2′-O-Methyl modified guide RNA promotes the single nucleotide polymorphism (SNP) discrimination ability of CRISPR–Cas12a systems

The CRISPR–Cas12a system has been widely applied to genome editing and molecular diagnostics. However, off-target cleavages and false-positive results remain as major concerns in Cas12a practical applications. Herein, we propose a strategy by utilizing the 2′-O-methyl (2′-OMe) modified guide RNA (gRNA) to promote the Cas12a''s specificity. Gibbs free energy analysis demonstrates that the 2′-OMe modifications at the 3′-end of gRNA effectively suppress the Cas12a''s overall non-specific affinity while maintaining high on-target affinity. For general application illustrations, HBV genotyping and SARS-CoV-2 D614G mutant biosensing platforms are developed to validate the enhanced Cas12a''s specificity. Our results indicate that the 2′-OMe modified gRNAs could discriminate single-base mutations with at least two-fold enhanced specificity compared to unmodified gRNAs. Furthermore, we investigate the enhancing mechanisms of the 2′-OMe modified Cas12a systems by molecular docking simulations and the results suggest that the 2′-OMe modifications at the 3′-end of gRNA reduce the Cas12a''s binding activity to off-target DNA. This work offers a versatile and universal gRNA design strategy for highly specific Cas12a system development.

This study illustrates that 2′-O-methyl modified gRNAs improve the specificity of the CRISPR–Cas12a system (mg-CRISPR) via suppressing the Cas12a''s affinity to off-target DNA and provides an efficient strategy for high-specificity gRNA design.     

Optical Control of a CRISPR/Cas9 System for Gene Editing by Using Photolabile crRNA

Currently CRISPR/Cas9 is a widely used efficient tool for gene editing. Precise control over the CRISPR/Cas9 system with high temporal and spatial resolution is essential for studying gene regulation and editing. Here, we synthesized a novel light-controlled crRNA by coupling vitamin E and a photolabile linker at the 5′ terminus to inactivate the CRISPR/Cas9 system. The vitamin E modification did not affect ribonucleoprotein (RNP) formation of Cas9/crRNA/tracrRNA complexes but did inhibit the association of RNP with the target DNA. Upon light irradiation, vitamin E-caged crRNA was successfully activated to achieve light-induced genome editing of vascular endothelial cell-growth factor A (VEGFA) in human cells through a T7E1 assay and Sanger sequencing as well as gene knockdown of EGFP expression in EGFP stably expressing cells. This new caging strategy for crRNA could provide new methods for spatiotemporal photoregulation of CRISPR/Cas9-mediated gene editing.     

CRISPR-Cas12a System for Biosensing and Gene Regulation

Clustered regularly interspaced short palindromic repeats (CRISPR) is a promising technology in the biological world. As one of the CRISPR-associated (Cas) proteins, Cas12a is an RNA-guided nuclease in the type V CRISPR-Cas system, which has been a robust tool for gene editing. In addition, due to the discovery of target-binding-induced indiscriminate single-stranded DNase activity of Cas12a, CRISPR-Cas12a also exhibits great promise in biosensing. This minireview not only gives a brief introduction to the mechanism of CRISPR-Cas12a but also highlights the recent developments and applications in biosensing and gene regulation. Finally, future prospects of the CRISPR-Cas12a system are also discussed. We expect this minireview will inspire innovative work on the CRISPR-Cas12a system by making full use of its features and advantages.     

