So far, few microclusters containing vanadium have been described in the literature. In this report, the synthesis protocol for the preparation of oxovanadium (IV) microclusters with 2-phenylpyridine is shown for the first time. Moreover, the crystal structure of these microclusters is also studied through the use of X-rays. The morphology of the prepared crystals is investigated using a field-emission Scanning Electron Microscope (SEM). The new compound, after activation by modified methylaluminoxane as the catalytic system, is investigated regarding the oligomerizations of 3-buten-1-ol, 2-chloro-2-propen-1-ol, allyl alcohol, and 2,3-dibromo-2-propen-1-ol. The products of oligomerization are tested by the TG-FTIR and MALDI-TOF-MS methods. Moreover, the values of catalytic activities for the new oxovanadium(IV) microclusters with 2-phenylpyridine are determined for the 3-buten-1-ol, 2-chloro-2-propen-1-ol, allyl alcohol, and 2,3-dibromo-2-propen-1-ol oligomerizations. Oxovanadium(IV) microclusters with 2-phenylpyridine are shown to be very highly active precatalysts for the oligomerization of allyl alcohol, 2,3-dibromo-2-propen-1-ol, and 3-buten-1-ol. However, in the case of 2-chloro-2-propen-1-ol oligomerization, the new microclusters are seen as highly active precatalysts. All oligomerization processes were carried out under a nitrogen atmosphere, atmospheric pressure, and temperature of 65 °C. The obtained products from the oligomerization of all the tested olefins, 3-buten-1-ol, 2-chloro-2-propen-1-ol, allyl alcohol, and 2,3-dibromo-2-propen-1-ol, were subjected to TG-FITR and MALDI-TOF-MS tests. The results obtained by the TG-FITR method are shown in The test results obtained by the TG-FTIR method show that the oligomers of 3-buten-1-ol, 2-chloro-2-propen-1-ol, allyl alcohol, and 2,3-dibromo-2-propen-1-ol consist of oligomer chains with a different content of mers; from 3 in the case of 2,3-dibromo- 2-propen-1-ol to 17 mers in the case of allyl alcohol. Oligomeric chains composed of the largest number of mers were obtained using allyl alcohol as the monomer. Thus, a mixture of oligomers consisting of 17, 14, and 11 mers was obtained. The oligomerization of 3-buten-1-ol led to a chain sequence with 16, 14, 12, and 9 mers. In the case of the oligomerization of 2-chloro-2-propen-1-ol, chains of 14, 13, 11, 9, and 7 mers were obtained. A distinctive case was the oligomerization of 2,3-dibromo-2-propen-1-ol, which resulted in the lowest number of mers—i.e., 5, 4, and 3—occurring in the oligomer chains.These studies on the catalytic activity of new oxovanadium(IV) microclusters with 2-phenylpyridine determined the values of catalytic activity for the compounds formed from the oligomerization of four different olefins: 3-buten-1-ol, 2-chloro-2-propen-1-ol, allyl alcohol, and 2,3-dibromo-2-propen-1-ol. The calculated catalytic activities are as follows in Based on the adopted division of catalytic efficiency introduced in 1999 by Britovsek, Gibson, and Wass , the fol10O28H2]4- cluster forms centrosymmetric dimers by interacting with O(cluster)–H···O(cluster). A SEM analysis showed that the morphology of the obtained crystals were irregular with a smooth surface. The bigger particles sizes ranged from 1 to 4.5 µm. The oxovanadium(IV) microclusters with 2-phenylpyridine were used after activation by MMAO-12 as catalytic systems in the oligomerizations of 3-buten-1-ol, 2-chloro-2-propen-1-ol, allyl alcohol, and 2,3-dibromo-2-propen-1-ol. The products of oligomerization were tested by the TG-FTIR and MALDI-TOF-MS methods. TG-FTIR method showed that the oligomer samples obtained from selected olefins release CO, CO2, and water. MALDI-TOF-MS data confirmed that all oligomers consist of chains with a different content of mers in the range of 3–17. This study shows the catalytic activities of oxovanadium(IV) microclusters with 2-phenylpyridine act, after activation by MMAO-12, as highly active catalytic systems for 3-buten-1-ol, allyl alcohol, and 2,3-dibromo- 2-propen-1-ol, and that they exhibit highly active catalytic properties for 2-chloro-2-propen-1-ol oligomerization.In this report, we described for the first time the synthesis protocol for the preparation of oxovanadium(IV) microclusters with 2-phenylpyridin, and the catalytic activity of the new compound for the oligomerizations of 3-buten-1-ol, 2-chloro-2-propen-1-ol, allyl alcohol, and 2,3-dibromo-2-propen-1-ol. The synthesis of new microclusters was carried out using vanadyl acetylacetonate, oxydiacetic acid, and 2-phenylpyridine in water. The crystallographic studies confirmed that a [V


Accordingly, we found that VdH1 silencing was reduced over time in recovered hyphae cultured in vitro, inferring that once the fungus got rid of the 35S-VdH1i cotton plants would gradually regain their pathogenicity. To explore whether continually exporting dsRNAs/siRNAs from transgenic plants into recipient fungal cells guaranteed the effectiveness and stability of HIGS, we created GFP/RFP double-labeled V. dahliae and transgenic Arabidopsis expressing dsGFP (35S-GFPi plants). Confocal images visually demonstrate the efficient silencing of GFP in V. dahliae that colonized host vascular tissues. Taken together, our results demonstrate that HIGS effectively triggers long-lasting trans-kingdom RNAi during plant vasculature V. dahliae interactions, despite no amplification or transitivity of RNAi being noted in this soil-borne fungal pathogen.Host-induced gene silencing (HIGS) based on trans-kingdom RNA interference (RNAi) has been successfully exploited to engineer host resistance to pests and pathogens, including fungi and oomycetes. However, revealing the mechanisms underlying trans-kingdom RNAi between hosts and pathogens lags behind applications. The effectiveness and durability of trans-kingdom silencing of pathogenic genes are uncharacterized. In this study, using our transgenic  Neurospora crassa  dCTP using the Rediprime II system (Amersham). For low molecular weight RNA gel blots, 40 µg of total RNA was separated by electrophoresis on 17% PAGE gels and electrically transferred to nylon N+ membranes. Then, [ɑ-32P] UTP-labeled gene-specific transcript sequences were used (New England Biolabs). For detection of the silencing of VdH1 in recovered hyphae, colonies recovered from the same infected plant at 20 days postinoculation were mixed for RNA isolation. For qRT–PCR, 2 μg of total RNA was reverse transcribed into cDNA using HiScript II Q RT SuperMix for qPCR (Vazyme). qRT–PCR analysis was performed with a 1000 series Thermal Cycling Platform (Bio-Rad) using SYBR qPCR Master Mix (Vazyme). The constitutively expressed elongation factor 1-α of V. dahliae (VdELF1) was used as an internal control. Gene-specific primers are listed below: VdELF1-qRT-F, CCATTGATATCGCACTGTGG and VdELF-qRT-R, TGGAGATACCAGCCTCGAAC; RFP-qRT-F, AGGACGGCTGCTTCATCTAC and RFP-qRT-R, CTTCAGGGCCTTGTGGGT; GFP-qRT-F, ATGGTGAGCAAGGGCGAGGAG and GFP-qRT-R, TAGGTCAGGGTGGTCACGAGG. At least three biological replicates and three technical replicates were performed in each experiment for each sample. Fungal isolates were grown in liquid Czapek–Dox medium for 3 days with shaking at 200 rpm and 26 °C in the dark, and the resulting mycelium was harvested for RNA isolation using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. For high molecular weight RNA gel blots, 20 µg of total RNA was separated on 1.2% agarose gels containing 6% formaldehyde and transferred to nylon N+ membranes. DNA probes were labeled with [a-5 conidia/mL in water solution) for 10 min and then transferred onto a 0.75% agar plate at 25 °C in the dark. Images were obtained under a confocal laser microscope  with 100× oil immersion objective lenses. The excitation wavelengths and emission filters were as follows: 488 nm/bandpass 500 to 550 nm for GFP and 561 nm/bandpass 570 to 670 nm for RFP. Confocal images were captured with a Leica hybrid detector and analyzed with Leica LAS AF software. A. thaliana roots were immersed in a conidial suspension (~10V. dahliae recovered from V592-infected cotton (VdWT−1st) and V592-infected 35S-VdH1i cotton (VdVdH1i−1st) were grown in liquid Czapek–Dox medium and harvested as described above for RNA extraction. 35S-VdH1i transgenic cotton plants grown at 26 °C with a 16 h light (8000 lux)/8 h dark cycle for approximately 3 weeks were subject to RNA extraction. RNA isolation, sRNA library construction, and sRNA sequencing were performed by BGI . The raw data were filtered to remove low-quality reads to obtain clean sequences. The sRNAs were aligned with VdH1 to the region used in designing hairpin RNA for creating 35S-VdH1i cotton plants. A Perl script was used to search for known miRNAs in cotton [n cotton ,63 with


