Double-stranded Oligonucleotides Research Articles

Modulation of G4 DNA sensing behavior by triphenylamine-imidazole based D-D-π-A-type organic fluorophores with subtle structural change

Two unsymmetrical D-D-π-A-type cyanine dyes TG and TGS using triphenylamine-imidazole as the electron donor moiety (D-D), ethylene unit as π conjugated spacer (π), and quinolinium as the electron acceptor (A) were developed. Their ground-state geometries, electronic properties as well as fluorescence responses to with various polynucleotide structures, including G-quadruplexes (G4s), double-stranded and single-stranded oligonucleotides were investigated by a combined experimental and theoretical study. The high structural similarity between the two dyes makes them undergo a similar internal conversion process to form an ICT state, giving significantly large Stokes shifts. However, they behave quite different polynucleotide structures sensing behaviors. That is, TG with a non-planar D-D moiety can selectively bind with G4s, and emit turn-on fluorescence possibly because of the restriction of its molecular rotations, while TGS with a planar D-D moiety failed to interact with G4s and remained the weak fluorescence signal. Moreover, TG has the potential to be used in visualizing the cellular nucleus in cells. Our findings suggest that a subtle structural change of dyes may lead to distinctly different fluorescence behaviors in G4s sensing event.

Ultraviolet C Irradiation-Induced Dehybridization of Double-Stranded Oligonucleotides: Mechanism Investigation and Label-Free Measurement of the Photodamage Level.

Elucidating the mechanism and estimating the extent of conformation change of double-stranded DNA (dsDNA) upon ultraviolet (UV) exposure are of vital importance for understanding the DNA photodamage process. The existing research was mainly focused on the lesions of single-stranded DNA (ssDNA) and involved off-site measurement of the photodamage level. In this work, short-wavelength UV (UVC) (254 nm) irradiation was demonstrated to induce the dehybridization of dsDNA due to the loss of paring capacity of photodamaged pyrimidine nucleobases. The intrinsic programmability of dsDNA enabled researchers to rationally design the on-demand dehybridization sites. The spatial conformation switch of dsDNA caused by UVC irradiation could be evolved into a label-free sensing platform for the on-site measurement of the DNA photodamage level.

Abstract A019: Targeting STAT3 for lung cancer prevention

Abstract Former smokers have an elevated risk of lung cancer and account for a large proportion of newly diagnosed lung cancer. Former smokers with airway dysplasia have received modest benefit in randomized chemoprevention trials, while active smokers have not. More effective chemoprevention agents for former smokers at risk for lung cancer are needed. We previously reported that a cyclic double-stranded DNA oligonucleotide STAT3 decoy (CS3D) administered intravenously (IV) had strong anti-tumor effects in lung cancer xenograft models and was also effective as a chemoprevention agent using a tobacco carcinogen (NNK)-induced lung cancer mouse model that mimics “ex-smokers”. CS3D prevents the binding of activated STAT3 dimers to the promoter of target genes and induces p-STAT3 degradation. Our goal was to develop a clinically applicable direct delivery method of CS3D to the lungs. Using fluorescently tagged CS3D, we evaluated the feasibility and determined the optimal dosing schedule of CS3D administered intratracheally (IT, a mimic of human inhalation) compared to IV in mice. IT delivery 3 times/week provided a high initial drug level that was cleared from the lungs within a few hours, while IV dosing provided lower initial drug levels but persisted up to 8 hrs, with CS3D accumulation over time in the lungs. While the pharmacodynamic effect was also greater with systemic dosing, IT delivery produced inhibition of the STAT3 pathway. Phosphorylated STAT3, VEGF, IL6 and Ki67 expression in NNK-induced lung preneoplasias was down-modulated by IT CS3D delivery after 4 weeks of treatment compared to the mutant oligonucleotide (CS3M), confirming our previous findings with IV delivery. We also demonstrated that CS3D delivered IT altered the pulmonary environment by promoting a pro-inflammatory, anti-tumor response in myeloid cells of the lung directly, or by acting on tumor and stromal components to establish an immune-reactive tumor-microenvironment. No toxicities were found during IT or IV treatment and no organ abnormalities were detected by either regimen. A long-term chemoprevention study of CS3D delivered IT in the ex-smoker lung cancer murine model is underway. Our findings suggest that blocking STAT3 may be a useful strategy for lung cancer prevention, and may involve both inhibition of oncogenic signaling and enhanced anti-tumor immunity. Citation Format: Jill M. Siegfried, Adam C. Soloff, Autumn Gaither Davis, Amy A. Powers, Seth H. Eisenberg, Laura P. Stabile. Targeting STAT3 for lung cancer prevention [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A019.

