Antisense oligonucleotides (ASOs) are an increasingly represented class of drugs. These small sequences of nucleotides are designed to precisely target other oligonucleotides, usually RNA species, and are modified to protect them from degradation by nucleases. Their specificity is due to their sequence, so it is possible to target any RNA sequence that is already known. These molecules are very versatile and adaptable given that their sequence and chemistry can be custom manufactured. Based on the chemistry being used, their activity may significantly change and their effects on cell function and phenotypes can differ dramatically. While some will cause the target RNA to decay, others will only bind to the target and act as a steric blocker. Their incredible versatility is the key to manipulating several aspects of nucleic acid function as well as their process, and alter the transcriptome profile of a specific cell type or tissue. For example, they can be used to modify splicing or mask specific sites on a target. The entire design rather than just the sequence is essential to ensuring the specificity of the ASO to its target. Thus, it is vitally important to ensure that the complete process of drug design and testing is taken into account. ASOs’ adaptability is a considerable advantage, and over the past decades has allowed multiple new drugs to be approved. This, in turn, has had a significant and positive impact on patient lives. Given current challenges presented by the COVID-19 pandemic, it is necessary to find new therapeutic strategies that would complement the vaccination efforts being used across the globe. ASOs may be a very powerful tool that can be used to target the virus RNA and provide a therapeutic paradigm. The proof of the efficacy of ASOs as an anti-viral agent is long-standing, yet no molecule currently has FDA approval. The emergence and widespread use of RNA vaccines during this health crisis might provide an ideal opportunity to develop the first anti-viral ASOs on the market. In this review, we describe the story of ASOs, the different characteristics of their chemistry, and how their characteristics translate into research and as a clinical tool. Antisense oligonucleotide (ASO) drug development is emerging as a powerful tool for targeting RNAs, and has revolutionized the field of drug discovery. A rational drug design for any disease requires the identification of a suitable target and the development of a drug with a specific affinity for that target.The main goal is to understand the molecular mechanisms responsible for diseases and to investigate whether downregulating or activating a certain component of the molecular pathway of a disease would produce the desired biological effects. This would then be useful to the development of a new drug to treat that disease.The ability of an ASO to specifically recognize any RNA (via Watson–Crick base-pairing) means that the only information required for their synthesis is the target RNA sequence. This is now possible given the publication of the human genome sequence. Therefore, it seems straightforward to design a low-cost, highly specific lead compound to selectively target almost any gene (both coding and non-coding). As such, ASOs represent a very powerful target-directed approach for identification and validation in vitro, and subsequentially in vivo. This can be accomplished in a rapid manner, limiting the use of KO gene mouse models. It also provides an opportunity to test a therapeutic approach in the early stage of a project. Moreover, ASOs are highly valuable as novel and personalized therapeutic strategies to treat a wide range of diseases linked to dysregulated gene expression . More iASOs are able to affect gene expression in several ways. Specifically, they can cause RNA cleavage or RNA steric blockage in both the cytoplasm and the nucleus , dependiIt is clear that ASOs are extremely flexible molecules that can be used to modulate most cellular processes. Their application in basic and translational research represents a potent tool which scientists can use to disentangle complex molecular mechanisms and to pave the way for new, and potentially revolutionary, therapeutic approaches for diseases that are currently considered incurable.Antisense oligonucleotides (ASO) are short synthetic DNA or RNA molecules typically designed to target both coding and non-coding RNA in order to correct their expression level and ultimately their activity. They act by sequence paring so they need to be complementary to the target RNA to ensure sequence specific hybridization. The length is usually between 12 to 25 nucleotides, with a vast proportion of them designed to be around 18–21 nucleotides. Statistical calculations demonstrate that given the size of the human transcriptome, a sequence of 13 nucleotides for RNA occurs only once. Considering this fact, the average length of ASOs is enough to target a unique region of the genome [The first evidence of an injection of DNA inhibiting RNA activity was shown back in 1977, by impairing the translation of mRNAs in a cell-free system . A year Initially, ASOs were developed and used primarily to achieve translational silencing. They induce silencing through mediation by mRNA decay which leads to a reduced expression of the protein coded from the specific mRNA. This process is mediated by RNase H activation. RNase H is a ubiquitous enzyme that is able to recognize and cleave RNA-DNA duplexes that are at least 5 nucleotides long. Some ASO-RNA duplexes can recruit and activate RNase H, which leads to the degradation of the target RNA with an efficacy of up to 80–90%. In order to design a potent ASO for such applications, it is necessary to consider that different chemical modifications have different intrinsic abilities in recruiting RNase H ,18,19.The first ASOs used were synthetic deoxyribonucleotides, but their efficacy and application were limited due to several parameters that were overcome with chemical modifications. First generation ASOs, based on naked RNA, were highly sensitive to the degradation by both endo and exo-nucleases. These molecules have a limited ability to permeate into the membrane, and their solubility is low. Furthermore, the product of their degradation (phosphodiester oligonucleotides to dNMP2 mononucleotides) can be cytotoxic . All of The first important advancement that led to the evolution of first-generation ASOs into second-generation ASOs was the substitution of one of the non-bridging oxygen atoms by different chemical groups which improved the stability of the molecule. Substituting the non-bridging oxygen with methyl, sulfur and amine groups led to the production of methyl-phosphonates, phosphorothioates (PS), and phosphoramidate respectively. These new backbones increased the overall oligo stability even though there were some substantial differences between them. The methylphosphonate backbone is a non-charged molecule for which the modification occurs on each nucleotide of the chain. The uncharged state of those molecules reduces the solubility and cellular uptake in that it permeates cells mainly by endocytosis and not by diffusion through the membrane. This chemical modification plays an important structural role because the absence of charges eliminates the charge-charge repulsions. This is what is normally observed in an RNA-DNA duplex leading to a less stable bond between the ASO and its target. Moreover, oligonucleotides with methylphosphonate backbones are not able to engage with RNAse H activity ,21,22,23PS-based oligonucleotides seem to have most of the characteristics that researchers are looking for in an ASO, and they have in fact been used as antisense therapies in multiple studies. The first characteristic is their ability to recruit RNase H and trigger its activity. These negatively charged ASOs are not fully resistant to nucleases but have a higher stability compared to phosphodiester linkages and they are highly soluble. Thus, PS ASOs are very useful for basic research studies, but more importantly, they are very suitable as therapeutic molecules.The third member of first-generation ASOs are the phosphoramidates. Members of this family are uncharged molecules that do not activate the RNase H activity ,25. TheyDespite the efficiency of some first-generation ASOs, PS backbone oligonucleotides are able to interact with both plasma and cellular proteins which lead to off-target effects. It is also important to highlight the fact that they are not fully resistant to RNases, a point that might be a strength in certain situations but may be a weakness in others. Furthermore, their chemistry is difficult to manipulate and functionalize, preventing the whole class of compounds to have the flexibility that scientists would like to see in a research tool. In order to overcome the weaknesses of the first-generation ASOs and develop ones that are excellent therapeutic molecules, scientists developed more advanced molecules that fall under the umbrella of second-generation ASOs.Members of the second generation are characterized by the replacement of the hydrogen atom at the 2′-position of the ribose by an O-alkyl group leading to the formation of 2′-O-methyl (2′-OMe) and 2′-O-methoxyethyl (2′-MOE) nucleotides . These mIn order to increase the resistance to nucleases, a phosphate group can replace an oxygen atom as in PS ASO: an 2′-O-methoxyethyl (2′-MOE) with a PS backbone. To improve the mRNA decay, it is possible to induce the RNase H activity using different strategies, which led to the third generation of ASOs.The third generation of ASOs have a high degree of heterogeneity and involve many types of chemical modifications. Here we will present the most common of these modifications. The main goal of developing the next generation of ASOs was to