Vitis vinifera L.) is the most cultivated fruit plant. It has a significant impact on the economies of many countries through wine and fresh and dried fruit production. In recent years, the grape and wine industry has been facing outbreaks of known and emerging viral diseases across the world. Although high-throughput sequencing (HTS) has been used extensively in grapevine virology, the application and potential of third-generation sequencing have not been explored in understanding grapevine viruses and their impact on the grapevine. Nanopore sequencing, a third-generation technology, can be used for the direct sequencing of both RNA and DNA with minimal infrastructure. Compared to other HTS methods, the MinION nanopore platform is faster and more cost-effective and allows for long-read sequencing. Due to the size of the MinION device, it can be easily carried for field viral disease surveillance. This review article discusses grapevine viruses, the principle of third-generation sequencing platforms, and the application of nanopore sequencing technology in grapevine virus detection, virus–plant interactions, as well as the characterization of viral RNA modifications. Among all economically important plant species in the world, grapevine ( V. vinifera from Eurasia [In 2020, over 7.3 million hectares of agricultural land worldwide were planted with grapevines. The economies of many countries are influenced by wine, table grape, raisin, seed oil, alcoholic beverage, and vinegar production , as well Eurasia . The eco Eurasia . In addi Eurasia .Grapevine red blotch virus and Grapevine Pinot gris virus, as well as new strains of known viruses, have been identified and detected through high-throughput sequencing (HTS) technologies. These viruses cause substantial negative impacts on grapevine production by reducing vine growth, fruit production, and fruit quality [More than 80 viruses from 17 families and 34 genera can infect grapevine . Approxi quality ,10. Early identification of viruses allows growers to take quick actions and effective sanitary measures, such as removing infected vines, limiting the movement of agricultural machinery, cleaning tools, controlling vectors to limit the spread of viruses, and providing improved tools for certification procedures of propagative materials before importing germplasm ,11. SomeGrapevine Syrah virus 1 (GSyV-1), through total RNA and dsRNA sequencing, Grapevine vein-clearing virus (GVCV) and Grapevine Pinot gris virus (GPGV), through small RNA sequencing, Grapevine virus F (GVF) and Grapevine red blotch virus (GRBaV), through dsRNA sequencing, and Grapevine Roditis leaf discoloration-associated virus (GRLDaV), through siRNA sequencing [With the advent of HTS, the characterization and investigation of known and novel viruses have been made possible. Although many viruses have been detected and identified by total RNA extraction and sequencing, the abundance of viral sequences relative to cellular RNAs is often very low, and the detection of low-titer viruses may be compromised. Some extractionmethods have been developed to concentrate viral nucleic acids, thus improving the abundance of viral sequences submitted to HTS. These methods include virion-associated nucleic acids (VANAs) and double-stranded RNA (dsRNA) extraction, small interfering RNA (siRNA), and rRNA depleted and polyadenylated RNA isolation. Many known and novel grapevine viruses have been detected and identified with these methods, such as quencing ,23,24,25quencing ,27.There are many unanswered questions about the biological and epidemiological features of individual grapevine viruses in mixed infections, the molecular basis of plant–virus interactions, and grapevine virome complexity. Therefore, in this review, we briefly introduce the principle of third-generation sequencing platforms and explain the application of nanopore technology in several areas of research in grapevine virology.Through the advent of third-generation sequencing technologies, such as PacBio single-molecule real-time (SMRT) sequencing and Oxford Nanopore sequencing, some of the aforementioned limitations of second-generation sequencing have been addressed. In SMRT sequencing, a double-stranded DNA template is circularized, called SMRTbell, through the ligation of hairpin adapters to both ends. The template and DNA polymerase are immobilized at the bottom of a chip, named the SMRT Cell, consisting of many photonic nanostructures, zero-mode waveguides (ZMWs). The latest generation of SMRT sequencer has one million ZMWs per SMRT Cell. Single-molecule real-time sequencing does not require an amplification step, and the template is sequenced based on its complementary strand synthesis by DNA polymerase with fluorescently labeled dNTPs . After lIn 2014, a pocket-sized commercial sequencer device, MinION, was introduced by Oxford Nanopore Technologies (ONT). The concept of nanopore sequencing was initially described in 1996 . NanoporNanopore sequencing technology has lower throughput and high error rates compared with second-generation sequencing technologies. However, several features, such as the small size of the sequencer, ease of library preparation, low sequencing cost, and short run times, make it an interesting tool for the surveillance of viruses and other pathogens . To dateIn recent years, ONT has improved the read quality of nanopore sequencing by changing the chemical reagents used in library preparation kits and flow cells and developing improved algorithms for basecalling. For example, higher raw read accuracy (98.3%) was achieved by a new basecaller software, Bonito . Additioplum pox virus detection in tobacco samples indicated that nanopore technology could effectively identify plant viruses [The potential of nanopore sequencing for the detection and identification of plant pathogens in field and lab environments has been shown in many different studies. Different library preparation kits with straightforward protocols are available and have been used to detect a number of different viruses from infected plant samples . For ins viruses . However viruses . TherefoThe positive features of nanopore sequencing, such as quick library preparation protocols, pocket-sized sequencer machines, producing many sequences in few hours, and being a cost- and time-effective method, have the potential to dramatically impact plant pathology, especially in the area of diagnostics. In particular, this technology can be used for the parallel sequencing of multiple samples through multiplex barcoding. Our research group at Agriculture and Agri-Food Canada’s Saint-Jean-sur-Richelieu Research and Development Centre has been working on optimizing this technology to detect grapevine viruses. Preliminary results indicate that not only is this technology cheaper than HTS but also the diagnostic capabilities are similar to Illumina (unpublished data) and could be an attractive alternative diagnostic tool for grapevine viruses.Below, we discuss different library preparation protocols that can be used for the detection and identification of grapevine viruses. In addition, we describe how nanopore sequencing can be used for the