DNA micelle-templated copper nanoclusters for fluorescent imaging of MUC1-positive cancer cells.

DNA micelles formed by hydrophobic, self-assembly of amphiphilic DNA monomers have enormous potential in biological imaging owing to its unique and programmable, three-dimensional nanostructure. Herein, we rationally design double-stranded DNA oligonucleotides with two cholesterols that can spontaneously form the lipid-mediated DNA micelles and generate the high fluorescence signal after the formation of DNA-templated copper nanoclusters (CuNCs). Furthermore, the DNA aptamer specific to MUC1 protein, aberrantly overexpressed on the surface of cancer cells, is attached to lipid-mediated DNA micelles to confer the selectivity towards the target cancer cells. With the well-defined DNA nanostructures, the cell membrane of MUC1-positive cancer cells are stained by CuNCs exhibiting an intense, red fluorescence signal, which are clearly distinguished from MUC1-negative cancer cells. This approach may not only expand the application scope of both DNA micells and CuNCs, especially in the area of cellular imaging, but also provides a basis for developing other types of DNA nanostructures to detect target biomarkers.

DNA-Protein-Interaction (DPI)-ELISA Assay for PPAR-γ Receptor Binding.

Dysregulation of peroxisome proliferator-activated receptor (PPAR)-γ has been described in a plethora of pathological conditions, such as diabetes, obesity, inflammatory-related diseases, and cancer. Therefore, identifying novel drugs that are able to restore PPAR-γ activity is a current challenge, which is however slowed down by the lack of a rapid and reproducible activity assay. To date, only a few methods are able to characterize PPAR-γ activity and most of them are expensive, time-consuming, and not always quantitative.Herein, we presented a sensitive multi-well colorimetric assay, termed DNA-Protein-Interaction enzyme-linked immunosorbent assay (DPI-ELISA). This method is based on the ELISA principle, except that it allows to detect only activated PPAR-γ because, unlike classical ELISA, PPAR-γ is not captured by an antibody but by a double-stranded oligonucleotide probe containing its peroxisome proliferator response elements (PPRE) consensus sequence. Thus, DPI-ELISA represents a useful assay for PPAR-γ studies, as well as for the identification of novel PPAR-γ ligands for the development of innovative therapeutic approaches to human diseases where PPAR-γ signaling is dysregulated.

The Usefulness of Autoradiography for DNA Repair Proteins Activity Detection in the Cytoplasm towards Radiolabeled Oligonucleotides Containing 5′,8-Cyclo-2′-deoxyadenosine