improve resistance against nucleases and to increase their specificity. Thus, they are almost fully resistant to degradation by nucleases and peptidases, and this gives them tremendous stability in biological fluids . In addiCommon among third-generation ASOs are Lock Nucleic Acids (LNAs) which are characterized by the presence of a methylene bridge connecting the 2′-oxygen and 4′-carbon of the ribose ring. This extra bond locks the flexibility of the parent furanose and increases the strength of their target interactions. The presence of a single LNA monomer is enough to increase the melting temperature of the RNA/ASO binding from +3 to +11 °C compared to a full DNA oligo. They can bind on a single- or double-strand DNA or RNA and form a stable duplex or triplex . DespiteSecond-generation LNAs can interact with PS DNA to enhance their properties or trigger specific mechanisms of action. LNAs were shown early on to be formidable compounds and have paved the way for all antisense therapies used today. The first ASO to receive approval by the Food and Drug Administration (FDA) in 1998 and by the European Medicines Agency (EMA) in 1999 was a member of this class, and was very effective in the treatment of cytomegalovirus (CMV) retinitis (the details of which will be discussed later in this manuscript). In immunodeficient individuals, CMV infection can be quite severe when compared to healthy individuals where the infection can often go unnoticed. Therefore, the indication of this treatment is specific to patients with acquired immunodeficiency syndrome (AIDS) to prevent a progressive destruction of retinal cells ,36,37. VAnother member of the third-generation ASOs is phosphorodiamidate morpholino oligonucleotides (PMO) which consists of a nucleic acid where the five-membered ribose heterocycle is replaced by a six-membered morpholine ring. As PMOs are non-charged, they are not able to recruit RNase H . PeptideBoth LNAs and PMOs are uncharged molecules which decreases their affinity to plasma protein and increases the distribution and elimination in urine . Only 10The chemical modifications made over the last decade have allowed ASOs to be highly specific, more stable, reliable, and safe. They can be selective for a single-nucleotide polymorphism (SNPs) using different modifications such as 2-thiothymidine, 3′-fluorohexitol nucleic acid, and cET in combination with a little gap to engage RNase H ,45. The As previously described, the activity of RNase H is not possible for every ASOs, and we are able to divide them based on that fact. However, while this inability may appear as an inconvenience, scientists have taken advantage of this characteristic by developing steric blockers to correct splicing or to compete with a miRNA binding on a specific target .The strength and stability of interactions between an ASO and the complementary target RNA depends on many factors. Therefore, several considerations must be taken into account in order to have a successful ASO design. These include prediction of RNA secondary structures ,48, idenmfold [sfold [Typically, the optimal length of an ASO to target a unique sequence is approximately 18–21 nucleotides. Shorter sequences increase the probability of off-target hybridization while longer sequences reduce the cache of cellular uptake , an aspemfold  and sfold [sfold , allows d [sfold , mapped d [sfold  or softwd [sfold . The seqd [sfold . As RNasd [sfold . MoreoveIn addition, to obtain a highly potent lead compound, the binding energy between ASOs and mRNAs needs to be considered, and indeed several programs are able to calculate their thermodynamic properties ,58. A BLAn unmodified ASO is susceptible to rapid break down by exo- and endonucleases in the body, and their overall charged properties confer a weak permeation across biological membranes. Therefore, ASO delivery represents the major translational challenge, unlike their use in preclinical research. In vitro, it is relatively easy to overcome all of these problems by enhancing ASOs’ internalization through transient permeabilization of plasma membrane by mechanical approach , or by means of a delivery cationic liposome system (Lipofectamin or Transfectam). Tiny ASOs, typically 15 nucleotides or less, exhibit the property of being internalized by gymnotic uptake. This aspect is double-sided because it does not require any further chemical modification, but at the same time, specificity to a given tissue or cell type is very difficult. These are some of the main reasons why, nowadays, most antisense therapeutics have focused on their topical use and delivery via the liver. However, chemical modifications represent some of the most effective approaches to improve their pharmacokinetic and pharmacodynamic properties. They include backbone modification, different ribose, base modification, and functionalization of the ASOs with a specific targeting motif, like Gal-Nac  or aptamm) which makes them inadequate for most therapeutic purposes. This can be overcome by combining additional chemical modifications, like the 2’ modification  and furanose ring modification , which allows for their comprehensive use in almost all ASO drugs or drug candidates in clinical settings. Moreover, while these modifications dramatically improve RNA-binding affinity, it is important to note that PS do not support RNase H cleavage, thus damping the efficacy of the ASO. Consequently, second- and third-generation sugar modifications are often implemented in a ‘gapmer’ design in which a central gap region, consisting of approximately 5 to 10 PS-modified 2’deoxynucleotides, allow RNase H to sit and promote RNA degradation. This gap is flanked on both 3’ and 5’ sides by approximately five modified nucleotides’ ‘wings’ (2’OMe or 2’-MOE) resistant to nuclease cleavage. Furthermore, the symmetry of the gapmer is not an indispensable condition. Indeed, nucleotides responsible for the RNase H engagement can be placed anywhere in the oligo sequence and they will still be able to engage RNase H enzymatic activity [As previously discussed, PS-ASOs are easy to synthetize and carry a negative charge which allows for cellular delivery. Therefore, they might be designed in a way that makes them capable of activating RNase H and providing optimum protein-binding, resulting in suitable pharmacokinetics. The interaction of PS-ASO with 95% of plasma proteins extend their tissue elimination half-life, thus supporting peripheral tissue distribution. However, they also trigger off-target effects such as immune stimulation and complementary activation. Despite these disadvantages, PS chemical modifications are widely used for loss-of-function studies in vitro and in vivo for gene target identification and validation. However, PS modifications decrease the melting temperature  and create PPMO , allowinAny base with PS or PMO substitution creates a chiral center, meaning that two possible stereoisomeric forms with different properties like hydrophobicity/ionic character, nuclease resistance, target affinity, and RNase activity  might exASOs can be used with different routes of administration: intravenous, subcutaneous, intravitreal, intrathecal or local. It depends primarily on the targeted cell types and the chemical attributes of the molecule . The phaGiven the localization of cells to be treated, local administration can be an effective means of treatment. Formirsen (described further), which is used to treat the retinal cells infected by Cytomegalovirus (CMV), was injected intraocularly into the vitreous humor. This route of administration offers the advantage of high cellular specificity, and reduces the elimination that occurs with systemic administration. However, in the case of Formirsen, there were severe localized adverse effects such as increased intraocular pressure and local inflammation . For theOther than implementing chemical modifications to improve drug-likeness, covalent conjugation to cell-targeting or cell-penetrating moieties and nanoparticles’ formulation were developed to address the delivery challenge. Implementing bioconjugates including lipids, peptides, antibodies, aptamers, and sugars allow for receptor-associated cellular- binding for specific tissue/cell uptakes. They are typically chemically well-defined and small, meaning that high-scale synthesis at a low cost is obtainable along with a favorable distribution profile. For instance, Gal-Nac conjugation is one of the leading strategies to enhance PS-ASO hepatocyte delivery given its high affinity to ASGR1 and ASPGR ,83,84,85Animal models have been essential for proof of principle studies as well as for pharmacokinetic and pharmacodynamic investigations. Treating mice model with ASO was proven very effective to collect information and data on drug metabolism and target engagement. If an ASO is designed to target a specific tissue, typically with one of the strategies just described, an animal model is important to provide information about safety and efficacy. However, when an ASO is designed for a non-coding target in humans, it might be ineffective in a mouse model since most of the non-coding RNA are not very well conserved between different species. In the specific case of miRNA inhibition, even when the target miRNA is conserved between species, the results of treatment with ASOs might be profoundly different because the miRNA networking can be completely different moving from one species to another. These targets are specific to the transcriptome of each species and will result in different effects (including side effects), therefore, safety might become a major concern.Thanks to the improved chemical modifications in these molecules which make them more stable and more specific, ASOs have become a very powerful therapeutic tool. Since the first ASO was developed for a