identification of different long non-coding RNAs and circular RNAs involved in grapevine virology, as well as its potential to study plant or viral RNA modification. Grapevine viruses are represented by many families, including viruses with RNA or DNA genomes . DiffereEighteen different reagent kits for the nanopore sequencing of various genetic materials have been commercialized, and four of them were used in different plant virus detection projects . The dirAnother nanopore sequencing kit that has recently attracted more attention is the direct RNA sequencing kit. The association of motor protein, as the regulator of the nucleic acid molecule translocation, with the RNA molecule indicates the low translocation velocity of RNA through the nanopore protein, which means that the passing speed of RNA within the nanopore is slow enough to be measured and sequenced. Consequently, using algorithms adapted to this platform, current intensity fluctuations induced by the passage of RNA molecules through the nanopore can be converted into sequence information ,51.In contrast to the direct cDNA sequencing kit, the cDNA synthesis step (the first strand of cDNA) is optional for direct RNA sequencing. After the ligation of a double-stranded RT Adapter (RTA) and RNA sequencing adapter (RMX), native RNA can be sequenced directly . NeverthAs a common limitation among all nanopore sequencing kits, high sequencing error rates limit the accurate characterization of quasi-species, new strains or subtypes of a virus, and the de novo assembly of viral reads ,55,56. ICowpea bright yellow mosaic virus (CoBYMV) and African cassava mosaic virus (ACMV), have been detected using ligation and rapid sequencing kits, respectively [Since several single-stranded DNA viruses, such as GRBV, and double-stranded DNA viruses, such as GVCV, can infect grapevines, two library preparation kits, ligation and rapid sequencing, are available for detecting them. Indeed, similar viruses, such as ectively ,38. Althectively . For the real-time detection of known viruses, adaptive sequencing by nanopore sequencing devices is possible. In this kind of sequencing, through partial mapping reads against desirable reference genomes, undesired reads can be ejected from the nanopore through provisionally reversing the voltage, and only targeted sequences are sequenced ,62. AdapThere are various infectious long non-coding RNAs that originated from viruses or viroids. Viroids are single-stranded circular non-coding RNAs that are the smallest infectious entities (between 246 and 401 nt in length) of plants and do not encode any protein. Viroids can replicate individually in the host plant nucleus or chloroplast by using host RNA polymerases through asymmetric and symmetric rolling-circle mechanisms . AlthougPospiviroidae family, such as Hop stunt viroid, Australian grapevine viroid, Grapevine latent viroid, and Grapevine yellow speckle viroid-1,2,3 and a tentative and an unclassified viroid, Grapevine hammerhead viroid-like RNA, have been identified in grapevines [As minor mutations in viroid sequences can dramatically affect the viroid movement , sequencapevines ,69. Grapapevines ,70. TherTobacco ringspot virus (TRSV) as a helper virus and attenuated the symptoms in infected tobacco samples [Sobemovirus, Nepovirus, and Polerovirus [As with viroids, virusoids  are other circular, non-coding RNAs ranging from 220 to 457 nt in length. Virusoids replicate through a rolling-circle mechanism based on RNA intermediates and are dependent on helper viruses or host plants. In contrast to viroids, virusoids are encapsidated by the helper virus coat protein ,71. The  samples . In contRecently, the function of long non-coding RNAs (lncRNA) and circular RNA (circRNA) as new RNA regulators in plant developmental processes and stress responses, especially in plant–virus interactions, has been studied . Long noCircular RNAs (circRNAs), as single-stranded and stress-inducible RNAs, are generated by rare back-splicing events from precursor mRNAs (Pre-mRNAs). In a back-splicing event, an upstream 3′ splice site is ligated to a downstream 5′ splice site through a 3′–5′ phosphodiester bond ,88 CircRAlthough many lncRNAs and circRNAs have been identified by the advent of short-read sequencing , the preConcurrent with the advancement in next-generation sequencing, many circRNAs in plants have been characterized by various algorithms and bioinformatics tools, such as CIRI , KNIFE , and CIR6A) modifications in moso bamboo (Phyllostachys edulis) by using nanopore direct RNA sequencing. Concisely, extracted total RNAs were treated with RNase R to remove linear RNAs. Since the digestion of linear RNAs was not entirely achieved by RNase R treatment, poly(A) tailing was performed to deplete the remaining linear RNAs through oligo (dT)25 beads. In the next step, rRNAs were depleted, and then the purified circRNAs were fragmented and dephosphorylated. Finally, after first-strand cDNA synthesis using a customized RT adapter containing degenerate primers, the sequencing adapter was ligated. Although this library preparation approach successfully identified circRNAs and their m6A modifications, the low sequencing depth of nanopore direct RNA sequencing remained the main obstacle for quantitative analysis [Two interesting library preparation methodologies for circRNA sequencing based on nanopore direct RNA and cDNA sequencing are introduced in the following section. A novel library preparation procedure was reported in a recent study to characterize 470 unique circRNAs and their N6-methyladenosine , 5-methylcytidine (m5C), or N4-acetylcytidine (ac4C) on eukaryote mRNAs [6A on HIV-1 mRNA can modulate HIV-1 gene expression and affects the HIV-1 RNA stability and exportation of its RNA from nucleus [6A not only can decrease virus pathogenicity in Influenza virus but also promotes infection kinetics [6A methylation of alfalfa mosaic virus RNA. This correlated with reduced systemic spread of the virus, suggesting that viral RNA methylation dynamics may play an important role in virus infections in plants [5C methylation sites on the human coronavirus genome through the bioinformatics tool called Tombo [6A modification sites on different types of RNAs by using nanopore sequencing data [Recently, a novel area in virology, viral RNA epitranscriptomics, has been investigated with the advent of new RNA modification site mapping techniques, such as nanopore direct RNA sequencing. Many questions about the functionality of these RNA modifications have been raised. RNA modifications appear through adding biochemical groups, such as a methyl group, to adenosine, cytosine, uracil, and guanosine nucleotides . Althougte mRNAs ,114, vir nucleus . In addikinetics ,116. Likn plants . Until ned Tombo . Moreoveing data ,120. In ing data . The conThrough the advent of nanopore sequencing, a new way to sequence and quantify RNA and DNA has been established. Soon, nanopore technology will have a tremendous impact on plant virus detection, RNA modification research, plant epigenomics, epitranscriptomics, and other aspects of plant biology and virology. The sequencing error rate of this technology initially was significantly higher than other high-throughput sequencers. However, the technology is being continuously improved to increase its accuracy by optimizing new nanopore proteins and motor proteins, designing new basecalling algorithms, changing the chemistry of library preparation kits, and testing a new sequencing orientation (Outy). This technology is a cost- and time-effective method for addressing various aspects of grapevine virology, such as massively parallel sequencing (MPS), grapevine virus detection, virus–plant interactions, and viral RNA modifications. Although a number of challenges, such as error correction and base modification detection, need to be resolved, the valuable potential of nanopore technology in grapevine virology should not be overlooked.