Autoradiography of 32P-radiolabeled oligonucleotides is one of the most precise detection methods of DNA repair processes. In this study, autoradiography allowed assessing the activity of proteins in the cytoplasm involved in DNA repair. The cytoplasm is the site of protein biosynthesis but is also a target cellular compartment of synthetic therapeutic oligonucleotide (STO) delivery. The DNA-based drugs may be impaired by radiation-induced lesions, such as clustered DNA lesions (CDL) and/or 5′,8-cyclo-2′-deoxypurines (cdPu). CDL and cdPu may appear in the sequence of STO after irradiation and subsequently impair DNA repair, as shown in previous studies. Hence, the interesting questions are (1) is it safe to combine STO treatment with radiotherapy; (2) are repair proteins active in the cytoplasm; and (3) is their activity different in the cytoplasm than in the nucleus? This unique study examined whether the proteins involved in the DNA repair are affected by the CDL while they are still present in the cytoplasm of xrs5, BJ, and XPC cells. Double-stranded oligonucleotides with bi-stranded CDL were used (containing AP site in one strand and a (5′S) or (5′R) 5′,8-cyclo-2′-deoxyadenosine (cdA) in the other strand located 1 or 4 bp in both directions). The results have shown that the proteins involved in the repair were active in the cytoplasm, but less than in the nucleus. The general trends aligned for cytoplasm and nucleus—lesions located in the 5′-end direction inhibited the course of DNA repair. The combination of STO with radiotherapy should be applied carefully, as unrepaired lesions within STO may impair their therapeutic efficiency.

STING Expression in Peripheral Sensory Neurons Underlies Virus Recognition and Pain

Pain is an early and common symptom of viral infections, and yet we have a limited understanding about the mechanisms through which viruses induce pain. Peripheral sensory neurons are well-studied for their role in pain, and are emerging as new actors in pathogen recognition and defense. It has been reported that peripheral sensory neurons express several proteins associated with virus recognition, including the stimulator of interferon genes (STING). Although STING and interferon signaling has been recently proposed as a critical regulator of physiological pain and a promising new target for treating chronic pain, whether and how STING in peripheral sensory neurons participate in virus recognition, defense and pain is still completely unknown. Here, we report that intradermal delivery of viral oligonucleotides induced pain responses in mice via the expression of STING and the transient receptor potential vanilloid subtype 1 (TRPV1) in sensory neurons. In particular, we found that intradermal injections in mouse hindpaw of double-stranded DNA oligonucleotides derived from the herpes simplex virus (HSV-60) induced immediate spontaneous pain responses (hindpaw lifting, shacking and licking), as well as transitory mechanical pain hypersensitivities lasting up to 4 h. Remarkably, mice treated with the TRPV1 antagonist AMG9810 or resiniferatoxin, an ultrapotent TRPV1 agonist leading to the peripheral denervation, abrogated both HSV-60 induced pain responses. STING expression is enriched in TRPV1+ peripheral sensory neurons, and similar abrogation of HSV-60 induced pain responses was observed in conditional knockout mice lacking the expression of STING uniquely in peripheral sensory neurons. In support of these data, we also found that STING agonists directly can activate sensory neurons in vitro calcium imaging analysis and elicited pain responses in wild-type, but not TRPV1 knockout mice. Thus, our initial findings provide a previously undiscovered mechanism by which viruses are recognized by peripheral sensory neurons and induce pain. Grant support from NINDS R21 NS121946.

Rapid and visual monitoring of alien sequences using crop wild relatives specific oligo-painting: The case of cucumber chromosome engineering

Wild species related to domesticated crops (crop wild relatives, or CWRs) represent a high level of genetic diversity that provides a practical gene pool for crop pre-breeding employed to address climate change and food demand challenges globally. Nevertheless, rapid identifying and visual tracking of alien chromosomes and sequences derived from CWRs have been a technical challenge for crop chromosome engineering. Here, a species-specific oligonucleotide (oligo) pool was developed by using the reference genome of Cucumis hystrix (HH, 2n = 2x = 24), a wild species carrying many favorable traits and interspecific compatibility with cultivated cucumber (C. sativus, CC, 2n = 2x = 14). These synthetic double-stranded oligo probes were applied to validate the assembly and characterize the chromosome architectures of C. hystrix, as well as to rapidly identify C. hystrix-chromosomes in diverse C. sativus-hystrix chromosome-engineered germplasms, including interspecific hybrid F1 (HC), synthetic allopolyploids (HHCC, CHC, and HCH) and alien additional lines (CC-H). Moreover, a ∼2Mb of C. hystrix-specific sequences, introduced into cultivated cucumber, were visualized by CWR-specific oligo-painting. These results demonstrate that the CWR-specific oligo-painting technique holds broad applicability for chromosome engineering of numerous crops, as it allows rapid identification of alien chromosomes, reliable detection of homoeologous recombination, and visual tracking of the introgression process. It is promising to achieve directed and high-precision crop pre-breeding combined with other breeding techniques, such as CRISPR/Cas9-mediated chromosome engineering.