therapeutic purpose (created by Zamecnik and Stephenson in 1978 ), ASOs h®) marketed by Ionis Pharmaceuticals (formerly Isis) and Novartis Ophthalmics received the first ASO FDA approval. A year later, the EMA gave the same authorization. It is the only first-generation ASO to have received approval. It is a PS oligonucleotide targeting the viral mRNA encoding the early -2 (IE-2) protein, required for viral replication. Its indication was for the treatment of the cytomegalovirus (CMV) retinitis. CMV infection is of great concern in immunosuppressed individuals, while in healthy individuals, it often goes unnoticed. So the treatment was recommended for acquired immunodeficiency syndrome (AIDS) patients because a CVM infection could lead to the progressive destruction of the retinal cells [In 1999, Formirsen  developed by Grenzyme. This ASO is composed of PS-modified nucleotides surrounded by 2′O-MOE-based nucleotides, so it is a gapmer that belongs to the second generation. It is used to treat homozygous familial hypercholesterolemia patients. This pathology is caused by the mutation of the LDL cholesterol receptor, the pro-protein convertase subtilisin/kexin 9 (PCSK9) or apolipoprotein B (apo B), leading to the elevation of the low-density lipoprotein (LDL). This deregulation of LDL level results in an increased risk of developing coronary heart disease and atherosclerosis in young people [Perhaps one of the most well-known ASOs, approved by the FDA in 2013, is Mipomersen , is the first siRNA-based molecule that received the AMM. Developed by Alnylam, it targets the transthyretin (TTR) protein involved in the transport of the thyroxin and retinol-binding protein vitamin A complex. Patients who suffer from the hereditary transtherthyretin-mediated amyloidosis (hATTR) present with a mutation in a TTR gene leading to the production of an abhorrent TTR which is more susceptible to misfolding. This generates TTR amyloid fibril in the extracellular space of the heart, liver, nerve, and gastrointestinal tract which can disrupt the normal working of the organs. After the release of this siRNA in the cytoplasm of cell, it interacts with TTR mRNA leading to the reduction of TTR protein expression and the inhibition of the fibrils’ formation [Patisiran  which is a genetic disorder characterized by the excessive production of delta-aminolevulinate syntethase 1 (ALAS1). This over-expression of ALAS1 leads to the accumulation of neurotoxins such as aminolevulinic acid (ALA), and porphobilinogen (PBG) which causes abdominal pain, nausea, and seizures . To imprCurrently, there are a number of candidates under investigation that are seeking FDA approval. We focus here on only a few examples that are in Phase 3 clinical trials and that are showing promising results.Vutrisiran is an ASO developed by Alnylam Pharmaceuticals with the same indication as Patisiran. It is under investigation in Phase 3 clinical trials for the treatment of hATTR. It is also an siRNA that targets TTR mRNA to decrease the production of the protein .Tominersen is a new promising drug for the treatment of Huntington’s disease. This neurodegenerative disease is caused by the mutation of the coding gene of the huntington (HTT) protein. It corresponds to an increase in the number of repetitions of the CAG triplet leading to a mutant HTT with extended polyglutamine tract. This mutated protein causes the accumulation in neurons and affects their normal functioning which results in dystonia, cognitive dysfunction and behavioral difficulties ,107. TorVolanesorsen was developed by Ionis Pharmaceuticals, and is indicated for familial chylomicronemia syndrome. This syndrome is caused by a mutation of the lipoprotein lipase (LPL) gene leading to a poor fat breakdown. It results in an accumulation of fat in the blood and causes abdominal pain, eruptive xanthoma, pancreatitis, hepatosplenomegaly, and lipemia retinalis. Volanesorsen targets APOC3 mRNA in order to decrease plasma triglyceride and apolipoprotein C-III levels . ResultsIn contrast to the previous examples, Miravirsen does not target an mRNA but rather a microRNA (miRNA). MiRNA are short non-coding molecules (22 nucleotides) that target RNA by sequence complementary. MiRNA-bonding induce mRNA decay and the inhibition of the translation, and because they are involved in numerous biological processes, their deregulation is implicated in many pathologies . MiravirAnother anti-miR-122 (RG-101) was developed by Regulus Therapeutics. The chemical modification of nucleotides differs from Miravirsen. It is composed of phosphorothioate coupled to an N-acetylgalactosamine group  in order to increase the hapatocytes’ uptake due to the presence of asialoglycoprotein receptor at the surface of the cells. Unfortunately, the development of the molecule was halted due to liver toxicity .Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of the ongoing coronavirus disease 2019 (COVID-19) pandemic, which is currently creating a world health emergency . AlthougThe potential for ASOs as virus inhibitors dates back to 1970, with the first antisense experiment utilizing ASOs to block Rous sarcoma virus replication and the first antisense molecule approved by the FDA, Fomivirsen . MoreoveTo rationally design an ASO, the knowledge of the viral genome sequence and structure is sufficient, and approaches that can inhibit the SARS-CoV-2 life cycle at different stages are important for COVID-19 management .SARS-CoV-2 has a 29,903 base of single-stranded, positive-sense RNA flanked by 5′ and 3′ untranslated regions (UTRs) and shares 89.1% homology with SARS-CoV (GenBank accessible number MN908947). After infection, the viral genome is translated either in non-structural proteins (NSPs) from two open reading frames (ORF1a and ORF1b) taking part in forming the replication/transcription complex (RTC), or structural proteins from subgenomic viral RNAs. Among these, the most important are the nucleocapsid (N) protein, the transmembrane (M) protein, the envelope (E) protein, and the spike (S) protein . As all However, the viral RNA genome has a high mutation rate enabling it to rapidly evolve and adapt. This allows it to evade the immunity in response to previous infections or vaccinations by slightly modifying its core proteins ,129, cauCertain RNA structures, crucial for the viral cell cycle, are well conserved amongst the genome. This makes them valuable ASO targets  to fightRecent advances in high-throughput structure probing strategies (SHAPE-MaP and DMS-MaPseq) allows for the characterization of the full-structural RNA landscape pinpointing a well-defined subset of highly-conserved and weakly-structured regions ideal for ASO design ,142. UsiOther LNA gapmer ASO candidates were also predicted to have excellent properties to target more key domains such as the 5’untranslated region (5’UTR), and the open reading frames 1a and 1b (ORF1a and ORF1b) involved in the expression of the RTC and gene N, which encodes the genome-associated nucleoprotein, with GAP1 candidate on 5’UTR being the most potent . FurtherAnother strategy to minimize the risk of escape-mutant occurrence is the inhibition of cellular genes that are unlikely to mutate, which the viruses depended on. The most forward approach is to silence the angiotensin-converting enzyme 2 (ACE2) as SARS-CoV-2 entry receptor ,151. UsiDegraded structural protein RNAs could offer a variable strategy as well. In a recent study, Xiaoxuan Su et al. raised the possibility of designing a chimeric oligonucleotide comprising a 2’-OMe-modified antisense oligonucleotide. The purpose would be to recognize the target viral RNA and a 5’-phosphorylated 2’-5’ poly (A)4 (4A2-5-ASO) for guided RNase L activation to sequence-specifically degrade the viral RNA in order to target envelope and spike proteins thus inhibiting SARS-CoV-2 infection . AdditioAs previously mentioned, the strong tendency to undergo frequent genetic mutations leads to a continuous emergence of new variants with enhanced infectivity, even among vaccinated people. Therefore, SARS-CoV-2 surveillance is also essential in controlling widespread transmission given the challenge in predicting the severity of illness in infected individuals . AlthougThe advent of sequencing and high throughput analysis in the last two decades has given us a better molecular understanding of pathologies and genetic characterization. This has notably led to the development of targeted therapies and a paradigm shift in how we view personalized medicine. Currently, genetic diseases, cancer, and neurodegenerative diseases are within the domain of ASO-based therapies, however, ASOs can also be used to fight viral infections. They are especially important now that we are in the midst of a major health crisis. ASOs are a powerful tool, and when combined with vaccinations, they have the potential ability to effectively fight the COVID-19 pandemic by providing a possible preventive and curative treatment.ASOs are mostly known for degrading an mRNA or for modifying the splicing process. The molecules currently authorized by the governmental authorities or which are undergoing clinical trials act by one of these mechanisms. However, the versatility and adaptability of these molecules have made them excellent candidates for more innovative and original therapeutic strategies . It is pSeveral studies have developed new therapeutic strategies which induce the degradation of mRNA mediated by ASO without the involvement of RNase H. No-go-decay (NGD) is involved in translational control as it acts when the ribosome progression is disrupted, and induces the mRNA decay by endonucleolytic . ASOs weTherefore, ASOs are an extremely versatile group of molecules used specifically to correct genetic alterations and propose a personalized therapy for each individual patient .