Carbon-ion radiotherapy (CIRT) for esophageal cancer has been receiving significant attention given its high local control rates and minimal damage to normal tissues. However, the efficacy and safety of salvage surgery after CIRT for esophageal cancer remain unclear. We report the case of a patient who underwent salvage thoracoscopic surgery after CIRT.A 51-year-old woman underwent upper gastrointestinal endoscopy and a type 0-IIa + 0-IIc esophageal squamous cell carcinoma located 27–29 cm from the patient’s incisors, classified as clinical stage I (T1bN0M0), was detected. She received CIRT  for localized esophageal cancer and achieved complete remission after 4 months. Six years after CIRT, follow-up endoscopic examination demonstrated a type 0-IIa + 0-IIc tumor in the previously treated area. In addition, a type 0-IIa lesion located 20–22 cm from the incisors was found. We diagnosed localized ESCC, classified as clinical stage I (T1bN0M0). Salvage thoracoscopic surgery was performed in the prone position with five access ports. Although the esophagus tightly adhered to the thoracic descending aorta and left main bronchus with severe fibrosis, the esophagus could be separated from the surrounding organs with careful forceps manipulation. The operation time and blood loss were 8 h 45 min and 253 mL, respectively. The patient was discharged from our hospital 17 days after the salvage surgery without any complications. Pathological findings revealed two squamous cell carcinomas. Both tumors were localized in the lamina propria mucosa, and lymph node metastasis was not detected. The tumors were diagnosed as pathological stage IA (pT1aN0M0) according to the TNM criteria. Moreover, pathological examinations showed severe fibrosis of the previously irradiated tissues compared to the normal esophagus located outside of the irradiation field. Following the surgery, the patient had no recurrence for 1 year and 6 months.Thoracoscopic radical esophagectomy can be performed as salvage surgery. Careful and discreet surgery is integral to perform salvage surgery after CIRT since CIRT may cause severe adhesions and fibrosis in the irradiated field. Esophageal cancer is the sixth most common cause of cancer-related deaths worldwide . EsophagIn light of advancements in irradiation machinery, carbon-ion radiotherapy (CIRT) has been increasingly used alongside stereotactic body radiation therapy. CIRT has higher local control rates and incurs minimal damage to normal tissues thanks to a higher local dose distribution than conventional radiotherapy using photons. Several studies have documented the effectiveness of CIRT for esophageal cancer , 6; howeHere, we document the case of a patient with ESCC who underwent thoracoscopic esophagectomy following CIRT as salvage surgery. To our knowledge, this is the first report of salvage esophagectomy following CIRT.18F-fluorodeoxyglucose positron emission tomography (FDG-PET) did not detect any lymph nodes or distant metastases. The patient was diagnosed with middle intrathoracic ESCC, classified as clinical stage I (T1bN0M0) according to the UICC-TNM classification 8th edition [A 51-year-old female underwent upper gastrointestinal endoscopy during a medical checkup. An examination identified a type 0-IIa + 0-IIc lesion located 27–29 cm from the patient’s incisors , is increasingly performed worldwide. According to a recent review article, the incidence rates of postoperative complications after MIE, such as pneumonia, arrhythmia, anastomotic leakage, and recurrent laryngeal nerve palsy, are approximately 10% each . SalvageAlthough several studies have demonstrated the treatment efficacy of CIRT for ESCC , 6, no cThere have been no randomized trials comparing clinical results of X-rays and CIRT. It is difficult to discuss the therapeutic results of CIRT when compared with those of X-rays because there have been few reports on their treatment outcomes and the number of cases treated is small. Enrollment for a phase I/II clinical trial of CIRT for patients with clinical stage I ESCC (UMIN000013552) has already been completed and the results of the analysis are awaited.Prior to surgery, we found that the tumor had infiltrated the submucosal layer according to the endoscopic ultrasonography (EUS) findings. However, a pathological examination revealed that the tumor was located in the LPM. Typically, it remains difficult to accurately predict the depth of the tumor following irradiation due to tissue fibrosis. Griffin et al. showed that the accuracy rate of EUS for esophageal cancer following neoadjuvant chemoradiotherapy was 36% . AlthougIn this case, 6 years after CIRT, a new esophageal cancer developed at the site of the primary tumor. It remains unknown whether a newly appearing cancer is a recurrent primary tumor or metachronous disease. Since we did not perform a genomic analysis, it was difficult to correctly answer this oncological question. However, Xi et al. reported that 88.3% of recurrences after chemoradiotherapy in patients with esophageal cancer occurred within 2 years . We specAlthough CIRT caused strong fibrosis and adhesion between the tumor and surrounding tissues, we performed a thoracoscopic radical esophagectomy as salvage surgery. Careful and discreet surgery is integral to perform salvage surgery after CIRT since CIRT may cause severe adhesions and fibrosis in the irradiated field.


Antisense sequence-specific knockdown of pathogenic RNA offers opportunities to find new solutions for therapeutic treatments. However, to gain a desired therapeutic effect, the multiple turnover catalysis is critical to inactivate many copies of emerging RNA sequences, which is difficult to achieve without sacrificing the sequence-specificity of cleavage. Here, engineering two or three catalytic peptides into the bulge–loop inducing molecular framework of antisense oligonucleotides achieved catalytic turnover of targeted RNA. Different supramolecular configurations revealed that cleavage of the RNA backbone upon sequence-specific hybridization with the catalyst accelerated with increase in the number of catalytic guanidinium groups, with almost complete demolition of target RNA in 24 h. Multiple sequence-specific cuts at different locations within and around the bulge–loop facilitated release of the catalyst for subsequent attacks of at least 10 further RNA substrate copies, such that delivery of only a few catalytic molecules could be sufficient to maintain knockdown of typical RNA copy numbers. We have developed fluorescent assay and kinetic simulation tools to characterise how the limited availability of different targets and catalysts had restrained catalytic reaction progress considerably, and to inform how to accelerate the catalytic destruction of shorter linear and larger RNAs even further.  Over recent decades, powerful and versatile sequence-specific interventions, such as siRNAs  RNA-cleaving constructs also showed multiple substrate turnover against model synthetic RNA  and furs siRNAs , with mas siRNAs  and leads siRNAs . Saturats siRNAs . CRISPR s siRNAs ,43, albes siRNAs . More des siRNAs , and mays siRNAs ,47. Artis siRNAs , but rems siRNAs ,54 and tetic RNA , but werdiseased’ to ‘normal’ to achieve therapeutic effect. In this context our pilot structures  sequence-specifically ,65–66,682-CO2H attached at the central point of the recognition motif, which hybridized with target RNA (tRNAPhe) in a sequence-specific manner and cut it at multiple positions  was purchased from Biomatik . Modified oligonucleotide sequences containing two or more internal abasic nucleotides, with aminohexyl linker attached at C1’ position in α or β-configuration were purchased from ATDBio Ltd . Alpha and beta aminohexyl phosphoramidite monomers were purchased from Link Technologies Ltd . Fluorescently-labelled, linear perfect-match target F-Q-RNA  was purchased from ATDBio Ltd . A scrambled conjugate  was synthesized following the previously reported protocol (The peptide Ac-Leu-Arg-Leu-Arg-Gly-Leu-Arg-Leu-Arg-Gly-OH (Ac-[LRLRG]protocol  and was 1H NMR spectra were recorded using Bruker Avance II+ spectrometers operating at proton frequencies of 400 MHz using BBI 1H/D-BB Z-GRD Z8202/0347 probe. Bruker Topspin 4.0.3 software was used for data processing. MALDI (matrix-assisted laser desorption ionization) mass spectra were collected on a Bruker Daltonics Ultraflex TOF/TOF mass spectrometer  at the Manchester Interdisciplinary Biocentre, University of Manchester.All chemicals and solvents were purchased from Sigma-Aldrich (UK) unless stated otherwise. Water was purified by a Milli-Q purification system . Reversed-phase