Amphiphilic pH-sensitive polypeptides for siRNA delivery

The effective delivery of nucleic acid therapeutics into tumor cells is of great importance in terms of cancer gene therapy. Successful application of siRNAs is restricted by the requirement to overcome several biological barriers, i.e. cell membrane penetration, endosomal escape, enzymatic degradation in cytosol, etc. Cationic polymers are promising delivery systems due to their biodegradability and effective binding with siRNA. A key characteristic of the particle trafficking into the cell is a noticeable change in pH during this process. This change can be used as a natural stimulus for pH-sensitive polymer particles. Such polymers are able to buffer the environment of a late endosome that leads to an abnormal flow of ions and water followed by endosomal swelling and disrupt and quick escape of the carriers into a cytoplasm. In present work, we synthesized a series of amphiphilic positively charged biocompatible pH-sensitive polypeptides forming polyplexes with nucleic acids. Four amino acids were used to compose the delivery systems, namely l-lysine, l-glutamic acid, l-phenylalanine and l-histidine. The number and the ratio of amino acids were varied in order to establish the optimal composition. The characteristics of polypeptides were studied by 1H NMR spectroscopy, high performance liquid chromatography and static light scattering. All synthesized polypeptides tended to a self-assembly in aqueous media and formed nanoparticles with hydrodynamic diameters ranging from 140 to 360 nm. The formation of polyplexes with short double-stranded oligonucleotides as a physicochemical siRNA model as well as the release of cargo in buffer media mimicking lysosome (pH∼5) and cytosol or bloodstream (pH 7.4) were studied. Cellular uptake of the particles was efficient and their cytotoxicity was negligible. Successful RNAi-mediated down-regulation of GFP gene expression was revealed in MDA-MB-231/GFP breast cancer cells. The amphiphilic polypeptides obtained can be considered as promising non-viral candidates for siRNA delivery.

Single-molecule conductance of double-stranded RNA oligonucleotides.

RNA oligonucleotides are crucial for a range of biological functions and in many biotechnological applications. Herein, we measured, for the first time, the conductance of individual double-stranded (ds)RNA molecules and compared it with the conductance of single DNA : RNA hybrids. The average conductance values are similar for both biomolecules, but the distribution of conductance values shows an order of magnitude higher variability for dsRNA, indicating higher molecular flexibility of dsRNA. Microsecond Molecular Dynamics simulations explain this difference and provide structural insights into the higher stability of DNA : RNA duplex with atomic level of detail. The rotations of 2'-OH groups of the ribose rings and the bases in RNA strands destabilize the duplex structure by weakening base stacking interactions, affecting charge transport, and making single-molecule conductance of dsRNA more variable (dynamic disorder). The results demonstrate that a powerful combination of state-of-the-art biomolecular electronics techniques and computational approaches can provide valuable insights into biomolecules' biophysics with unprecedented spatial resolution.

Spatial Separation of Plasmonic Hot-Electron Generation and a Hydrodehalogenation Reaction Center Using a DNA Wire.