Hollow sphere structures with perforations (PHSSs) in three different arrangements (simple cubic (SC), body-centred cubic (BCC), and face-centred cubic (FCC)) were fabricated through three-dimensional (3D) printing, and the mechanical behaviours of these PHSSs under quasi-static compression were investigated experimentally and numerically. The results indicated that under uniaxial compression, the PHSSs mainly undergo three stages, i.e., a linear elastic stage, a large deformation or plateau stage, and a densification stage. During the stage of large deformation, the SC and BCC PHSSs experience a preliminary compaction sub-stage after layer-by-layer buckling, while for the FCC PHSS, layer-by-layer collapse and compaction are the dominant deformation behaviours. A numerical simulation was employed to study the mechanical properties of PHSSs with different geometric parameters under quasi-static compression and to explore the effect of the wall thickness, hole diameter, and sphere arrangement on the first peak stress, plateau stress, and specific energy absorption (SEA) of the PHSSs. The results reveal that the geometric parameters have a significant impact on the large deformation behaviour and energy absorption capacity of PHSSs. The presented PHSS is also proven to be much lighter than traditional metallic hollow sphere structure (MHSS) and has higher specific strength and SEA. With abundant pores in the matrix material and an internal microstructure, cellular metal or metal foam has unique functional and structural properties. This material is ultralightweight and is characterised by good sound absorption, high heat resistance, energy absorption, and high specific stiffness and strength . It has Based on the regular atomic arrangement concept, Sanders and Gibson performed a numerical simulation analysis of the elasticity modulus and initial yield stress of MHSSs in different packing patterns, such as simple cubic (SC), body-centred cubic (BCC) and face-centred cubic (FCC) patterns, and found that MHSSs have better mechanical properties than cellular materials with random porous structures ,5. GasseMany experimental and numerical studies have also been performed to reveal the large deformation process and quasi-static and dynamic responses of hollow sphere structures in detail. Gao et al. numerically studied the static and dynamic deformation behaviours and mechanical properties by using simplified geometric models of sintered MHSSs and proposed formulas for the elastic modulus, yield strength, and plateau stress of the FCC packing material under quasi-static compression . VesenjaWith ping-pong balls as the experimental object, Yu et al. carried out a series of static and dynamic compression experiments on single-ball and multi-ball arrays, theoretically derived the compressive deformation pattern of a stacking of spheres, and verified the theories through various two-dimensional (2D) ping-pong ball stackings; some findings were beneficial to understanding the deformation mechanism of a real MHSS ,18,19. TAlthough some understanding of the static and dynamic mechanical properties of hollow sphere structures/materials has been gained through these studies, which were mostly conducted under ideal stacking and connection conditions by using the finite element method, the obtained theoretical formulas are not consistent with the experimental results in some cases. Although not impossible, it is at least difficult for traditional manufacturing technology to ensure that the stacking pattern of hollow spheres is kept in line as well as the connection mode between spheres; moreover, the mechanical properties of hollow sphere materials are extremely sensitive to the arrangement pattern of the spheres and the connection geometry between the spheres.Additive manufacturing/3D printing, as an emerging manufacturing technology, can be used to directly produce more complex components regardless of the structure or shape, and this technique offers a new approach for fabricating hollow sphere materials. Recently, researchers have used stainless steel, photosensitive resin, polylactic acid (PLA) plastics, and nylon to fabricate different hollow sphere structures through additive manufacturing techniques, such as fusion selective laser melting (SLM) ,24, sterUnderstanding the mechanism of large deformation or plastic behaviour of perforated hollow sphere structures (PHSSs) is important for designers to more fully utilise the energy absorption characteristics. In this work, multi-cell PHSSs in different arrangements were prepared by using the MJF 3D printing technology and the large deformation behaviour of PHSSs under quasi-static compression was then investigated experimentally and numerically. The experimental results verified the effectiveness of the associated FEMs. Furthermore, the effect of geometric parameters, such as wall thickness, hole diameter and packing pattern, on the structural deformation mode, first peak stress, plateau stress and specific energy absorption (SEA) was analysed by using the finite element method, which offers a reference for further lightweight engineering applications of 3D printed hollow sphere structures.mR, wall thickness t, opening or hole diameter md, and distance d between adjacent hollow spheres. Since the perforations are located between spheres or in connections, for different sphere spatial arrangements, the numbers of openings in a single hollow sphere are different: there are 6, 8 and 12 openings corresponding to 6, 8 and 12 connections for one single hollow sphere in the SC, BCC and FCC packing structures, respectively. Rm = 9.75 mm, d = 20.5 mm, dm = 5.5 mm, and t = 0.5 mm, and the relative density is 0.0654, 0.0833, and 0.0882, respectively. The relative density can be calculated by Equation (1) [For the traditional soldered or adhesive bonded metallic hollow-sphere structure a, the cotion (1) .(1)ρrel3; this material can be simplified as an ideal elastoplastic material with a Young’s modulus of 1700 MPa and a yield stress of 48 MPa [In this paper, HP MJF 3D printing technology is used to produce the experimental samples, and HP nylon P12 powder is used as the base material. Under the standard printing process, the MJF 3D printing accuracy is 0.1 mm, and the density of the printed solid material is 1.01 g/cmf 48 MPa ,28.To ensure the reliability of the experimental results, three samples of each packing pattern were prepared and tested in this study. After printing, all the hollow sphere structures stood for 10 days in a dry environment at room temperature (25 °C) and were then subjected to a quasi-static compression experiment. The samples were placed between the upper and lower plates of a hydraulic universal testing machine  with an accuracy of 0.5% in the experiment, and the downward loading rate of the upper plate was set to 10 mm/min.Numerical simulations were performed for the structures under the corresponding experimental conditions using ABAQUS 6.14 finite element software. The geometric models of PHSSs in different arrangements were simulated with automatically generated four-node shell element (S4R) meshes, which significantly reduced the modelling time. An average length of the elements 0.8 mm, approximately 4% of the sphere diameter, was used following a mesh convergence analysis. For the SC, BCC, and FCC PHSSs with 3 × 3 × 3 cells, the FEMs contained 53,352, 96,336, and 189,216 elements, respectively. The FEM of the BCC PHSS is shown in σ was defined as the plate pressure divided by the projected area of the structure on the plane, and the nominal strain ε was defined as the ratio of the loading displacement to the initial height of the structure. It is necessary to note that all the “stress” and “strain” mentioned in following sections indicate the nominal stress and nominal strain respectively, if not indicated otherwise.The ABAQUS/Explicit method was then employed to simulate the mechanical behaviour of different structures under quasi-static compression, and a comparison was made between the numerical simulations and the experimental results. For ease of analysis, the nominal stress t and hole diameter md on the compressive mechanical