Microwave-Assisted Extraction of Anticancer Flavonoid, 2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethyl Chalcone (DMC), Rich Extract from Syzygium nervosum Fruits

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethyl chalcone (DMC) is a biological flavonoid that is present in the fruits of Syzygium nervosum (Ma-Kiang in Thai). Microwave-assisted extraction (MAE), which utilizes microwave radiation to heat the extraction solvent quickly and effectively, was used to recover DMC-rich extract from Syzygium nervosum fruit. To determine the DMC content, a highly accurate and precise HPLC technique was developed. The influences of MAE conditions, including the solid–liquid ratio, microwave power, and microwave duration on the content of DMC, were sequentially employed by a single factor investigation and response surface methodology (RSM) exploratory design. The predicted quadratic models were fitted due to their highly significant (p < 0.0001) and excellent determination coefficient (R² = 0.9944). The optimal conditions for producing DMC-rich extract were a ratio of sample to solvent of 1:35 g/mL, a microwave power of 350 W, and a microwave time of 38 min. Under the optimal MAE setting, the DMC content reached 1409 ± 24 µg/g dry sample, which was greater than that of the conventional heat reflux extraction (HRE) (1337 ± 37 µg/g dry sample) and maceration (1225 ± 81 µg/g dry sample). The DMC-rich extract obtained from MAE showed stronger anticancer activities against A549 (human lung cancer cells) and HepG2 (human liver cancer cells) than the individual DMC substance, which makes MAE an effective method for extracting essential phytochemicals from plants in the nature.     

Sustainable Protocol for the Synthesis of 2′,3′-Dideoxynucleoside and 2′,3′-Didehydro-2′,3′-dideoxynucleoside Derivatives

An improved protocol for the transformation of ribonucleosides into 2′,3′-dideoxynucleoside and 2′,3′-didehydro-2′,3′-dideoxynucleoside derivatives, including the anti-HIV drugs stavudine (d4T), zalcitabine (ddC) and didanosine (ddI), was established. The process involves radical deoxygenation of xanthate using environmentally friendly and low-cost reagents. Bromoethane or 3-bromopropanenitrile was the alkylating agent of choice to prepare the ribonucleoside 2′,3′-bisxanthates. In the subsequent radical deoxygenation reaction, tris(trimethylsilyl)silane and 1,1′-azobis(cyclohexanecarbonitrile) were used to replace hazardous Bu₃SnH and AIBN, respectively. In addition, TBAF was substituted for camphorsulfonic acid in the deprotection step of the 5′-O-silyl ether group, and an enzyme (adenosine deaminase) was used to transform 2′,3′-dideoxyadenosine into 2′,3′-dideoxyinosine (ddI) in excellent yield.     

Synthesis and Biological Evaluation of 5′-O-Fatty Acyl Ester Derivatives of 3′-Fluoro-2′,3′-dideoxythymidine as Potential Anti-HIV Microbicides

A number of 5′-O-fatty acyl derivatives of 3′-fluoro-2′,3′-dideoxythymidine (FLT, 1) were synthesized. These conjugates were evaluated for their potential as topical microbicides with anti-HIV activity against cell-free (X4 and R5), cell-associated, and multidrug-resistant viruses. Compared to FLT and 3′-azido-2′,3′-dideoxythymidine (AZT), 5′-O-(12-azidododecanoyl) (5), 5′-O-myristoyl (6), and 5′-O-(12-thioethyldodecanoyl) (8) derivatives of FLT were found to be more active against both cell-free viruses (lymphocytotropic and monocytotropic strains) with EC₅₀ values of 0.4 μM, 1.1 μM, and <0.2 μM, respectively, as well as cell-associated virus with EC₅₀ values of 12.6, 6.4, and 2.3 μM, respectively. Conjugates 5, 6, and 8 exhibited >4 and >30 times better antiviral index than FLT and AZT, respectively. Conjugates 5 and 8 were significantly more potent than FLT against many multidrug-resistant strains. A comparison of the anti-HIV activity with the corresponding non-hydrolyzable ether conjugates suggested that ester hydrolysis to FLT and fatty acids is critical to enable anti-HIV activity. Cellular uptake studies were conducted using fluorescent derivatives of FLT attached with 5(6)-carboxyfluorescein through either β-alanine (23) or 12-aminododecanoic acid (24) spacers. The lipophilic fluorescent analog with a long chain (24) showed more than 12 times higher cellular uptake profile than the fluorescent analog with a short chain (23). These studies further confirmed that the attachment of fatty acids improved the cellular uptake of nucleoside conjugates. In addition, 5, 6, and 8 were the least cytotoxic and did not alter vaginal cell and sperm viability compared to the positive control, a commercial topical spermicide (N-9), which significantly decreased sperm and vaginal cell viability inducing the generation of proinflammatory cytokines.     

