity (KD) of all these four binders against CPV was evaluated by ELONA indicating relatively higher affinity of 6A and 10A than remaining two DNA sequences. Out of which, aptamer 6A displayed cross-reactivity with canine distemper virus and canine corona virus. Hence, aptamer 10A was considered as better binding sequence having high specificity and affinity for CPV. The study confirms the future utility of selected aptamers in development of a reliable diagnostic for rapid detection of CPV. KEY POINTS: • Canine parvovirus-specific ssDNA aptamers were identified with nanomolar affinity (100-150 nM). • Three aptamers displayed negligible cross-reactivity with other related viruses. • Aptamer 10A displayed high binding affinity and specificity to CPV.


Assuntos
Aptâmeros de Nucleotídeos , Parvovirus Canino , Animais , DNA de Cadeia Simples/genética , Cães , Biblioteca Gênica , Parvovirus Canino/genética , Técnica de Seleção de Aptâmeros
8.
Biochemistry ; 60(41): 3086-3097, 2021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34613715

RESUMO

Understanding the thermodynamic mechanisms of adaptation of biomacromolecules to high hydrostatic pressure can help shed light on how piezophilic organisms can survive at pressures reaching over 1000 atmospheres. Interaction of proteins with nucleic acids is one of the central processes that allow information flow encoded in the sequence of DNA. Here, we report the results of a study on the interaction of cold shock protein B from Bacillus subtilis (CspB-Bs) with heptadeoxythymine template (pDT7) as a function of temperature and hydrostatic pressure. Experimental data collected at different CspB-Bs:pDT7 ratios were analyzed using a thermodynamic linkage model that accounts for both protein unfolding and CspB-Bs:pDT7 binding. The global fit to the model provided estimates of the stability of CspB-Bs, ΔGProto, the volume change upon CspB-Bs unfolding, ΔVProt, the association constant for CspB-Bs:pDT7 complex, Kao, and the volume changes upon pDT7 single-stranded DNA (ssDNA) template binding, ΔVBind. The protein, CspB-Bs, unfolds with an increase in hydrostatic pressure (ΔVProt < 0). Surprisingly, our study showed that ΔVBind < 0, which means that the binding of CspB-Bs to ssDNA is stabilized by an increase in hydrostatic pressure. Thus, CspB-Bs binding to pDT7 represents a case of linked equilibrium in which folding and binding react differently upon an increase in hydrostatic pressure: protein folding/unfolding equilibrium favors the unfolded state, while protein-ligand binding equilibrium favors the bound state. These opposing effects set a "maximum attainable" pressure tolerance to the protein-ssDNA complex under given conditions.


Assuntos
Proteínas de Bactérias/metabolismo , DNA de Cadeia Simples/metabolismo , Bacillus subtilis/química , Proteínas de Bactérias/química , DNA de Cadeia Simples/química , Pressão Hidrostática , Ligação Proteica , Desdobramento de Proteína , Temperatura , Termodinâmica
9.
Nat Commun ; 12(1): 5966, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34645815

RESUMO

The BRCA2 tumor suppressor protects genome integrity by promoting homologous recombination-based repair of DNA breaks, stability of stalled DNA replication forks and DNA damage-induced cell cycle checkpoints. BRCA2 deficient cells display the radio-resistant DNA synthesis (RDS) phenotype, however the mechanism has remained elusive. Here we show that cells without BRCA2 are unable to sufficiently restrain DNA replication fork progression after DNA damage, and the underrestrained fork progression is due primarily to Primase-Polymerase (PRIMPOL)-mediated repriming of DNA synthesis downstream of lesions, leaving behind single-stranded DNA gaps. Moreover, we find that BRCA2 associates with the essential DNA replication factor MCM10 and this association suppresses PRIMPOL-mediated repriming and ssDNA gap formation, while having no impact on the stability of stalled replication forks. Our findings establish an important function for BRCA2, provide insights into replication fork control during the DNA damage response, and may have implications in tumor suppression and therapy response.