properties and energy absorption capacity of the structures. The relationships between the geometric parameters and the relative density are shown in mR was fixed to 9.75 mm, and the distance between adjacent hollow spheres was fixed to 20.5 mm. As t varied (t = 0.195~0.975 mm), md was fixed to 5.5 mm. As md varied (md = 3.5~7.5 mm), t was fixed to 0.5 mm. As shown in t value has a significant effect on the relative density, while the md value has a small effect on the density. In addition to the relative density, the thickness-to-radius ratio (mt/R) is also an important parameter of hollow spheres [md = 5.5 mm, there is an approximately linear relationship between mt/R and the relative density by using the linear fitting method: A parameter study was conducted to assess the effect of wall thickness  spheres . AccordiIn this study, compression tests were conducted for three groups of structures with different arrangements, and the results of each group reveal favourable consistency. A comparison between the experimental results and the numerical simulation results shows certain differences between the two, mainly because some errors and defects inevitably occurred during 3D printing and post-processing, thereby resulting in changes in the mechanical properties of the structure. The presence of errors and defects is also a common problem to be solved in the engineering application of 3D printing technologies. Overall, the deformation patterns of the PHSSs in different compression stages and the corresponding stress–strain curves were substantially consistent; therefore, the FEM established in this study is reliable.YP is defined as the first local maximum on the nominal stress–strain curve and serves as one of the indicators for predicting the large deformation behaviour; the mean stress in the plateau stage  in the nominal stress (σ)–strain dE(ε)In the calculation of SEA, because the densification strain varies with geometric parameters, the strain Based on finite element simulations, the effect of geometric parameters, such as wall thickness, hole diameter, and arrangement pattern, on the mechanical response, deformation mode, and energy absorption properties of PHSSs under quasi-static compression was investigated. Because of the localization of large deformations in hollow sphere structures, the representative volume element (RVE) under periodical or symmetric boundary conditions ,11,12,15t, the t value was set as 0.195 mm, 0.39 mm, 0.585 mm, 0.78 mm, and 0.975 mm; the md value was fixed to 5.5 mm; other parameters were unchanged; and the corresponding t/Rm was 0.02, 0.04, 0.06, 0.08, and 0.1. YP, plateau stress σpl, and SEA with different wall thicknesses for the SC, BCC, and FCC PHSSs, respectively. The results indicate that the YP, σpl and SEA values of the different PHSSs increased as the wall thickness increased, and the differences were as follows: for the SC PHSS, YP was always less than σpl; for the BCC PHSS, YP was essentially equal to σpl; and for the FCC PHSS, YP was always greater than σpl. When investigating the influence of wall thickness t/Rm values were 0.02 and 0.04), the sphere wall near each opening in the PHSS was prone to stress concentration; as the displacement increased, for two adjacent hollow spheres, one could undergo unstable buckling deformation, and the other could collapse into it; and for the different PHSSs, such a local deformation mechanism could cause different changes in the lateral dimensions of the structure. For the SC PHSS, the hollow spheres collapsed into each other in the same direction as the loading, and the hollow spheres squeezed each other vertically, which could increase the lateral size. When the wall was very thin, the lateral dimension did not change significantly; as the t value increased (t/Rm reached 0.06), the deformation mechanism of the mutual depression of the SC PHSS became stable, and the lateral dimension after compression gradually increased as t increased , layer-by-layer deformation developed first to reach a preliminary compaction state , and then the hollow spheres entered another stage of large deformation featuring layer-by-layer compaction  under uniaxial compression. As the t value increased , such deformation mode for the SC and BCC PHSSs tended to be stable, while the large deformation of the FCC PHSS was characterised by layer-by-layer continuous collapse and compaction , YP began to decrease, and the curve reflecting the variation in YP of the FCC PHSS with the change in md was parabolic, which is probably because the FCC PHSS had the most openings; therefore, the larger md was, the greater the loss of mass was, and when md reached approximately 6 mm, the strength began to decrease due to an excessively high mass loss. Similar conclusions have been drawn for the mechanical properties in the elastic stage and the initial yield stage of PHSSs [YP value of PHSSs in different arrangements, but it had a relatively small effect on the σpl and SEA values in the large deformation stage of various PHSSs (due to the average effect in the stress–strain curves). The SEA results show that for the SC PHSS, the SEA value decreased slightly as md increased, but the energy absorption did not fluctuate significantly. For the BCC and FCC PHSSs, when md = 7.5 mm, the SEA value was significantly higher than the values of structures with smaller hole diameters . This may be because the BCC and FCC PHSSs had more openings than the SC PHSS; when the md value was very large, the decrease in the density was greater, and according to Equation (3), the SEA value of the BCC and FCC PHSSs could be higher for large md values. When investigating the effect of hole diameter of PHSSs . In addimd = 3.5 mm) and largest (md = 7.5 mm) hole diameters. The influence mechanism of hole diameter md was similar to that of wall thickness t, i.e., the sphere wall near the opening in the different PHSSs was prone to stress concentration and local buckling/depression when md was very small; the minor changes in the lateral dimensions of the SC PHSS could almost be ignored (zero expansion)  and energy absorption characteristics (SEA) of PHSS are compared with those of the traditional MHSS made of mild steel, as shown in ρ of the porous structure or material is the product of the relative density and solid material density. This comparison focuses on the results from FCC structures with different wall thicknesses according to Reference [3, and the traditional MHSS has a higher density ranging from 173 to 1600 kg/m3, while the specific strength and SEA capacities of the 3D-printed PHSS are higher than those of the traditional MHSS. The reason can be attributed to two aspects: using lightweight solid material with good mechanical properties and perforating appropriately in hollow sphere structures for further dead weight reduction.The specific strength t or md value was extremely small; and the layer-by-layer collapse-compaction behaviour was dominant for the large deformation of the FCC PHSS.With different geometric parameters, the SC and BCC PHSSs underwent a preliminary compaction sub-stage after layer-by-layer buckling in the stage of large deformation; the FCC PHSS did not exhibit a similar deformation behaviour unless the (2)Among the PHSSs with the same relative density, the FCC PHSS exhibited the best compressive mechanical properties and energy absorption capacity; the SC PHSS exhibited the lowest; and the BCC PHSS was in between.(3)md had a significant effect on the first peak stress of different PHSSs but a relatively small effect on the plateau stress and SEA.The first peak stress, plateau stress and SEA of the PHSSs increased as the wall thickness t increased, and the hole diameter (4)When the wall thickness was very thin or the hole diameter was very small, the local depression of hollow spheres caused the BCC and FCC PHSSs to exhibit obvious transverse contraction under axial compression, and this behaviour weakened as the wall thickness or hole diameter increased.(5)The presented 3D-printed PHSS was much lighter than the traditional MHSS made of mild steel and had higher specific strength and SEA than the traditional MHSS.For this study, PHSSs in three different arrangements were fabricated using MJF 3D printing technology, and the mechanical behaviours of these PHSSs under quasi-static compression were studied through experiments and finite element simulations. The experimental results verify the effectiveness of the FEM. The results show that the deformation process of the 3D-printed PHSSs was similar to that of traditional hollow sphere structures and consisted of three stages, i.e., an elastic deformation stage, a large deformation stage, and a densification stage. Special attention was given to the large deformation stage, which is directly related to the energy absorbing capacity. Based on the numerical simulation, the effects of the geometric parameters of the PHSSs, such as the sphere arrangement, wall thickness, and hole diameter, on the compressive mechanical behaviours and energy absorption characteristics were investigated. The following conclusions can be drawn:


Kink deformation is often observed in materials with laminated layers. Graphite composed of stacked graphene layers has the unique laminated structure of carbon nanomaterials. In this study, we performed the interlayer deformation of graphite under compression using a simulation of molecular dynamics and proposed a differential geometrical method to evaluate the kink deformation. We employed “mean curvature” for the representativeness of the geometrical properties to explore the mechanism of kink deformation and the mechanical behaviors of graphite in nanoscale. The effect of the number of graphene layers and the lattice chirality of each graphene layer on kink deformation and stress–strain diagrams of compressed graphite are discussed in detail. The results showed that kink deformation occurred in compressed graphite when the strain was approximately equal to 0.02, and the potential energy of the compressed graphite proportionately increased with the increasing compressive strain. The proposed differential geometric method can not only be applied to kink deformation in nanoscale graphite, but could also be extended to solving and predicting interlayer deformation that occurs in micro- and macro-scale material structures with laminated layers. Graphite that is composed of stacked graphene layers has the unique laminated structure of carbon nanomaterials. Although graphene that consists of a hexagonal pattern of carbon atoms has extremely excellent mechanical behavior, natural graphite with laminated graphene is soft and easy to deform under high pressure due to the weak van der Waals (vdW) interaction force between the adjacent graphene layers . When dec axis. However, the deformation of graphite has not been described by exact quantitative figures, and there is no explanation of the relationship between the shape of it and force. Especially, the deformation mechanism of laminated structures was still not clarified clearly enough up until now.According to the unique deformation phenomena in laminated structures, the concept of the kink band was proposed by , where tThe establishment of a general deformation theory based on a differential geometric method is expected to elucidate the deformation mechanism in laminated structures, e.g., graphite in the present work. To investigate kink deformation in micro-buckled graphite with laminated graphene layers, in the following parts, we build two kinds of atomic simulation models: armchair graphite and zigzag graphite. Both kinds of graphite incorporate 20, 30, 40, 50, or 60 atomic layers, and we performed the compression test on each type of graphite using MD simulation at first. Then, we proposed a differential geometric method to evaluate the mean curvature of each graphene layer in each type of graphite. Next, we discussed the geometrical properties of unique deformation and studied the relationship between the distributions of mean curvature and potential energy. Lastly, we summarized the remarkable conclusions.x direction . The interaction was evaluated using the adaptive intermolecular reactive empirical bond-order (AIREBO) potential , where ton force ; the thiS. We introduced mean curvature, defined by atomic configuration, so that we could characterize out-of-plane deformation with quantification. It should be sufficient to approximate the normal vector m using the normal vector of the plane I–J–K. Here we assumed local Cartesian coordinates O–w was parallel to the normal vector m.To evaluate the out-of-plane deformation of graphene from plane to curved surface, we proposed a differential geometry method to calculate the mean curvature tensor using the atomistic configuration. During the compression process, each graphene layer, modeled as a 2D plane, initially deformed to a 3D curved surface due to the out-of-plane deformation. Thus, this 2D–3D transition generated folds and kink deformations in the graphite. However, in general, the mean curvature of the differential was used to describe the shape of a folding non-individual body. The structure of graphene is made up of discrete carbon atoms, in which each atom is connected with three adjacent atoms via C–C covalent bonds. As shown in S could be expressed as:Thus, the curved surface S, we have the following conditions.R with rotation angle w direction was introduced.R, if only the rotation angle in the principle direction was rotated, the curvature tensor Since the atom O ditions.κ11uI2+2κEquation  helped uEquation .(6)κP=κH is the average value of the principal curvature components The mean curvature The deformation behavior of graphite under compression depends on the external force and its internal structure. In this section we discuss the effect of compressive strain, the number of graphite layers, and the lattice chirality of the graphite within the layered structure mentioned in z direction to become obvious; the mountain and valley shapes could be observed clearly when the compressive strain From the results obtained using armchair graphite with 40 graphene layers under compression, as shown in To deeply discuss the effect the number of graphene layers has on micro-buckled graphite, we listed and compared the deformation shapes of armchair graphite with different number of graphene layers when the compressive strain was n = 20, as shown in n = 50, as shown in n = 60 as shown in According to the derived Equation , we calcDuring the compressive process, each graphene layer was generated by out-of-plane deformation. We picked the bottom layer (To discuss the relationship between the mean curvature and site potential energy in detail, we plotted In this work, in order to investigate the interlayer deformation of micro-buckled graphite and to establish a general methodology for evaluating the kink deformation of compressed laminated structures, we performed compression tests on armchair and zigzag graphite with different numbers of laminated graphene layers using MD simulation. We proposed a differential geometric method using mean curvature to explore the relationship between geometrical and mechanical properties. From the compression tests, not only the kink deformation but also the delamination were observed, and different kinds of deformation patterns appeared in the graphite according to the different chiralities of the graphite and different number of graphene layers. We concluded that the kink deformation in graphite started from the position of delamination and the growth of kink deformation weakened the growth of delamination. We also showed the relationships between compressive stress and compressive strain, and between site potential energy and compressive strain to investigate the structural effect on the material properties of graphite. Moreover, according to the calculated mean curvature, we found that the concentration of compressive stress and site potential energy were generated at almost the same locations in all of the analytical models. Lastly, we should note that the calculation of site potential energy and mean curvature using the proposed differential geometric method could be extended and used for solving and predicting the interlayer deformation that occurs in micro and macro materials with laminated layers.