Targeted Synthesis of 3,3′-, 3,4′- and 3,6′-Phenylpropanoid Sucrose Esters

In this study, we report on an orthogonal strategy for the precise synthesis of 3,3′-, 3,4′-, and 3,6′-phenylpropanoid sucrose esters (PSEs). The strategy relies on carefully selected protecting groups and deprotecting agents, taking into consideration the reactivity of the four free hydroxyl groups of the key starting material: di-isopropylidene sucrose 2. The synthetic strategy is general, and potentially applies to the preparation of many natural and unnatural PSEs, especially those substituted at 3-, 3′-, 4′- and 6′-positions of PSEs.     

Effects of 2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone from Syzygium nervosum Seeds on Antiproliferative,DNA Damage,Cell Cycle Arrest,and Apoptosis in Human Cervical Cancer Cell Lines

2′,4′-Dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC), a natural product derived from Syzygium nervosum A. Cunn. ex DC., was investigated for its inhibitory activities against various cancer cell lines. In this work, we investigated the effects of DMC and available anticervical cancer drugs (5-fluorouracil, cisplatin, and doxorubicin) on three human cervical cancer cell lines (C-33A, HeLa, and SiHa). DMC displayed antiproliferative cervical cancer activity in C-33A, HeLa, and SiHa cells, with IC₅₀ values of 15.76 ± 1.49, 10.05 ± 0.22, and 18.31 ± 3.10 µM, respectively. DMC presented higher antiproliferative cancer activity in HeLa cells; therefore, we further investigated DMC-induced apoptosis in this cell line, including DNA damage, cell cycle arrest, and apoptosis assays. As a potential anticancer agent, DMC treatment increased DNA damage in cancer cells, observed through fluorescence inverted microscopy and a comet assay. The cell cycle assay showed an increased number of cells in the G₀/G₁ phase following DMC treatment. Furthermore, DMC treatment-induced apoptosis cell death was approximately three- to four-fold higher compared to the untreated group. Here, DMC represented a compound-induced apoptosis for cell death in the HeLa cervical cancer cell line. Our findings suggest that DMC, a phytochemical agent, is a potential candidate for antiproliferative cervical cancer drug development.     

Nucleotide Analogues Bearing a C2′ or C3′-Stereogenic All-Carbon Quaternary Center as SARS-CoV-2 RdRp Inhibitors

The design of novel nucleoside triphosphate (NTP) analogues bearing an all-carbon quaternary center at C2′ or C3′ is described. The construction of this all-carbon stereogenic center involves the use of an intramoleculer photoredox-catalyzed reaction. The nucleoside analogues (NA) hydroxyl functional group at C2′ was generated by diastereoselective epoxidation. In addition, highly enantioselective and diastereoselective Mukaiyama aldol reactions, diastereoselective N-glycosylations and regioselective triphosphorylation reactions were employed to synthesize the novel NTPs. Two of these compounds are inhibitors of the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, the causal virus of COVID-19.     

The Influence of 5′,8-Cyclo-2′-deoxypurines on the Mitochondrial Repair of Clustered DNA Damage in Xrs5 Cells: The Preliminary Study