Assuntos
Proteína BRCA2/genética , DNA Primase/genética , DNA de Neoplasias/genética , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/genética , Proteínas de Manutenção de Minicromossomo/genética , Enzimas Multifuncionais/genética , Reparo de DNA por Recombinação , Proteína BRCA2/antagonistas & inibidores , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Dano ao DNA , DNA Helicases/antagonistas & inibidores , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Primase/antagonistas & inibidores , DNA Primase/metabolismo , Replicação do DNA , DNA de Neoplasias/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Proteínas de Manutenção de Minicromossomo/antagonistas & inibidores , Proteínas de Manutenção de Minicromossomo/metabolismo , Enzimas Multifuncionais/antagonistas & inibidores , Enzimas Multifuncionais/metabolismo , Osteoblastos/metabolismo , Osteoblastos/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
Elife ; 102021 10 19.
Artigo em Inglês | MEDLINE | ID: mdl-34665130

RESUMO

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA-repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5'-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.


Assuntos
Caenorhabditis elegans/genética , Reparo do DNA , DNA de Cadeia Simples/genética , DNA/genética , Edição de Genes/métodos , Camundongos/genética , Peixe-Zebra/genética , Animais , Células HEK293 , Humanos , Células K562
11.
Chem Commun (Camb) ; 57(71): 8977-8980, 2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34486619

RESUMO

This study reports a photoelectrochemical biosensor for dopamine-loaded liposome-encoded magnetic beads cleaved by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas 12a system for the quantification of human papilloma virus (HPV)-related DNA using neodymium-doped BiOBr nanosheets (Nd-BiOBr) as a photoactive matrix. Magnetic beads and dopamine-loaded liposomes are covalently attached to the both ends of ssDNA to construct dumbbell-shaped dopamine-loaded liposome-encoded magnetic bead (DLL-MB) probes. When the guide RNA binds to the target HPV-16, the ssDNA will be cleaved by Cas12a, thereby degrading the double dumbbell probes. After magnetic separation, the dissolved DLLs are treated with Triton X-100 to release the dopamine (as an electron donor), which was then detected by an amplified photocurrent using the Nd-BiOBr-based photoelectrode.


Assuntos
Proteínas de Bactérias/química , Técnicas Biossensoriais/métodos , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , DNA Viral/análise , Endodesoxirribonucleases/química , DNA de Cadeia Simples/química , Dopamina/química , Técnicas Eletroquímicas/métodos , Fluoresceínas/química , Corantes Fluorescentes/química , Papillomavirus Humano 16/química , Lipossomos/química , Fenômenos Magnéticos , Processos Fotoquímicos
12.
Talanta ; 235: 122749, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517617

RESUMO

Signal output mode is the important part of biosensor. In general, "signal on" and "signal off" are two common output modes. The development of dual signals-based ratio analysis as a powerful diagnostic tool has attracted widespread attention in the biosensor field in recent years. Dual signals ratio sensors with "signal on" and "signal off" are more favored because of their low background signal and better sensitivity and selectivity. In this study, inspired by the idea that EcoR V can cut specific sites of DNA to produce two corresponding fragments, and by using the capturing probe as guy wires, a reliable and sensitive method for EcoR V assay is developed based on the ratio of dual chemiluminescence (CL) signals for the first time. In particular, in the existence of the objective EcoR V, the substrate DNA would be degraded into two double stranded oligonucleotides with blunt ends which include the sequence I and the sequence II, then they can separately compete with two different corresponding capture probes on magnetic beads (MBs). One of capture probe hybridized with the sequence I containing more guanine (G) bases that reacted with the phenylglyoxal (PG) to produce chemical reaction which triggered a positive CL signal output I + CL as "signal-on"; another capture probe is priority to hybridize the sequence II, which triggered the weaker reporter DNA linked with horseradish peroxidase (HRP) probe to fall off the MBs, thereby outputting a negative CL signal I-CL as "signal-off". By comparing the linear relation and the correlation coefficient, the I-CL/I + CL ratio method has better linear relation (0.01-10 U/mL) and higher sensitivity (0.0045 U/mL). In addition, this developed strategy of high selectivity which can directly detect low concentration of target EcoR V in human serum, and thus this dual ratio biosensor might offer a promising detection approach for clinical diagnostics.