Using hot charge carriers far from a plasmonic nanoparticle surface is very attractive for many applications in catalysis and nanomedicine and will lead to a better understanding of plasmon-induced processes, such as hot-charge-carrier- or heat-driven chemical reactions. Herein we show that DNA is able to transfer hot electrons generated by a silver nanoparticle over several nanometers to drive a chemical reaction in a molecule nonadsorbed on the surface. For this we use 8-bromo-adenosine introduced in different positions within a double-stranded DNA oligonucleotide. The DNA is also used to assemble the nanoparticles into nanoparticles ensembles enabling the use of surface-enhanced Raman scattering to track the decomposition reaction. To prove the DNA-mediated transfer, the probe molecule was insulated from the source of charge carriers, which hindered the reaction. The results indicate that DNA can be used to study the transfer of hot electrons and the mechanisms of advanced plasmonic catalysts.

1052. Characterisation of the DNA binding properties of ridinilazole, a selective antibiotic currently in phase III trials for the treatment of Clostridioides difficile

Background Clostridioides difficile infection (CDI) is recognised by the CDC as an “urgent threat” in the USA, responsible for nearly 13,000 deaths, and carries an economic burden ranging from &5.4 to &6.3 billion per year. In a phase II study, ridinilazole was shown to be effective at treating CDI and decreasing subsequent recurrence compared to vancomycin. However, the precise mechanism of action of ridinilazole has yet to be fully elucidated. We now present data that reveals ridinilazole clearly co-localises with DNA in C. difficile and binds with high affinity to the minor groove of DNA. These interactions are predicted to have consequences on cellular functions within C. difficile.MethodsHigh resolution confocal microscopy was used to track the intracellular localisation of ridinilazole in C. difficile. Fluorescence intensity was used to characterise the DNA binding properties of ridinilazole; sequence specificity was demonstrated with AT- or GC-rich DNA polymers, and tight binding was shown using short double-stranded oligonucleotides. Hanging drop vapour diffusion enabled co-crystallisation and subsequent structural determination of DNA-bound ridinilazole. ResultsConfocal microscopy revealed clear co-localisation of ridinilazole to the DNA within C. difficile. Ridinilazole demonstrated a dose-dependent increase in fluorescence in response to increasing concentration of target DNA. Fluorescence binding studies revealed that ridinilazole shows a preference towards AT-rich DNA sequences. Tight binding characteristics were demonstrated by ridinilazole in complex with short double-stranded oligonucleotides, returning dissociation constants (Kd) of 20 – 50 nM. Crystallisation enabled co-structures of ridinilazole bound to the minor groove of double-stranded DNA oligonucleotides to be solved.ConclusionRidinilazole demonstrates tight binding with sequence specificity within the minor groove of DNA and co-localises with DNA in C. difficle. Further analysis is ongoing to fully understand this novel mechanism of action, the downstream consequences of these interactions and how they contribute to the bactericidal activity of ridinilazole.Disclosures Clive Mason, PhD, Summit Therapeutics (Employee, Shareholder) Tim Avis, n/a, Summit therapeutics (Shareholder) Chris Coward, PhD, Summit Therapeutics (Employee, Scientific Research Study Investigator, Shareholder) David Powell, PhD, Summit Therapeutics (Employee) Kevin W. Garey, Pharm.D., M.S., FASHP, Summit Therapeutics (Research Grant or Support)

Prognostic value of microRNAs in heart failure: A meta-analysis.

Background:Reported studies have shown that expression levels of microRNAs (miRNAs) are related to survival time of patients with heart failure (HF). A systematic review and meta-analysis were conducted to study circulating miRNAs expression and patient outcome.Methods:Meta-analysis estimating expression levels of circulating miRNAs in HF patients from January 2010 until June 30, 2018, through conducting online searches in Pub Med, Cochrane Database of Systematic, EMBASE and Web of Science and reviewed by 2 independent researchers. Using pooled hazard ratio with a 95% confidence interval to assess the correlation between miRNAs expression levels and overall survival.Results:Four relevant articles assessing 19 circulating miRNAs in 867 patients were included. In conclusion, the meta-analysis results suggest that HF patients with low expression of serum miR-1, miR-423-5p, miR-126, miR-21, miR-23, miR-30d, miR-18a-5p, miR-16-5p, miR-18b-5p, miR-27a-3p, miR-26b-5p, miR-30e-5p, miR-106a-5p, miR-233-3P, miR-301a-3p, miR-423-3P, and miR-128 have significantly worse overall survival (P < .05). Among them, miR-18a-5p, miR-18b-5p, miR-30d, miR-30e-5p, and miR-423-5p are strong biomarkers of prognosis in HF.