The 5′,8-cyclo-2′-deoxypurines (cdPus) affect the DNA structure. When these bulky structures are a part of clustered DNA lesions (CDL), they affect the repair of the other lesions within the cluster. Mitochondria are crucial for cell survival and have their own genome, hence, are highly interesting in the context of CDL repair. However, no studies are exploring this topic. Here, the initial stages of mitochondrial base excision repair (mtBER) were considered—the strand incision and elongation. The repair of a single lesion (apurinic site (AP site)) accompanying the cdPu within the double-stranded CDL has been investigated for the first time. The type of cdPu, its diastereomeric form, and the interlesion distance were taken into consideration. For these studies, the established experimental model of short oligonucleotides (containing AP sites located ≤7 base pairs to the cdPu in both directions) and mitochondrial extracts of the xrs5 cells were used. The obtained results have shown that the presence of cdPus influenced the processing of an AP site within the CDL. Levels of strand incision and elongation were higher for oligos containing RcdA and ScdG than for those with ScdA and RcdG. Investigated stages of mtBER were more efficient for DNA containing AP sites located on 5′-end side of cdPu than on its 3′-end side. In conclusion, the presence of cdPus in mtDNA structure may affect mtBER (processing the second mutagenic lesion within the CDL). As impaired repair processes may lead to serious biological consequences, further studies concerning the mitochondrial repair of CDL are highly demanded.     

Design,Synthesis and Biological Evaluation of (2′,5′ and 3′5′-Linked) cGAMP Analogs that Activate Stimulator of Interferon Genes (STING)

Stimulator of interferon genes (STING) is an endoplasmic reticulum adaptor transmembrane protein that plays a pivotal role in innate immune system. STING agonists, such as endogenous cyclic dinucleotide (CDN) cyclic GMP-AMP (cGAMP), have been used in diverse clinical research for immunogenic tumor clearance, antiviral treatments and vaccine adjuvants. CDNs containing noncanonical mixed 3′-5′ and 2′-5′ phosphodiester linkages show higher potency in the activation of the STING pathway. In this study, a series of 2′3′-CDNs were designed and synthesized through a modified one-pot strategy. We then established a surface plasmon resonance (SPR)-based binding assay to quantify the binding affinities of synthesized CDNs for human STING, which requested a minuscule amount of sample without any pretreatment. Using this assay, we identified compound 8d (K_D = 0.038 μM), a novel CDN that showed higher binding affinity with hSTING than cGAMP (K_D = 0.543 μM). Cellular assays confirmed that 8d could trigger the expression of type I IFNs and other proinflammatory cytokines more robust than cGAMP. 8d also exhibited more resistant than cGAMP to enzymatic cleavage in vitro, indicating the successful improvement in drug availability. These findings provide guidelines for the design and structural optimization of CDNs as STING agonists.     

Heartwood of Dalbergia cochinchinensis: 4,7,2′-Trihydroxy-4′-methoxyisoflavanol and 6,4′-Dihydroxy-7-methoxyflavane Reduce Cytokine and Chemokine Expression In Vitro

Dalbergia cochinchinensis has been widely used in traditional medicine because of its flavonoids; however, the impact of the flavonoids to modulate the inflammatory response to oral cells remains to be described. For this aim, we isolated 4,7,2′-trihydroxy-4′-methoxyisoflavanol (472T4MIF) and 6,4′-dihydroxy-7-methoxyflavane (64D7MF) from the heartwood of D. cochinchinensis and confirmed the chemical structure by nuclear magnetic resonance. We show here that both flavonoids are inhibitors of an inflammatory response of murine RAW 264.7 inflammatory macrophages stimulated by LPS. This is indicated by interleukin (IL)1, IL6, and chemokine CCL2 production besides the phosphorylation of p65. Consistently, in primary murine macrophages, both flavonoids decreased the inflammatory response by lowering LPS-induced IL1 and IL6 expression. To introduce oral cells, we have used human gingival fibroblasts and provoked the inflammatory response by exposing them to IL1β and TNFα. Under these conditions, 472T4MIF, but not 64D7MF, reduced the expression of chemokines CXCL1 and CXCL2. Taken together, we identified two flavonoids that can reduce the expression of cytokines and chemokines in macrophages and fibroblastic cells.     

CRISPR-Cas System for RNA Detection and Imaging

The system of clustered regularly interspaced short palindromic repeats(CRISPR)and CRISPR-associated endonucleases(Cas)have been widely used in gene editing,disease treatment,molecular diagnosis and chromosome imaging.On account of the programmable target recognition of CRISPR-Cas system and the specific targeting function toward RNA of type Ⅵ class Ⅱ Cas proteins,CRISPR-Cas system has been deployed as RNA recognition and detection tools,exhibiting promising application potentials in the field of RNA detection and imaging.In this review,we summarize the latest research progresses as well as development prospects of CRISPR-Cas system in RNA diagnosis and live cell RNA imaging.     