HPLC purification of synthesized peptide and conjugates was carried out on PerkinElmer  and Agilent 1100  HPLC systems equipped with diode array detector and Rheodyne (3725i) manual injector on a semi-preparative Phenomenex Luna C-18 column . NAP disposable columns prepacked with Sephadex G-25 DNA grade  were used to desalt peptidyl-oligonucleotide conjugates. Oligonucleotide concentrations were calculated using extinction coefficients see  for absoThe analytical data and full characterization of the generated peptides and peptidyl-oligonucleotide conjugates are given in the Supplementary Information see .3G) was synthesized manually using solid-phase peptide synthesis on Boc-Gly-PAM resin . The Boc protective group was removed by the addition (2 × 15 ml for 2 and 25 min) of 50% (v/v) trifluoroacetic acid in dichloromethane . The resin was subsequently washed with DCM (4 × 15 ml) and neutralized with 5% DIPEA in DCM. Before each coupling reaction, the resin was washed with DCM (3 × 15 ml) and DMF (1 × 15 ml). The amino acid coupling reactions were achieved by pre-activating either Boc-Arg(Tos)-OH  or Boc-Leu-OH  in DMF (10 ml) with DIPEA  and HBTU . The coupling reaction proceeded for 45 min at room temperature under mechanical shaking. Following synthesis, the N-terminus was acetylated through the addition of acetic anhydride (1.80 mmol) with DIPEA (1.8 mmol) in DMF (15 mL). The peptide was cleaved from the resin by treatment with HF in the presence of p-cresol (0.75 g) and thiocresol (0.25 g) scavenger mixture for 3 h in an ice bath under constant stirring. After removal of HF, the crude peptide was precipitated and washed with cold diethyl ether (2 × 30 ml). Finally, the peptide was extracted with 10% acetic acid and 80% acetonitrile/0.1% TFA and then freeze dried.Ac-Leu-Arg-Leu-Arg-Leu-Arg-Gly  and mass spectrometry ). Analytical data for the Acetyl-[LRLRG]2-CO2H peptide has been previously reported in (1H NMR spectra can be seen in Supplementary Information (see The synthesized crude peptide was dissolved in 30% (v/v) acetic acid and purified by reverse-phase (RP) HPLC  using 0.1% TFA in HPLC-grade water (eluent A) and 0.1% TFA in acetonitrile (eluent B) with a linear gradient (0–100%) over 45 min at a flow rate of 8 ml/min. Following purification, the peptide was characterized using orted in ; MALDI-Ttion see .m/z = 926 [M+H]+,  : δ 0.86–0.94 , 1.49–1.94 , 2.02 , 3.21 , 3.84–4.37 .MALDI-TOF-MS: 6N16O9]) . 1H NMR  (1H NMR  (400 MHz4 in acetone (1.8 ml) for 36 h as described  salt (50 nmol) soluble in DMSO by dropwise addition of 4% (w/v) CTAB as previously described . This eaescribed , then ex4 in water as eluent A and 0.05 M LiClO4 in acetonitrile as eluent B with 2.0 ml/min flow rate of 0% B for 3 min, and 0–100% B over 30 min with absorbance monitored at 260 nm. The identity and purity of conjugates were confirmed by RP-HPLC, 1H NMR spectroscopy and mass spectrometry.Crude conjugates were purified using RP-HPLC with a 0.05 M LiClOm/z = 11189 [M+Na]+ , 400 MHz): δ 0.79–0.86 , 1.16–2.22 , 2× acetyl-CH3), 3.16 , 3.66–4.41 , 4.99–6.27 , 7.23–8.47 , dA(H8 × 8), dA(H2 × 8), dC(H6 × 5) and dT(H6 × 5)). H3’ sugar ring protons (3.6–4.4 ppm) were not analysed due to suppression of the residual water signal at 4.10 ppm.RP-HPLC fraction at around 28 min was collected and lyophilized to give a white powder  . Excess O187P28] .1H NMR  (1H NMR (2O with TSP (0.01 mM), 400 MHz): δ 0.78–0.85 , 1.15–2.43 , 2× acetyl-CH3), 3.17 , 3.57–4.35 , 4.96–6.25 , 7.25–8.46 , dA(H8 × 8), dA(H2 × 8), dC(H6 × 5) and dT(H6 × 5)). H3’ sugar ring protons (3.6–4.4 ppm) were not analysed due to suppression of the residual water signal at 4.12 ppm.RP-HPLC fraction at around 28 min was collected and lyophilized to give a white powder  . Excess 187P28]) .1H NMR . 1H NMR (D2O with TSP (0.01 mM), 400 MHz): δ 0.79–0.86 , 1.13–2.38 , 2× acetyl-CH3), 3.17 , 3.51–4.45 , 4.95–6.32 , 7.25–8.50 , dA(H8 × 8), dA(H2 × 8), dC(H6 × 5) and dT(H6 × 5)). H3’ sugar ring protons (3.6–4.4 ppm) were not analysed due to the suppression of residual water signal at 4.10 ppm.RP-HPLC fraction at around 28 min was collected and lyophilized to give a white powder  . Excess m/z = 11189 [M+Na]+  (1H NMR (2O with TSP (0.01 mM), 400 MHz): δ 0.79–0.86 , 1.13–2.38 , 2× acetyl-CH3), 3.17 , 3.51–4.45 , 4.95–6.32 , 7.25–8.50 , dA(H8 × 8), dA(H2 × 8), dC(H6 × 5) and dT(H6 × 5)). H3’ sugar ring protons (3.6–4.4 ppm) were not analysed due to suppression of the residual water signal at 4.10 ppm.RP-HPLC fraction at around 28 min was collected and lyophilized to give a white powder  . Excess 187P28]) . 1H NMR   (1H NMR (2O with TSP (0.01 mM), 400 MHz): δ 0.78–0.88 , 1.91–2.39 , 3 × acetyl-CH3), 3.17 , 3.62–4.33 , 5.03–6.35 , 7.25–8.47 , dA(H8 × 8), dA(H2 × 8), dC(H6 × 5) and dT(H6 × 5)). H3’ sugar ring protons (3.6–4.4 ppm) were not analysed due to the suppression of residual water signal at 4.27 ppm.RP-HPLC fraction at around 30 min was collected and lyophilized to give a white powder  . Excess 194P29]) .1H NMR  was incubated with one of the ‘bis’ or ‘triple’ conjugates in excess (up to 20 μM).Hybridization assays and data analysis were carried out as previously reported , here 3’Phe (1 μM) and one of the ‘bis’ or ‘triple’ conjugates in variable excesses  of single turnover conditions in Tris Buffer (50 mM Tris–HCl pH 7.0 supplemented with 0.2 M KCl and 1 mM EDTA).Cleavage studies were also carried out according to the previously reported protocols . The reaF-Q-RNA (1 μM) with fluorescein (F) and dabcyl quencher (Q) covalently attached to the internal deoxythymidine residues and one of the peptidyl-oligonucleotide conjugates (20 μM). Aliquots (10 μl) were taken at regular intervals  during incubation of the reaction mixture at 37°C in 50 mM Tris−HCl pH 7.0 supplemented with 0.2 mM KCl and 1 mM EDTA. The combined effect of dilution (10-fold) of the collected aliquots with water and elevated temperature  achieved complete dissociation of the cleaved RNA fragments from the conjugate. The extent of cleavage was evaluated by measuring the increase in fluorescence at λem of 522 nm (following excitation at 496 nm) at 37°C in microwell plates using a Tecan Safire plate reader operated under Magellan Data Analysis Software (V.7). The fluorescence of untreated F-Q-RNA was subtracted at each time-point to eliminate the background signal and normalized here to the complete (100%) cleavage of the same F-Q-RNA substrate achieved by RNase A (20 nM) in parallel control incubates. As controls, target was incubated both in the presence and absence of unconjugated oligonucleotide, and parallel measurements were conducted to rule out spontaneous cleavage. In addition, the target was incubated with a non-complementary scrambled conjugate (5’-luc-h-9/14) (The reaction mixture (100 μl) contained fluorescently-labelled linear target RNA -h-9/14)  to assessingle’ conjugates (BC5-α or BC5-L-β) and one of the most active ‘bis’ conjugates (BC5-L-ββ) against linear fluorescently-labelled F-Q-RNA target present at 2-fold, 5-fold, 10-fold, 20-fold or 30-fold molar excess over conjugate. These reaction mixtures (100 μl) containing one of the 5-nt bulge–loop inducing conjugates (5 μM) and the corresponding molar excess of F-Q-RNA, which was present at 10 μM, 25 μM, 50 μM, 100 μM or 150 μM concentration, were incubated at 37°C over 96 h in 50 mM Tris buffer pH 7.0 supplemented with 0.2 mM KCl and 1 mM EDTA. Aliquots (10 μl) of each reaction mixture were taken after the same time intervals  and similarly subjected to dilution (10-fold) in water and high temperature (80°C for 3 min) to dissociate reaction complexes. The extent of F-Q-RNA target cleavage by individual conjugates  was evaluated by measuring the increase in fluorescence intensity at each time point and normalizing it to the fluorescence of the fully-cleaved (100%) F-Q-RNA substrate by RNase A, as described in the previous section. These data were then used to estimate product concentration (or amount) to quantify the accumulation of the F-Q-RNA cleavage products. Kinetic parameters  were estimated by analysis with a non-linear model to simulate change in initial reaction velocity (V0) with available initial substrate [S0] allowing for the formation of unreactive substrate and catalytic complexes  bulge–loop in the RNA upon conjugate-RNA hybridization, in order to provide several possible cleavage points in the exposed single-stranded RNA region. The opportunity to cut RNA at many positions is re-enforced further by incorporating several catalytic peptides into the same conjugate molecule to allow multiple attacks on the induced bulge–loop region  to cleave many