Assuntos
Técnicas Biossensoriais , DNA de Cadeia Simples , DNA/genética , DNA de Cadeia Simples/genética , Peroxidase do Rábano Silvestre , Humanos , Luminescência
13.
Talanta ; 235: 122763, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517624

RESUMO

The disease diagnosis by detecting single microRNAs (miRNAs) can produce high false positive rate. Herein, a novel fluorescence biosensor method for one-step simultaneous detection of multiple miRNAs was proposed by using single-stranded DNA (ssDNA) functionalized double quantum dots (QDs) and black hole quencher (BHQ)-decorated magnetic nanobeads (MNs). MNs were linked with two black hole quenchers (BHQ1 and BHQ3) via a complementary DNA (cDNA). The ssDNA/cDNA hybridization contributed to the fluorescence quenching of double QDs due to the fluorescence resonance energy transfer (FRET) between double QDs and BHQ. In the presence of target miRNA-33 (miR-33) and miRNA-125b (miR-125b), the ssDNA1 and ssDNA2 were respectively hybridized with miR-33 and miR-125b to form more stable duplexes. Thus, the double QDs were released into supernatant after the magnetic separation, leading to the fluorescence signals recovery at 537 nm and 647 nm. A wide linear range (0.5 nM-320 nM for miR-33 and 0.1 nM-250 nM for miR-125b) and low limits of detection (0.09 nM for miR-33 and 0.02 nM for miR-125b) were achieved. Moreover, our approach has been demonstrated to simultaneously detect miR-33 and miR-125b in cell extracts. With advantages of high sensitivity, strong specificity, low background and low cost, the strategies show great potentials for the detection of various targets in bioanalysis and disease diagnosis.


Assuntos
Técnicas Biossensoriais , MicroRNAs , Pontos Quânticos , DNA de Cadeia Simples/genética , Transferência Ressonante de Energia de Fluorescência , MicroRNAs/genética , Hibridização de Ácido Nucleico
14.
Talanta ; 235: 122783, 2021 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-34517641

RESUMO

As the light-harvesting "antenna", G-rich oligonucleotides (such as the G-quadruplex) can interact with lanthanide (III) to bring a luminescent enhancement response. In this study, phenomenon of luminescent enhancement of G-triplex/terbium (III) (G3/Tb3+) and interaction between G3 and Tb3+ were first reported and characterized. Based on G3/Tb3+ luminescence, a label-free aptasensor for the detection of ofloxacin (OFL) residues in the food was developed. The OFL triggered the action of rolling circle amplification (RCA) allowed for the amplification product of G3-forming sequences in the single-stranded DNA, which promoted the conformational transition of the G3/Tb3+ complexes once the addition of Tb3+. Under the optimal conditions, the logarithmic correlation between the G3/Tb3+ luminescence intensity and the concentration of OFL was found to be linear in the range of 5-1000 pmol L-1 (R2 = 0.9949). The limit of detection was 0.18 pmol L-1 (3σ/slope). Additionally, the good recoveries of 90.19-108.89 % and the relative standard deviations values of 0.59-5.87 % were obtained in the application of the aptasensor detecting OFL in the practical samples. These results confirmed that the present aptasensor has a good analytical performance and bright prospect for detecting ofloxacin residues in food.


Assuntos
Aptâmeros de Nucleotídeos , Técnicas Biossensoriais , DNA de Cadeia Simples , Limite de Detecção , Luminescência , Ofloxacino , Térbio
15.
J Phys Chem Lett ; 12(37): 9132-9141, 2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34523927

RESUMO

Different nucleotides generate specific ionic currents that discriminate between the nucleotides while they are passing through the nanopore constriction. MspA is a commonly used nanopore for DNA sequencing. However, the reasons of the current variation remain ambiguous. Our work unveils the microscopic mechanism of current variation for an ssDNA passing through the MspA nanopore by all-atom molecular dynamic simulations. Besides the physical rigidity and dimensions of the nucleotides, nucleotide orientation is observed to induce nonignorable current variation. Besides the generally considered MspA nanopore constriction, it is also found that the region below constriction could be used to detect and differentiate single nucleotides when the single-stranded DNA translocates in the form of base-constriction-base meshing and ratcheting across the nanopore constriction compared to other regions. The work provides a novel insight into facilitating the development of low-cost and high-throughput nanopore DNA sequencing.