5,2′-Dibromo-2,4′,5′-trihydroxydiphenylmethanone Inhibits LPS-Induced Vascular Inflammation by Targeting the Cav1 Protein

Vascular inflammation is directly responsible for atherosclerosis. 5,2′-Dibromo-2,4′,5′-trihydroxydiphenylmethanone (TDD), a synthetic bromophenol derivative, exhibits anti-atherosclerosis and anti-inflammatory effects. However, the underlying pathways are not yet clear. In this study, we first examined the effects of TDD on toll-like receptor-4 (TLR4) activity, the signaling receptor for lipopolysaccharide (LPS), and found that TDD does not inhibit LPS-induced TLR4 expression in EA.hy926 cells and the vascular wall in vivo. Next, we investigated the global protein alterations and the mechanisms underlying the action of TDD in LPS-treated EA.hy926 cells using an isobaric tag for the relative and absolute quantification technique. Western blot analysis revealed that TDD inhibited NF-κB activation by regulating the phosphorylation and subsequent degradation IκBα. Among the differentially expressed proteins, TDD concentration-dependently inhibited Caveolin 1(Cav1) expression. The interaction between Cav1 and TDD was determined by using biolayer interference assay, UV-vis absorption spectra, fluorescence spectrum, and molecular docking. We found that TDD can directly bind to Cav1 through hydrogen bonds and van der Waals forces. In conclusion, our results showed that TDD inhibited LPS-induced vascular inflammation and the NF-κB signaling pathway by specifically targeting the Cav1 protein. TDD may be a novel anti-inflammatory compound, especially for the treatment of atherosclerosis.     

Chemical synthesis of stimuli-responsive guide RNA for conditional control of CRISPR-Cas9 gene editing

CRISPR-Cas9 promotes changes in identity or abundance of nucleic acids in live cells and is a programmable modality of broad biotechnological and therapeutic interest. To reduce off-target effects, tools for conditional control of CRISPR-Cas9 functions are under active research, such as stimuli-responsive guide RNA (gRNA). However, the types of physiologically relevant stimuli that can trigger gRNA are largely limited due to the lack of a versatile synthetic approach in chemistry to introduce diverse labile modifications into gRNA. In this work, we developed such a general method to prepare stimuli-responsive gRNA based on site-specific derivatization of 2′-O-methylribonucleotide phosphorothioate (PS-2′-OMe). We demonstrated CRISPR-Cas9-mediated gene editing in human cells triggered by oxidative stress and visible light, respectively. Our study tackles the synthetic challenge and paves the way for chemically modified RNA to play more active roles in gene therapy.

Conditional control of CRISPR-Cas9 activity by reactive oxygen species and visible light is achieved using stimuli-responsive guide RNA synthesized by a general method based on RNA 2′-O-methylribonucleotide phosphorothioate.     

2′-O-Trifluoromethylated RNA – a powerful modification for RNA chemistry and NMR spectroscopy