copies of RNA substrate. This can only be achieved through rapid release of the cleaved RNA fragments after each cleavage event, to liberate the catalytic conjugate from the hybridized complex, to allow a new attack on the next RNA molecule Figure  and achicascades  to enablbis’ and ‘triple’ bulge–loop inducing conjugates that bind tRNAPhe in a sequence-specific manner and form a single-stranded 5-nt bulge–loop 61C-62A-63C-64A-G65 in the TΨC-stem region, while maintaining Watson–Crick base pairing with the 3’-acceptor stem, TΨC-arm and variable loop , the thermodynamic parameters  and Tm values for each potential complex between the recognition oligonucleotide and every 26-mer segment from the 5’-part of tRNAPhe sequence. Some representative structures from this screening, along with the corresponding Tm, ΔG, ΔH and ΔS values are shown in via aminohexyl linker attached to the anomeric C1’ carbon of the internal abasic sugar residues (X) to generate ‘bis’ or ‘triple’ conjugates, respectively, as illustrated in Figure We engineered here a series of the ‘ble loop , as prev BC5-L-β . As a pa-arm see . We calcbis’ conjugates  in αα, ββ, αβ or βα-configuration. This provided us with the opportunity to explore several possible conformational arrangements to enhance the probability of identifying orientations of the key players, vital for achieving potent RNA cleavage. A relatively long catalytic peptide Acetyl-[LRLRG]2-CO2H (Pep1) was chosen for ‘bis’ conjugation. This contained 10 amino acids, including four catalytically-important arginine residues scattered along the peptide chain  was chosen to generate a ‘triple’ conjugate via attachment to the three abasic sugar moieties located in the middle of the antisense recognition motif  to allow fair comparison of their catalytic performance against RNA. Given the slight superiority of the β-configuration in terms of RNA cleavage within the bulge–loops, previously observed for the ‘single’ conjugates  and 30.3 min (‘triple’ conjugate) was observed, which was reproducible in all conjugation reactions . However, careful signal integration, along with comparison between the NMR spectra of starting oligonucleotide and conjugates, allowed us to verify successful conjugation. For example, characteristic peptide signals, generated by Leu-Hδ and Arg-Hδ in resonance areas of 0.8–1.2 ppm and 3.1–3.4 ppm, respectively, are visible in the 1H NMR spectra of conjugates , as well as H1’ sugar ring protons (5.4–6.4 ppm), confirmed 2:1 and 3:1 stoichiometric ratios of the peptide content to the oligonucleotide component in ‘bis’ and ‘triple’ conjugates, respectively.A shift in HPLC retention time from 17.5 min (average for starting oligonucleotides) to 28.5 min  were estimated from the curve of the relative proportion of tRNAPhe bound to conjugate as a function of conjugate concentration, and are given in Table bis’ and ‘triple’ conjugate nomenclature and composition.The catalytic performance of ‘nditions . First, ssay see , as the Phe in a concentration-dependent manner  was reached at a conjugate concentration around or slightly above 5 μM for the ‘bis’ conjugates and above 10 μM for the ‘triple’ conjugate  enhanced their association constants in comparison to those of their ‘single’ counterparts  (single’ counterpart BC5-L-β (Ka = 0.5 ± 0.2 × 106 M–1) , thus suggesting that the attachment of the peptide(s) in the β-configuration may lead to less favourable binding conformations, which seem to annul the stabilizing effect arising from any additional interactions.All conjugates were capable of binding to tRNAnner see . The useonjugate . Compari106 M–1) . The difbis’ and ‘triple’ conjugates were first assessed against 3’-FITC-tRNAPhe under physiological conditions  over 4, 8, 24, 48 and 72 h and analysed by 12% PAGE electrophoresis under denaturing conditions. A large 20-fold excess of conjugates (20 μM) over tRNAPhe (1 μM) ensured that even the conjugates with the lowest binding affinity (e.g. BC5-L-ββ and BC5-L-βββ) reached full hybridization. The cleavage sites were identified through comparison with RNase T1 and 2 M imidazole tRNAPhe hydrolysis ladders , and in terms of cleavage measured exclusively at bulge–loop regions . No spontaneous cleavage was seen in the absence of conjugate  degradation of the target RNA in 24 h  were able to attack the entire loop and cleave it at five different positions, with a predominance of the 65G-66A site, followed by the 63C-64A > 64A-65G > 62A-63C >> 61C-62A positions. Overall, BC5-L-αα and BC5-L-ββ conjugates seemed to be more versatile, both in terms of their ability to produce cuts at multiple sites and to catalyse cleavage of a variety of phosphodiester linkages, including G-X, C-A, A-C and even A–G. In contrast, the attachment of two catalytic peptides in opposite configurations (either αβ or βα) in BC5-L-αβ and BC5-L-βα ‘bis’ conjugates led to a relatively narrow, but sharp cleavage with only two major cuts in the target region 61C-62A-63C-64A-65G. It is evident from Figures 63C-64A position became the most vulnerable when tRNAPhe was attacked by either the BC5-L-αβ or BC5-L-βα conjugate, whereas the 62A-63C site was less favourable. The other two sites  showed only minor cleavage contributions, not exceeding 8% even when taken together. Cleavage at A–C and especially at C-A phosphodiester bonds showed strong predominance over that at G-X linkages.Two distinctive cleavage patterns were observed amongst ‘n Figure . BC5-L-αbis’ conjugates suggested that the spatial orientation of the two catalytic peptides against each other seems to control whether they act independently or jointly in the catalysis. RNA cleavage requires the coordinated attack of two guanidinium groups from the opposite sides of the RNA backbone , one acure 8 in . In the  Figures  and 4. P BC5-L-β  showed striple’ conjugate BC5-L-βββ, with all three peptide moieties attached in the same configuration showed similar cleavage patterns to those seen for BC5-L-αβ and BC5-L-βα ‘bis’ conjugates, thus suggesting possible joint action of the separate peptide moieties in the catalysis. Presumably, the shorter and less flexible Acetyl-(LR)3G-CO2H peptide restricts the possibility for each chain to form a guanidine-guanidinium dyad for independent catalytic action. The attachment of the peptide moieties at the three different abasic nucleotides seems to separate them sufficiently in space, which is further reinforced by a helical turn of the RNA-DNA hybrid duplex. This seems to allow these short peptides to approach the RNA backbone from opposite directions and supply arginine guanidinium groups for the synchronized catalytic action.Interestingly, the ‘bis’ and ‘triple’ conjugates was also reflected by full cleavage of 3’-FITC-tRNAPhe (up to 100%) at lower conjugate concentrations (10 μM and 5 μM) with lower molar excess of the conjugate over the target . As expected, BC5-L-αα and BC5-L-ββ were able to produce more cuts at nearby folded locations in addition to those seen for the BC5-L-αβ, BC5-L-βα and BC5-L-βββ conjugates, which included two extra cuts in the anticodon and variable loops . In agreement with the available X-ray structure of yeast tRNAPhe (2-CO2H peptide in its unfolded conformation (45.1 Å) or became conformationally accessible to the catalytic guanidinium groups (3G-CO2H peptide (34.2 Å) incorporated into the BC5-L-βββ structure was mainly able to reach the D-arm upon hybridization with tRNAPhe, but not the less proximal regions of the anticodon loop. Remarkably, these minor products of the nearby cleavage were accumulated during the first few hours of the cleavage reaction, and started to diminish rapidly after 8 h, followed by their complete disappearance at around 48 h (for BC5-L-βα) or 72 h . This was accompanied by faster accumulation of the cleavage products at the induced bulge–loop 61C-62A-63C-64A-65G .Due to the flexible attachment and overall length of the catalytic constructs, the ‘ Figures –E, where tRNAPhe , all idem groups . The shoG Figure –E in a cG Figure , ultimatPhe structure, but rapidly accelerates as the structure becomes more relaxed and flexible. As clearly illustrated by Figure ex-bulge’ cleavage), the cleavage behavior of tRNAPhe by all conjugates at different concentrations  followed the same distinctive path. Remarkably, up to 30–40% of nearby cleavage outside the bulge appeared necessary, in all cases, before reaching the point where bulge cleavage emerged and then progressively took over. The same cleavage path was followed at all concentrations  were retained, whereas more ‘conformationally demanding’ conjugates  had low initial velocities but, with few cuts