Assuntos
Nanoporos , Porinas/química , Análise de Sequência de DNA/métodos , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Simulação de Dinâmica Molecular , Mycobacterium smegmatis/metabolismo , Porinas/metabolismo
16.
J Chem Phys ; 155(9): 094305, 2021 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-34496579

RESUMO

DNA strands are polymeric ligands that both protect and tune molecular-sized silver cluster chromophores. We studied single-stranded DNA C4AC4TC3XT4 with X = guanosine and inosine that form a green fluorescent Ag10 6+ cluster, but these two hosts are distinguished by their binding sites and the brightness of their Ag10 6+ adducts. The nucleobase subunits in these oligomers collectively coordinate this cluster, and fs time-resolved infrared spectra previously identified one point of contact between the C2-NH2 of the X = guanosine, an interaction that is precluded for inosine. Furthermore, this single nucleobase controls the cluster fluorescence as the X = guanosine complex is ∼2.5× dimmer. We discuss the electronic relaxation in these two complexes using transient absorption spectroscopy in the time window 200 fs-400 µs. Three prominent features emerged: a ground state bleach, an excited state absorption, and a stimulated emission. Stimulated emission at the earliest delay time (200 fs) suggests that the emissive state is populated promptly following photoexcitation. Concurrently, the excited state decays and the ground state recovers, and these changes are ∼2× faster for the X = guanosine compared to the X = inosine cluster, paralleling their brightness difference. In contrast to similar radiative decay rates, the nonradiative decay rate is 7× higher with the X = guanosine vs inosine strand. A minor decay channel via a dark state is discussed. The possible correlation between the nonradiative decay and selective coordination with the X = guanosine/inosine suggests that specific nucleobase subunits within a DNA strand can modulate cluster-ligand interactions and, in turn, cluster brightness.


Assuntos
DNA de Cadeia Simples/química , Guanosina/química , Inosina/química , Prata/química , Sítios de Ligação , Fluorescência
17.
Nat Commun ; 12(1): 5545, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34545070

RESUMO

The RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA (ssDNA) and mediates invasion and strand exchange with homologous duplex DNA (dsDNA) during homologous recombination (HR), as well as protection and restart of stalled replication forks. Strand invasion by RAD51-ssDNA complexes depends on ATP binding. However, RAD51 can bind ssDNA in non-productive ADP-bound or nucleotide-free states, and ATP-RAD51-ssDNA complexes hydrolyse ATP over time. Here, we define unappreciated mechanisms by which the RAD51 paralog complex RFS-1/RIP-1 limits the accumulation of RAD-51-ssDNA complexes with unfavorable nucleotide content. We find RAD51 paralogs promote the turnover of ADP-bound RAD-51 from ssDNA, in striking contrast to their ability to stabilize productive ATP-bound RAD-51 nucleoprotein filaments. In addition, RFS-1/RIP-1 inhibits binding of nucleotide-free RAD-51 to ssDNA. We propose that 'nucleotide proofreading' activities of RAD51 paralogs co-operate to ensure the enrichment of active, ATP-bound RAD-51 filaments on ssDNA to promote HR.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Nucleotídeos/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Homologia de Sequência de Aminoácidos , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , DNA de Cadeia Simples/metabolismo , Fluorescência , Interferometria , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Especificidade da Espécie
18.
Sci Rep ; 11(1): 17682, 2021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34480058