New RNA modifications are needed to advance our toolbox for targeted manipulation of RNA. In particular, the development of high-performance reporter groups facilitating spectroscopic analysis of RNA structure and dynamics, and of RNA–ligand interactions has attracted considerable interest. To this end, fluorine labeling in conjunction with ¹⁹F-NMR spectroscopy has emerged as a powerful strategy. Appropriate probes for RNA previously focused on single fluorine atoms attached to the 5-position of pyrimidine nucleobases or at the ribose 2′-position. To increase NMR sensitivity, trifluoromethyl labeling approaches have been developed, with the ribose 2′-SCF₃ modification being the most prominent one. A major drawback of the 2′-SCF₃ group, however, is its strong impact on RNA base pairing stability. Interestingly, RNA containing the structurally related 2′-OCF₃ modification has not yet been reported. Therefore, we set out to overcome the synthetic challenges toward 2′-OCF₃ labeled RNA and to investigate the impact of this modification. We present the syntheses of 2′-OCF₃ adenosine and cytidine phosphoramidites and their incorporation into oligoribonucleotides by solid-phase synthesis. Importantly, it turns out that the 2′-OCF₃ group has only a slight destabilizing effect when located in double helical regions which is consistent with the preferential C3′-endo conformation of the 2′-OCF₃ ribose as reflected in the ³J (H1′–H2′) coupling constants. Furthermore, we demonstrate the exceptionally high sensitivity of the new label in ¹⁹F-NMR analysis of RNA structure equilibria and of RNA–small molecule interactions. The study is complemented by a crystal structure at 0.9 Å resolution of a 27 nt hairpin RNA containing a single 2′-OCF₃ group that well integrates into the minor groove. The new label carries high potential to outcompete currently applied fluorine labels for nucleic acid NMR spectroscopy because of its significantly advanced performance.

The new 2′-OCF₃ label for nucleic acid NMR spectroscopy carries high potential to outcompete currently applied fluorine labels because of significantly advanced performance.     

Synthesis and Anti-Hepatitis B Activities of 3′-Fluoro-2′-Substituted Apionucleosides

Nucleoside analogues have excellent records as anti-HBV drugs. Chronic infections require long-term administration ultimately leading to drug resistance. Therefore, the search for nucleosides with novel scaffolds is of high importance. Here we report the synthesis of novel 2′-hydroxy- and 2′-hydroxymethyl-apionucleosides, 4 and 5, corresponding triphosphates and phosphoramidate prodrugs. Triphosphate 38 of 2′-hydroxymethyl-apionucleoside 5 exhibited potent inhibition of HBV polymerase with an IC₅₀ value of 120 nM. In an HBV cell-based assay, the phosphoramidate prodrug 39 demonstrated potent activity with an EC₅₀ value of 7.8 nM.     

Substrate Specificity of Chimeric Enzymes Formed by Interchange of the Catalytic and Specificity Domains of the 5′-Nucleotidase UshA and the 3′-Nucleotidase CpdB

The 5′-nucleotidase UshA and the 3′-nucleotidase CpdB from Escherichia coli are broad-specificity phosphohydrolases with similar two-domain structures. Their N-terminal domains (UshA_Ndom and CpdB_Ndom) contain the catalytic site, and their C-terminal domains (UshA_Cdom and CpdB_Cdom) contain a substrate-binding site responsible for specificity. Both enzymes show only partial overlap in their substrate specificities. So, it was decided to investigate the catalytic behavior of chimeras bearing the UshA catalytic domain and the CpdB specificity domain, or vice versa. UshA_Ndom–CpdB_Cdom and CpdB_Ndom–UshA_Cdom were constructed and tested on substrates specific to UshA (5′-AMP, CDP-choline, UDP-glucose) or to CpdB (3′-AMP), as well as on 2′,3′-cAMP and on the common phosphodiester substrate bis-4-NPP (bis-4-nitrophenylphosphate). The chimeras did show neither 5′-nucleotidase nor 3′-nucleotidase activity. When compared to UshA, UshA_Ndom–CpdB_Cdom conserved high activity on bis-4-NPP, some on CDP-choline and UDP-glucose, and displayed activity on 2′,3′-cAMP. When compared to CpdB, CpdB_Ndom–UshA_Cdom conserved phosphodiesterase activities on 2′,3′-cAMP and bis-4-NPP, and gained activity on the phosphoanhydride CDP-choline. Therefore, the non-nucleotidase activities of UshA and CpdB are not fully dependent on the interplay between domains. The specificity domains may confer the chimeras some of the phosphodiester or phosphoanhydride selectivity displayed when associated with their native partners. Contrarily, the nucleotidase activity of UshA and CpdB depends strictly on the interplay between their native catalytic and specificity domains.