outside the bulge–loop, appeared to show an increase in velocity over time, despite the decline in the concentration of tRNAPhe substrate  with BC5-L-βα, the overall cleavage extent reached 100%, 66% or 37% when the conjugate concentration was 20, 10 or 5 μM, respectively, under otherwise identical experimental conditions. A similar trend was seen for the other four bulge–loop inducing conjugates BC5-L-αα, BC5-L-ββ, BC5-L-αβ and BC5-L-βββ, as well as for the previously studied ‘linear’  to the RNA target, although the ‘triple’ conjugate achieved this level of binding at slightly higher (10 μM) concentration. One possible explanation of this phenomenon is that in aqueous solutions such supramolecular conjugates co-exist in multiple conformational forms, with only some being ‘catalytically active’ against RNA  cleavage was achieved. An enhanced probability to form the catalytic guanidine-guanidinium dyads, essential for catalysis, combined with the high degree of conformational freedom of the cleaving constructs flexibly linked to the RNA-recognizing motifs, seem to be crucial for efficient destruction of long RNA sequences with complex secondary and tertiary structural elements. However, the complexity of biological RNA substrates makes it difficult to assess the possibility of the catalytic turnover reaction, especially when they form supramolecular complexes with the peptidyl-oligonucleotide conjugates. Indeed, multiple-turnover catalysis usually requires a significant excess of RNA substrates (up to 30-fold) over the catalytic molecules, thus resulting in a high concentration of RNA sequences in solution. This may trigger some level of substrate aggregation, which can be further facilitated by formation of multiple peptide bridges between different RNA-conjugate hybrids, stabilized by electrostatic interactions between the positively-charged peptides and the negatively-charged sugar-phosphate backbones of nucleic acids  had fluorophore (F) and quencher (Q) at the positions flanking the bulge–loop region (rArCrArGr--C) composed of the ribonucleotide residues, while the rest of the F-Q-RNA sequence comprised deoxyribonucleotide residues, which are resistant to cleavage by ribonucleases, including the chemical (arginine-mediated) catalysts studied here. This design allowed us to monitor cleavage exclusively in bulge–loops by eliminating cuts outside this region. The nucleotide sequence of this 31-mer model target matched precisely the region corresponding to the 3’-acceptor stem, TΨC-arm and variable loop of tRNAPhe, in order to allow comparisons with our tRNAPhe studies. The bulge–loop inducing conjugate  in a sequence-specific manner, thus forcing formation of the bulge–loop region in the middle of the sequence. The size of the induced loop could be varied by the length of the non-complementary sequence in the oligonucleotide recognition motifs, so that either the entire RNA region (rArCrArGrC) was vulnerable for cleavage or lesser parts (<5-nt) could be exposed to catalytic attack  and ‘triple’ (BC5-L-βββ) conjugates were compared with previously studied , 3-nt (BC3-α and BC3-β), 4-nt (BC4-α and BC4-β) or 5-nt . The number in the nomenclature of ‘single’ conjugates indicated the size of the induced bulge. Since ‘single’ conjugates were also designed to target the 3’-acceptor stem, TΨC-arm and/ variable loop of tRNAPhe  was confirmed by a sharp decline in fluorescence, which reached a plateau of full binding at around 10 μM conjugate concentration and had association kinetics similar to that of unconjugated oligonucleotide  conjugate was used against F-Q-RNA, nor from unconjugated complementary oligonucleotide, verifying the absence of non-specific cleavage  bulge–loop, all ‘d Figure  and 6E. d Figure . A simile target , where t earlier . This incleavage .Phe  was investigated using the same fluorescent assay, but with up to 30-fold excess of the linear target F-Q-RNA over the conjugate, and was compared with that of the most active representatives of ‘single’ conjugates with the shortest (BC5-α) and longest (BC5-L-β) antisense recognition motifs. Each conjugate (5 μM) was incubated with 2-, 5-, 10-, 20- or 30-fold excess of the F-Q-RNA substrate present at 10, 25, 50, 100 or 150 μM concentration, respectively, when cleavage kinetics was followed over 96 h  raised the number of cleaved F-Q-RNA molecules initially, which later declined with the further increase in substrate concentration. Surprisingly, the highest excess of the RNA substrate (20-fold and/or 30-fold) appeared to be detrimental for cleavage, showing lower than expected number of cleaved RNA substrates (Table F-Q-RNA concentrations (100 and/or 150 μM), when the molar excess of the RNA substrate exceeded 20- or 30-fold  approximated to a more hyperbolic profile of initial velocities reminiscent of Michaelis–Menten kinetics, but again with deviations at lower substrate excesses and the strongest decline in velocities with reaction progress at 30-fold substrate excess  suffered from lower velocities at larger substrate excesses (20-fold and 30-fold), resulting in the atypical parabolic kinetic profiles Figure  and I. Hs Figure , E and HAn explanation of this discrepancy was found in the likelihood of self-association of the target substrate and conjugates into hairpin and duplex structures. Hairpin structures involving intramolecular folding are particularly stable and can be formed rapidly see . When doThe RNA substrate, which is expected to be complexed ved data .e.g. inhibition of the conjugate by the generated product). The activation route of the self-associated (and thus unavailable) conjugate  Figure , which w) Figure . The two) Figure . The les) Figure  was consonjugate  proceedsavailable substrate Michaelis–Menten kinetics for initial velocities . However, beyond 10-fold substrate excess, the extent of cleavage declined, presumably from conjugate inactivation  phosphoramidate analogues  were successfully synthesized and purified by RP HPLC in good yields (30–40%) and characterized by NMR and MS, confirming the desired structures. All conjugates showed sequence-specific saturation binding, when hybridizing with the biological tRNA target, which was not affected by multiple catalytic peptides incorporated into the recognition motif.bis’ and ‘triple’ conjugates compared to 70–80% by ‘single’ conjugates in 72 h. More dense and rigid arrangement of shorter ‘triple’ peptide offered no advantage over the longer more flexible ‘bis’ peptides. Peptides in the same configuration offered a broader attack from the same side of the RNA backbone and cut tRNAPhe at five positions, whereas peptides in opposing configurations offered narrower attack from opposite sides of the RNA backbone, coinciding at only 2 positions.Multiple peptide attachment considerably increased catalytic cleavage with 90–100% degradation in 24 h by ‘bis’ and ‘triple’ conjugates also showed considerable cleaving activity outside the region directly engaged in hybridization between the tRNAPhe and conjugate recognition motif, which may in the future provide potential advantage for effective demolition of long non-coding RNA or viral genomic RNA with the complex secondary and tertiary structural elements.In addition to the cleavage at the induced bulge–loops, flexibly anchored catalytic peptides within the ‘Phe) to relax this structure before cleavage within the bulge emerged and took over.RNA secondary and tertiary structure dominated reaction kinetics, with initial velocities up to 100 nM/h, with a common pathway of first cutting folds nearby the bulge loop to a threshold level of cleavage , but with turnover number increasing with the number and configuration of catalytic peptides, reaching Complex parabolic behaviour of the cleavage kinetics was observed. A simulation model was developed to reveal the dynamic nature of available substrate and active conjugate, after complexation into unavailable and inactive complexes, particularly at large excesses of target. The underlying Michaelis-Menton kinetics then emerged, with KFull cleavage of 10–20-fold excesses of available target demonstrated that each catalyst molecule destroyed 5–10 targets, even with only <30% of the conjugates in the active state.via thorough structural design guided by the discoveries of this work.When more fully activated by physiological conditions, each conjugate molecule may destroy over 30 target molecules, when a few conjugate molecules could knockdown RNA copy numbers typical biologically. Further improvement in the activation of such conjugates can be achieved gkab1273_Supplemental_FileClick here for additional data file.