RESUMO

The concept of Molecular Crowding depicts the high density of diverse molecules present in the cellular interior. Here, we determine the impact of low molecular weight and larger molecules on binding capacity of single-stranded DNA (ssDNA) to the cold shock protein B (CspB). Whereas structural features of ssDNA-bound CspB are fully conserved in crowded environments as probed by high-resolution NMR spectroscopy, intrinsic fluorescence quenching experiments reveal subtle changes in equilibrium affinity. Kinetic stopped-flow data showed that DNA-to-protein association is significantly retarded independent of choice of the molecule that is added to the solution, but dissociation depends in a nontrivial way on its size and chemical characteristics. Thus, for this DNA-protein interaction, excluded volume effect does not play the dominant role but instead observed effects are dictated by the chemical properties of the crowder. We propose that surrounding molecules are capable of specific modification of the protein's hydration shell via soft interactions that, in turn, tune protein-ligand binding dynamics and affinity.


Assuntos
Proteínas e Peptídeos de Choque Frio/metabolismo , DNA de Cadeia Simples/metabolismo , Modelos Moleculares , Espectroscopia de Ressonância Magnética , Ligação Proteica
19.
Anal Methods ; 13(37): 4314-4319, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34476425

RESUMO

A key challenge for the discrete distribution-based Förster resonance energy transfer system (D-FRET) is the reduced intensity and stability of signal probes in complex biological matrices. Here, we present a spatially confined FRET (SC-FRET) probe with a stable structure and strong signal output. It consists of multivalent FRET pairs labeled with FAM or TAMRA. In this assay, p53 DNA was chosen as a model hairpin probe (HP), and two kinds of branched DNA probes (ssDNA-FAM, ssDNA-TAMRA) were involved. Under the action of p53 DNA, the unfolded HP acts as a primer to initiate polymerization extension of KFP polymerase and cleavage of Nb.BbvCI endonuclease, which produces plenty of ssDNA (primer-DNA). The branched DNA is designed to have the same binding core and different sticky ends, the core part of which can self-assemble to form X-shaped branched DNA (X-FAM or X-TAMRA), and the sticky ends of which are complementary to the primer-DNA. Therefore, the primer-DNAs released during the polymerization cleavage process will combine a large number of X-FAM and X-TAMRA in a limited space through complementary base pairing. Fluorescence was transferred from FAM to TAMRA, and a strong FRET response was generated by the locational effects. The proposed SC-FRET system based on the multivalent assembly of branched DNA exhibited a strong FRET response with an LOD of 0.01394 pM. Importantly, it also showed a high-contrast and stable FRET response in HeLa cells. Its superior biological stability is attributed to the large steric hindrance of the compact and rigid frame of the SC-FRET probe, which helps prevent intracellular degradation and provides a powerful tool for biomedical research.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteína Supressora de Tumor p53 , DNA/genética , DNA de Cadeia Simples/genética , Células HeLa , Humanos , Proteína Supressora de Tumor p53/genética
20.
J Biosci Bioeng ; 132(6): 552-559, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34518106

RESUMO

Various diseases, including cancer, are caused by genetic mutations. A 5'-tailed duplex (TD) DNA, consisting of a long single-stranded (ss) editor DNA and a short (∼35-base) ss assistant oligodeoxyribonucleotide, can introduce a base-substitution in living cells and thus correct mutated genes. Previously, several hundred-base DNAs were employed as the editor DNAs. In this study, 5'-TDs were prepared from various editor DNAs with different lengths and examined for their gene correction abilities, using plasmid DNA bearing a mutated copepod green fluorescent protein (copGFP) gene, in human cells. High-throughput analysis was performed by the reactivated fluorescence of the wild-type protein encoded by the corrected gene as the indicator. The analysis revealed that 5'-TDs with ∼100-base ss editor DNAs enabled gene editing at least as efficiently as those with longer editor DNAs. Moreover, the antisense strand was more effective as the editor than the sense strand, in contrast to the 5'-TDs with longer editor strands. These results indicated that the 5'-TD fragments with shorter editor strands than those used in previous studies are useful nucleic acids for gene correction.


Assuntos
DNA de Cadeia Simples , Oligodesoxirribonucleotídeos , Sequência de Bases , DNA/genética , Humanos , Oligodesoxirribonucleotídeos/genética , Plasmídeos
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