Saccharomyces cerevisiae ORC-related Rif2 protein protects telomere. We show that the small N-terminal Rif2 BAT motif (Blocks Addition of Telomeres) previously known to limit telomere elongation and Tel1 activity is also sufficient to block NHEJ and 5’ end resection. The BAT motif inhibits the ability of the Mre11-Rad50-Xrs2 complex (MRX) to capture DNA ends. It acts through a direct contact with Rad50 ATP-binding Head domains. Through genetic approaches guided by structural predictions, we identify residues at the surface of Rad50 that are essential for the interaction with Rif2 and its inhibition. Finally, a docking model predicts how BAT binding could specifically destabilise the DNA-bound state of the MRX complex. From these results, we propose that when an MRX complex approaches a telomere, the Rif2 BAT motif binds MRX Head in its ATP-bound resting state. This antagonises MRX transition to its DNA-bound state, and favours a rapid return to the ATP-bound state. Unable to stably capture the telomere end, the MRX complex cannot proceed with the subsequent steps of NHEJ, Tel1-activation and 5’ resection.Specific proteins present at telomeres ensure chromosome end stability, in large part through unknown mechanisms. In this work, we address how the  S. cerevisiae the interaction between Rif2 and Rad50 inhibits MRX functions at telomeres.Different proteins localised at telomeres ensure chromosome end stability to prevent double strand-end break recognition. Here the authors provide new insight into how in  The repressed pathways are non-homologous end joining (NHEJ), 5′ end resection, homologous recombination and the DNA damage checkpoint. In addition, telomeres in association with telomerase solve the problem of replicating chromosome ends by semiconservative DNA replication. They also control telomere length homoeostasis to avoid the occurrence of excessively short or long telomeres. These core telomere functions are established by a relatively small number of proteins specifically present or enriched at telomeres. How each factor acts at a molecular level is only partially deciphered19.Telomeres are protein–DNA complexes ensuring that native chromosome ends escape the pathways acting on broken DNA endsORC4 gene22  in N-terminal position (residues 37–48)22. Both epitopes interact with Rap1 with similar affinities (id ~30–50 µM) and synergise to ensure an efficient Rif2 recruitment to telomeres. They may also allow Rif2 molecules to interconnect adjacent telomere-bound Rap1 molecules22. Rif2 contributes to several telomere functions. Rif2 limits telomere elongation by telomerase30. It also represses NHEJ, preventing telomere-telomere fusions31 and inhibits 5′ end resection, homologous recombination and checkpoint activation37.Telomere proteins are usually not restricted to a unique telomeric function and can control several pathways. In this regard, the budding yeast telomere factor Rif2 is paradigmatic. Rif2 is a globular 46 kDa protein with a single folded AAA+ domain originating from a duplication of the e22 Fig. . It is rNBS1 complex (MRXMRN), an ATPase related to the SMC family . At double-strand breaks, the MRXMRN complex is recruited early to the broken ends where it has multiple roles. First, it promotes NHEJ repair through tethering of the broken ends and through interactions with the NHEJ factors KU, Lif1XRCC4 and Nej1XLF44. In contexts where NHEJ does not occur, MRXMRN promotes 5′ end resection, Mec1ATR/Rad3 checkpoint kinase activation and repair by homologous recombination through its nuclease activities46 and through interactions with chromatin remodellers and regulation of long-range resection actors49. At telomeres, MRXMRN is also essential to Tel1ATM kinase recruitment and activation, a key factor for chromosome ends maintenance by telomerase54.A shared feature of these pathways targeted by Rif2 is the involvement of the Mre11–Rad50–Xrs255.The intersection between Rif2 and MRX functions suggests that the MRX complex is a Rif2 target at telomeres. Supporting this model, Rif2 inhibits the recruitment of the MRX complex to DNA ends either directly or by inhibiting Tel1, which stabilises the complex interaction with DNABlocks Addition of Telomeres) present in the N-terminal position (residues 1–36) just upstream of the RBM motif28 21. To test BAT ability to block NHEJ, we targeted Rif2 N-terminal region (1–60) to telomeres by fusing it to endogenous Rap1 C-terminal end. A 10-Glycine linker connects the two sequences . As expected28, fusing the BAT to Rap1 shortens telomeres  allows us to target them at a double-strand end to test their impact on NHEJ efficiency. Targeting full-length Rif2 through Rap1 C-terminal domain (Rap1Cter) inhibits NHEJ in these assays21. Targeting Rif2 N-terminal region (1-60) or a smaller Rif2 peptide only including the BAT motif, not the RBM (1-36) also lowers NHEJ-dependent survival relative to control situations where unfused Gal4DBD is bound to the Gal4 sites and where Gal4DBD-Rif21-36/60 proteins remain untargeted to broken end   induced by the continuously expressed I-SceI endonuclease Fig.  (see figte) Fig. . NHEJ inte) Fig.  or Rap1 te) Fig. . This inIn the previous experiment, the edge of the Gal4 sites is 23 bp away from the broken end. To assess how far Rif2 BAT can act, we inserted sequences between the Gal4 sites and the I-SceI site. Strikingly, BAT ability to inhibit NHEJ rapidly weakens with increasing distances from the broken end Fig. . DistancNext, we determined if smaller N-terminal Rif2 fragments retain the ability to inhibit NHEJ. C-terminal truncations down to position 26 have no significant effect    Fig.  and targ60) Fig. . This mu60) Fig. . Then we8A) Fig.  and at t8A) Fig. . As prevS. cerevisiae and some other post-WGD (whole genome duplication) Saccharomycetaceae species, Rif2 and Orc4 are orthologs and only Rif2 possesses the BAT motif30 . These results show that Rif2 BAT limits 5′ end resection both in G1 and G2/M phases.Since Rif2 antagonizes 5′ DNA end resection55, we next tested BAT ability to oppose this interaction using a ChIP approach. As shown in Fig. DBD alone). Since MRX functionality requires its three subunits, this result shows that the BAT motif antagonises MRX stable interaction with DNA ends. This property can explain its ability to inhibit telomere elongation28, NHEJ  and Rap1-binding motif (RBM) are distinct and functionally separable.To assess the significance of this interaction between the BAT motif and Rad50, we tested its sensitivity to mutations that impact BAT ability to inhibit NHEJ. Mutations in the F8 and R12 residues, C-terminal truncation past residue 26 and a N-terminal truncation (6-31) weaken or abolish the interaction with Rad50 Fig. . These d62. Its N-terminal and C-terminal domains interact to form a globular ATPase Head. The rest of the protein assembles into a long coiled-coil with a median Zn-hook. Our attempt to arbitrarily truncate the protein into distinct domains failed in the Two-Hybrid assay. For this reason, we screened a library of random truncations within the Rad50 coiled-coil region and selected for clones still interacting with the BAT motif in Two-Hybrid. The shortest clone we obtained is a truncation of the coiled-coil region that connects the N-terminal and C-terminal Head domains   Fig. . Its intcc) Fig. . The intsay Fig. .Rif2 BAT mutants defective for NHEJ inhibition are also defective for interaction with Rad50. Therefore, we expect Rad50 mutants specifically defective for interaction with Rif2 to be insensitive to Rif2 BAT inhibition. Finding such mutants will inform us on where the BAT motif interacts on Rad50, information that may give insights into the mechanism of inhibition. To this end, we first performed a genetic screen to identify Rad50 mutants that would be prone to telomere fusions, that is to say defective for telomere protection by Rif2.CEN6) to select and rescue unstable dicentric chromosome fusions  and increases in cells lacking Rif2 and/or Sir4. To sensitise the assay, we choose to screen Rad50 mutants in cells lacking Sir4, where telomere protection relies more on Rif2   Fig. .Fig. 5Idm1 to m6, m11, m12) or mildly short telomeres  , indicating that NHEJ is still partially functional in these mutants. Targeting Rif2 BAT (1-36) to the break still represses NHEJ in the m8, m9 and m10 rad50 clones, indicating that these mutants are still sensitive to BAT inhibition. In the m7 and m13 clones, survival to I-SceI expression is not reduced by BAT targeting. This result