Drosophila and that this is more efficient for DNA breaks in an intron-containing gene, consistent with the higher damage-induced siRNA levels downstream of an intron. The spliceosome thus stimulates recruitment of RNA polymerase II to the break, rather than merely promoting the annealing of sense and antisense RNA to form the siRNA precursor. In contrast, RNA polymerase III nascent RNA libraries did not contain reads corresponding to the cleaved loci and selective inhibition of RNA polymerase III did not reduce the yield of damage-induced siRNAs. Finally, the damage-induced siRNA density was unchanged downstream of a T8 sequence, which terminates RNA polymerase III transcription. We thus found no evidence for a participation of RNA polymerase III in dilncRNA transcription in cultured Drosophila cells.DNA double-strand breaks are among the most toxic lesions that can occur in a genome and their faithful repair is thus of great importance. Recent findings have uncovered local transcription that initiates at the break and forms a non-coding transcript, called damage-induced long non-coding RNA (dilncRNA), which helps to coordinate the DNA transactions necessary for repair. We provide nascent RNA sequencing-based evidence that RNA polymerase II transcribes the dilncRNA in  Drosophila helps to fend off viral infections  . There wspection , the reaCG15098 gene stimulates antisense transcription by RNA polymerase II. For the intronless Tctp gene, we detected none or only few antisense reads and statistical analysis is not appropriate. Our observations are thus consistent with the notion that a lower antisense transcription activity for the intronless gene (this study) correlates with fewer DNA-damage induced siRNAs [We conclude that induction of a DNA double-strand break in the intron-containing d siRNAs . It therin vivo or whether the polymerase stalls when it encounters the break. As long as the transcript is not cleaved and removed, this creates an R-loop behind the polymerase with concomitant exposure of the non-template strand. This stretch of single-stranded DNA could also serve as a landing site for another RNA polymerase complex and transcription thus initiates in the antisense orientation [Our findings also have important mechanistic implications since it could be the very same polymerase that synthesizes both sense and antisense transcript. In this most rudimentary form of ‘recruitment’, stalling of the splicing reaction could e.g. contribute to post-transcriptional modifications on RNA polymerase II that promote direct re-initiation upon a run-off at the break – a ‘U-turn’ movement, essentially. However, it is currently unclear whether a run-off will occur at a DSB entation . In thisentation  could foIn the absence of DNA damage, the spliceosome protects the genome from R-loop mediated instability in transcribed regions  and intrp = 0.05, t-test, unequal variance, n = 3), while PFM-01 (25 µM) had essentially no effect  – did not lead to a de-repression of ciferase . This isCG15098 locus revealed a serendipitous stretch of eight adenosines in the second intron. For an RNA polymerase acting in antisense orientation, this corresponds to a T8-sequence preceded by a potential secondary structure element . Taken together, it is unlikely that RNA polymerase III functionally contributes to dilncRNA transcription in Drosophila. However, our observations cannot exclude that RNA polymerase III is recruited to sites of DNA damage without subsequently engaging in processive transcription of the dilncRNA.We had previously determined that the damage-induced siRNA response starts in close proximity to the break and extends all the way until the transcription start site ,4,34. Thment see , which sment see . We do nin vitro analysis with inhibitors [and RNA polymerase III–in dilncRNA generation, the recent description of RNA polymerase III as the exclusive source of dilncRNA in cultured human cell lines is surprising [Previously, several publications have provided independent evidence of RNA polymerase II as an enzyme capable of transcribing the dilncRNA. This includes biochemical reconstitutions , in vitrhibitors , ChIP wihibitors , detectihibitors  and metahibitors . A singlhibitors , but thehibitors . While nrprising . We now rprising . The sitrprising . A paralDrosophila S2-cells with stable expression of cas9 protein (clone 5–3) were cultured and transfected as previously described [CG15098 or Tctp. The sgRNA expression cassettes were first generated by PCR, then blunt-end cloned into pJet1.2 to yield pRB59 (CG15098) and pRB60 (Tctp). The target sites were 5ʹ- TCCAGTGTAGCTTCCCGTT-3ʹ for CG15098 and 5ʹ- ATATCTAATTTCTTTTTAC-3ʹ for Tctp as described [escribed . We furtescribed . For theescribed .6 cells/ml), resuspended in 500 µl of lysis buffer  supplemented with proteinase inhibitors (Roche complete without EDTA)) and incubated for 10 minutes on ice. Then nuclei were pelleted by centrifugation at 5000xg for 5 minutes and the supernatant (mostly cytosol) was discarded. The pellet was resuspended in lysis buffer without EDTA but containing 1 M urea, incubated for 5 minutes on ice and again pelleted at 5000xg for 5 minutes. The urea washing step was carried out twice in total, then the nuclei were resuspended in 110 µl of lysis buffer without EDTA and without urea. To digest the chromatin, 250 U of benzonase  were added and the resuspended nuclei were incubated at 37°C for 3 minutes in a heating block. The digestion was stopped by adding EDTA and NaCl to a concentration of 10 mM and 500 mM, respectively. The insoluble fraction was pelleted by centrifugation at 16000xg for 5 minutes and the supernatant was used as input material for the immunoprecipitation.48 or 36 hours after transfection, the cells were harvested  supplemented with Roche complete proteinase inhibitors without EDTA), then 1 µl of V5 antibody was coupled by rotation at 4°C over night. On the following day, the beads were washed 3x with 300 µl of IP buffer, then the input material was added and incubated with agitation for 60 minutes at 4°C. After separation of the unbound supernatant, the beads were washed 5x with 200 µl of IP-buffer. The immunopurified RNA polymerase complexes were the digested with proteinase K to liberate the associated nucleic acids and RNA was prepared by TRIZOL extraction and precipitation.20 µl of magnetic beads  were washed 3 times with 200 µl of IP buffer  and 40 ng of pRB4  were transfected per well of a 96-well plate using Fugene-HD (Promega). Inhibitors were added 2 hr prior to transfection in a volume of 1 µl DMSO . The luciferase assay was performed 96 hrs after transfection using the Dual-Glo Luciferase assay system (Promega E2920) in a Tecan M-1000 plate reader. Data analysis was carried out using Microsoft Excel.The luciferase assay for the detection of DNA-break induced siRNAs has been previously described . BrieflyClick here for additional data file.