Efficient and Economical Targeted Insertion in Plant Genomes via Protoplast Regeneration.

Versatile genome editing can be facilitated by the insertion of DNA sequences into specific locations. Current protocols involving CRISPR and Cas proteins rely on low efficiency homology-directed repair or non-homologous end joining with modified double-stranded DNA oligonucleotides as donors. Our simple protocol eliminates the need for expensive equipment, chemical and enzymatic donor DNA modification, or plasmid construction by using polyethylene glycol-calcium to deliver non-modified single-stranded DNA oligonucleotides and CRISPR-Cas9 ribonucleoprotein into protoplasts. Plants regenerated via edited protoplasts achieved targeted insertion frequencies of up to 50% in Nicotiana benthamiana and 13.6% in rapid cycling Brassica oleracea without antibiotic selection. Using a 60 nt donor containing 27 nt in each homologous arm, 6/22 regenerated N. benthamiana plants showed targeted insertions, and one contained a precise insertion of a 6 bp HindIII site. The inserted sequences were transmitted to the next generation and invite the possibility of future exploration of versatile genome editing by targeted DNA insertion in plants.

The Two Faces of the Guanyl Radical: Molecular Context and Behavior.

The guanyl radical or neutral guanine radical G(-H)• results from the loss of a hydrogen atom (H•) or an electron/proton (e–/H+) couple from the guanine structures (G). The guanyl radical exists in two tautomeric forms. As the modes of formation of the two tautomers, their relationship and reactivity at the nucleoside level are subjects of intense research and are discussed in a holistic manner, including time-resolved spectroscopies, product studies, and relevant theoretical calculations. Particular attention is given to the one-electron oxidation of the GC pair and the complex mechanism of the deprotonation vs. hydration step of GC•+ pair. The role of the two G(-H)• tautomers in single- and double-stranded oligonucleotides and the G-quadruplex, the supramolecular arrangement that attracts interest for its biological consequences, are considered. The importance of biomarkers of guanine DNA damage is also addressed.

RNA interference activity of single-stranded oligonucleotides linked between the passenger strand and the guide strand with an aryl phosphate linker

Recently, we demonstrated that asymmetrical 18 base-paired double-strand oligonucleotides comprised of alternately combined 2’-O-methyl RNA and DNA, termed MED-siRNAs, show high RNase resistance, efficient cleavage of target mRNA, and the subsequent reduction of target protein expression. The 5’-terminal phosphate group and the 3’-overhang of the guide strand were required to fully activate the RNAi activity of MED-siRNAs. Here, we evaluated MED-siRNAs modified with aryl phosphate groups at the 5’-end of the guide strand. The 5’-aryl phosphorylated MED-siRNAs showed highly efficient reduction of target protein expression comparable to 5’-phosphorylated MED-siRNAs. Moreover, 5’-aryl phosphorylated MED-siRNAs linked between the aryl phosphate group at the 5’-end of the guide strand and the hydroxyl group at the 3’-end of the passenger strand with alkyl amide linkers or peptides (e.g., DL-Ser-L-Ala-L-Tyr), resulted in single-stranded MED-siRNAs with a highly efficient cleavage activity of target mRNA with binding to Argonaute 2 via an RNA interference mechanism. These linker techniques could also be used to create siRNAs composed of naturally-occurring molecules such as amino acids. These findings suggest the possibility of using these single-stranded MED-siRNAs as siRNA reagents. Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.1927077 .