shows that these two mutants lead to NHEJ resistance to the BAT in this assay.We expected two classes of mutants: (i) mutants whose increased fusion frequency is simply a consequence of a telomere shortening caused by a partial loss of function of MRX and (ii) mutants more specifically impacting Rif2 inhibition and better retaining MRX functions and the ability to elongate telomeres. Therefore, as a secondary screen, we determined the telomere length in the selected mutants. They display either very short telomeres (13) Fig. . We discrad50-m7 allele carries three mutations, K81E, E242V and Q284L. The last two are within the N-terminal coiled-coil region. K81 is on a ß-sheet at the surface of Rad50 Head domain . The m13 allele carries five mutations, three in the Head  and two in the coiled-coil region (I292M and I382V). T22 and I93 are near K81 and part of the same ß-sheet at the surface of the Head. I43 is within the domain below T22. The clustering of the selected mutations in the Head suggests that this Rad50 ß sheet surface is important for Rif2 BAT function.The 65 to perform the simulation search for the binding site, allowing full flexibility of the peptide and small fluctuations of the Rad50 backbone. To limit the combinatorial explosion due to the mainchain peptide flexibility, we used a short BAT sequence containing the most conserved amino acids . These results show a specific requirement of K6, K81 and I93 for the Rad50–BAT interaction.To validate this docking result, as well as to define more precisely the location of the BAT peptide, we used the Rad50eet Fig. . Mutatiorad50 mutants remain NHEJ proficient, with one exception Q115A . In other words, they may not be perfect separation-of-function alleles. Rif1 is another repressor of telomere elongation that acts independently of Rif271. As expected for mutations impacting Rif2 function, the K6A and K81E mutations still result in longer telomeres in cells lacking Rif1 . Together with the previous finding that rif2-F8A and rif2-R12A BAT mutants elongate telomeres28, these results show that telomere length homoeostasis relies in part on the Rad50–BAT interaction and its inhibitory function.Since Rif2 BAT shortens telomeresion Fig. . Telomer72. In this method, ambiguous distance restraints that represent the spatial proximity between specific residues of the two partners are used to guide the docking. Residues K6, K81 and I93 of Rad50 and F8, R12, R13 and S14 of the BAT4-14 peptide were selected as the interacting residues. The HADDOCK calculation led to four clusters. The ambiguous character of the restraints and the fully flexible treatment of the BAT4-14 peptide explain the residual conformational spreading of the BAT peptide in these clusters. They all share the occupancy of a common region of the ß-sheet binding surface close to residue K6, K81 and I93. We then examined the consequences of this binding on the MRX complex structure.Next, we addressed the mechanism by which BAT binding on Rad50 Head inhibits MRX complex functions. First, we took advantage of the experimental results to build more accurate BAT-Rad50 models using the information-driven flexible docking approach HADDOCKEscherichia coli Rad50–Mre11SbcC-SbcD complex shows that it can adopt at least two states61. In an ATP-bound resting state, the Rad50 coiled-coils appear flexible and open. Upon DNA binding and consecutive ATP hydrolysis, the two coiled coils zip up into a rod and clamp DNA on the Rad50 Head. In this DNA-bound active state, Mre11 moves to the side of Rad50 to bind the DNA end. We used these structures to establish models of S. cerevisiae Rad50–Mre11 complex in the ATP-bound and DNA-bound states  (69% similarity/52% identity). To build the ATP-bound (resting state) and DNA-bound ScRad50-Mre11 models, we superimposed the ScRad50 and ScMre11 models on their E. coli homologues in the cryo-EM structures61 (PDB:6SV6 and 6S85).How the MRX complex operates at a molecular level is not fully resolved yet but a recent cryo-EM study of tes Fig. . To this4-14 peptide on Rad50 obtained with HADDOCK, some BAT residues occupy the same region where helix 181–192 of Mre11 lies, as illustrated in Fig. In the predicted ATP-bound state, K6, K81 and I93, the three Rad50 residues essential to BAT interaction, are exposed and located away from Mre11. However, the DNA-bound state brings helix 181–192 of Mre11 near the ß-sheet surface including residues K6, K81 and I93. Furthermore, in the docking models of the BAT43,61,75,76. MRX does not extrude loops but captures broken ends60, 150 mM NaCl, 1 mM DTT, 30 mM glutathione. Proteins bound to the beads were resolved by 12% SDS-PAGE and detected by silver staining.His-SUMO-Rad50−7 M α-factor for 4 h. I-SceI expression was induced for 1.5 h by galactose addition (2%). Cells were crosslinked for 10 min with formaldehyde (1%) at 30 °C. Cell lysis and chromatin sonication were performed using a Bioruptor. Immunoprecipitation was performed using the 4A6 anti-myc antibody. Input and immunoprecipitated DNA concentrations were determined by qPCR.Cells grown to OD 0.4 in synthetic glycerol lactate medium lacking uracil were arrested in G1 with 10S. cerevisiae Rad50 (Sc-Rad50) were modelled separately. Both domains were built by homology using Modeller 9.1773. We identified Rad50 from Chaetomium thermophilum (Ct-Rad50) as highly homologous to Sc-Rad50. The similarity between N-terminal domains of Sc-Rad50 (residue 2–189) and Ct-Rad50 is very high (62.8% identity/78% similarity). The sequence of the C-terminal domain of Sc-Rad50 (residue 1105–1312) is also highly similar to the one of Ct-Rad50 . This made it possible to use the structure 5DAC64 as a structural template for modelling both N- and C-terminal domains of S. cerevisiae Rad50. The best scoring models returned by Modeller were used for further investigations. Then, we build a model of Sc-Rad50 using the models obtained for its N- and C-terminal domains. The sequence used corresponds to the Rad50∆CC fragment lacking the coiled-coil region.The N-terminal and C-terminal domains of S. cerevisiae Mre11 (residue 1–415) involved in the interaction with Rad50. We used HMMER89 in the bioinformatics toolkit90 to identify a structural template in the protein database91. The X-ray structure 4YKE corresponding to the structure of Mre11 from Chaetomium thermophilum (Ct-Mre11) possesses a high sequence similarity with Mre11 from S. cerevisiae (Sc-Mre11) (52.2 % identity/69.7% similarity). Thus, 4YKE was selected as template to build the Sc-Mre11 model. 100 models were generated with Modeller 9.17 and the best scoring models were retained.A similar approach was used to model the catalytic domain of S. cerevisiae Rad50-Mre11 dimer models, we used the recently solved E. coli Rad50–Mre11 dimer complexes61 in the resting state (structure 6S6V) and in the cutting state (structure 6S85). These EM structures allowed us to orient the two partners and to build the Rad50-Mre11 dimer in the resting state and the cutting one. As the sequence homology between Sc-Rad50 and Ec-Rad50 and between Ec-Mre11 and Sc-Mre11 is low (below 15%), the RMSD on the coordinates after superimposition of the whole structures is high (>2.5 Å). However, this permitted to obtain initial Sc-Rad50-Mre11 dimer models in the ATP-bound state and DNA-bound state. These initial complexes were refined by molecular dynamics in explicit water solvent. The starting structure was immersed in a cubic water box. The box was set using the Tcl plugin Solvate of VMD92 so that the water layer around the solute (protein or DNA) was at least 12 Å. Then the system was neutralised with Na+ or Cl- ions using the autoionize Tcl plugin of VMD. All MD calculations were carried out with the NAMD software93 in the charmm36 forcefield94. The first step of the MD calculation consisted in a 1 ns restrained molecular dynamics simulation in the NPT ensemble. To this end, in addition to the standard energy terms , we applied a harmonic potential  restraining the CA atoms of the protein to their initial position while optimising sidechain atoms and water position. Then, a production step of 15 ns MD in the NPT ensemble was run without positional restraints. Frames were extracted from the last 5 ns MD trajectory and analysed. During MD, pressure control was achieved by a Nose-Hoover Langevin piston. MD trajectories were analysed with VMD v 1.9.4. Modelling figures were produced with PyMol95.To build Further information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Reporting Summary