Programmable site-selective labeling of oligonucleotides based on carbene catalysis

Site-selective modification of oligonucleotides serves as an indispensable tool in many fields of research including research of fundamental biological processes, biotechnology, and nanotechnology. Here we report chemo- and regioselective modification of oligonucleotides based on rhodium(I)-carbene catalysis in a programmable fashion. Extensive screening identifies a rhodium(I)-catalyst that displays robust chemoselectivity toward base-unpaired guanosines in single and double-strand oligonucleotides with structurally complex secondary structures. Moreover, high regioselectivity among multiple guanosines in a substrate is achieved by introducing guanosine-bulge loops in a duplex. This approach allows the introduction of multiple unique functional handles in an iterative fashion, the utility of which is exemplified in DNA-protein cross-linking in cell lysates.

Non-specific interactions of antibody-oligonucleotide conjugates with living cells

Antibody-Oligonucleotide Conjugates (AOCs) represent an emerging class of functionalized antibodies that have already been used in a wide variety of applications. While the impact of dye and drug conjugation on antibodies’ ability to bind their target has been extensively studied, little is known about the effect caused by the conjugation of hydrophilic and charged payloads such as oligonucleotides on the functions of an antibody. Previous observations of non-specific interactions of nucleic acids with untargeted cells prompted us to further investigate their impact on AOC binding abilities and cell selectivity. We synthesized a series of single- and double-stranded AOCs, as well as a human serum albumin-oligonucleotide conjugate, and studied their interactions with both targeted and non-targeted living cells using a time-resolved analysis of ligand binding assay. Our results indicate that conjugation of single strand oligonucleotides to proteins induce consistent non-specific interactions with cell surfaces while double strand oligonucleotides have little or no effect, depending on the preparation method.

Versatile CRISPR/Cas9 Systems for Genome Editing in Ustilago maydis.

The phytopathogenic smut fungus Ustilago maydis is a versatile model organism to study plant pathology, fungal genetics, and molecular cell biology. Here, we report several strategies to manipulate the genome of U. maydis by the CRISPR/Cas9 technology. These include targeted gene deletion via homologous recombination of short double-stranded oligonucleotides, introduction of point mutations, heterologous complementation at the genomic locus, and endogenous N-terminal tagging with the fluorescent protein mCherry. All applications are independent of a permanent selectable marker and only require transient expression of the endonuclease Cas9hf and sgRNA. The techniques presented here are likely to accelerate research in the U. maydis community but can also act as a template for genome editing in other important fungi.

Synthesis of sea urchin-shaped Au nanocrystals by double-strand diblock oligonucleotides for surface-enhanced Raman scattering and catalytic application

It is of great significance to construct specially designed gold nanocrystals (AuNCs) with precisely controllable size and morphology to achieve an excellent physicochemical performance. In this work, sea urchin-shaped AuNCs with tunable plasmonic property were successfully synthesized by the hybridized double-strand poly adenine (dsPolyA) DNA-directed self-assembly technique. Hybridized dsPolyA as the directing template had suitable rigidity and upright conformation, which benefited the controllable formation of these anisotropic multi-branched AuNCs with the assistance of surfactant. The effects of essential conditions influencing the synthesis and precise morphology control were investigated in detail. COMSOL simulation was used to evaluate their electromagnetic field distribution according to their morphologies, and the result suggested that sea urchin-shaped AuNCs had abundant ‘hot spots’ for surface-enhanced Raman scattering (SERS) detection due to their regular nanoprotuberance structure. Finally, sea urchin-shaped AuNCs with excellent SERS and catalytic performance were applied for the quantitative analysis of food colorant and catalytic degradation of potential pollutants. The SERS enhancement factor of sea urchin-shaped AuNCs was up to 5.27 × 106, and the catalytic degradation rate for 4-NP by these AuNCs was up to −0.13min−1.