Single-stranded DNA Molecules Research Articles

Dynamic behavior of the single-strand DNA molecules from the hydrophilic to hydrophobic regions on graphene oxide surface driven by heating.

Heating affects the interfacial properties of two-dimensional nanomaterials, especially when they interact with biomolecules. Here, we theoretically studied the dynamic processes driving single-strand DNA (ssDNA) molecules from the hydrophilic to hydrophobic regions on the graphene oxide (GO) surface by heating, as reported by recent experiments. This was accomplished by using multi-sample molecular dynamics simulations in the NVT ensemble, with the temperature increasing from 300 K to 350 K. When the temperature increased, the lifetime of hydrogen bonds between water molecule and oxygen-containing groups on the GO surface decreased from 10.04 ps to 6.86 ps, and the end-to-end distance of 4-mer and 8-mer ssDNA molecules also decreased. This indicated that heating facilitated the breaking/formation of hydrogen bonds and enhanced the flexibility of ssDNA molecules. By heating, active hydrogen bonding first led to unbalanced interactions between the ssDNA molecule and GO surface, and the enhanced flexibility allowed the ssDNA molecule to release stress by moving on the GO surface and relaxing its structure. The ssDNA molecule constantly adjusted its structure by a competition between intra and inter π-π stacking structures. With dynamic cooperation of hydrogen bonding and π-π stacking, the ssDNA molecule moved from the hydrophilic to hydrophobic regions. Our results offer fundamental interfacial science insights into the effects of heating on the interactions between biomolecules and two-dimensional nanomaterials.

The primase subunits of DNA polymerase α, PRIM1 and PRIM2, are required for the replication of the geminivirus tomato yellow leaf curl virus in the host plant.

Geminiviruses are causal agents of devastating diseases in crops. Geminiviral genomes are single-stranded (ss) circular DNA molecules that replicate in the nucleus of the infected cell through double-stranded (ds) intermediates by co-opting the plant DNA replication machinery. However, the identity of the plant DNA polymerases enabling geminiviral replication has remained largely elusive. Recently, we showed that DNA polymerase α mediates the ss-to-ds conversion of tomato yellow leaf curl virus (TYLCV), and is therefore essential for the infection. Here, we provide data indicating that the primase subunits of DNA polymerase α, PRIM1 and PRIM2, are also required for TYLCV replication.

Potential Therapeutic Use of Aptamers against HAT1 in Lung Cancer.

Lung cancer is one of the leading causes of death worldwide and the most common of all cancer types. Histone acetyltransferase 1 (HAT1) has attracted increasing interest as a potential therapeutic target due to its involvement in multiple pathologies, including cancer. Aptamers are single-stranded RNA or DNA molecules whose three-dimensional structure allows them to bind to a target molecule with high specificity and affinity, thus making them exceptional candidates for use as diagnostic or therapeutic tools. In this work, aptamers against HAT1 were obtained, subsequently characterized, and optimized, showing high affinity and specificity for HAT1 and the ability to inhibit acetyltransferase activity in vitro. Of those tested, the apHAT610 aptamer reduced cell viability, induced apoptosis and cell cycle arrest, and inhibited colony formation in lung cancer cell lines. All these results indicate that the apHAT610 aptamer is a potential drug for the treatment of lung cancer.

Development of DNA aptamer selection approach based on membrane ultrafiltration of aptamer/target complex

Background. Aptamers are small single-stranded DNA or RNA molecules that have an affinity for a specific target molecule. The main method of aptamers construction is the technology of systematic evolution of ligands with exponential enrichment (SELEX). However, the exact approach depends on the nature of target molecules, and is selected and optimized by each researcher independently. The article describes the technique of production of aptamers to the tick-borne encephalitis virus (TBEV) using membrane ultrafiltration with a molecular weight cut-off of 100 kDa. As a result, the pool of aptamers with observable affinity for TBEV is successfully selected and enriched.The aim. To develop the technique suitable for selection of specific DNA aptamers to a live, crude TBEV suspension directly in cell culture supernatant.Materials and methods. The selection of aptamers was carried out using a modified SELEX DNA aptamer technology in combination with semipermeable membrane ultrafiltration using Vivaspin 6 (Sartorius, Germany) concentrators of molecular weight cut-off of 100 kDa. Enrichment of a specific pool of aptamers was performed using real time polymerase chain reaction. Aptamers were sequenced with automated Sanger sequencing method. The direct virucidal effect of the aptamers was determined by the decrease in the titer of the infectious virus after incubation with the aptamer.Results. The pool of aptamers to TBEV was selected and enriched. This aptamer pool expressed affinity both to the infectious TBEV and to the TBEV antigen. Sixteen aptamers were sequenced from this pool and four of them were synthesized and tested for antiviral activity against TBEV. No antiviral activity was observed.Conclusions. The technique developed that can be successfully used to select aptamers to a live virus culture for the viruses comparable in size to TBEV or larger.

DNA-Directed Protein Anchoring on Oligo/Alkanethiol-Coated Gold Nanoparticles: A Versatile Platform for Biosensing Applications

Herein, we report on a smart biosensing platform that exploits gold nanoparticles (AuNPs) functionalized through ssDNA self-assembled monolayers (SAM) and the DNA-directed immobilization (DDI) of DNA-protein conjugates; a novel, high-sensitivity optical characterization technique based on a miniaturized gel electrophoresis chip integrated with online thermal lens spectrometry (MGEC-TLS), for the high-sensitivity detection of antigen binding events. Specifically, we characterized the physicochemical properties of 20 nm AuNPs covered with mixed SAMs of thiolated single-stranded DNA and bio-repellent molecules, referred to as top-terminated oligo-ethylene glycol (TOEG6), demonstrating high colloidal stability, optimal binder surface density, and proper hybridization capacity. Further, to explore the design in the frame of cancer-associated antigen detection, complementary ssDNA fragments conjugated with a nanobody, called C8, were loaded on the particles and employed to detect the presence of the HER2-ECD antigen in liquid. At variance with conventional surface plasmon resonance detection, MGEC-TLS characterization confirmed the capability of the assay to titrate the HER2-ECD antigen down to concentrations of 440 ng/mL. The high versatility of the directed protein-DNA conjugates immobilization through DNA hybridization on plasmonic scaffolds and coupled with the high sensitivity of the MGEC-TLS detection qualifies the proposed assay as a potential, easily operated biosensing strategy for the fast and label-free detection of disease-relevant antigens.

Reversible Assembly of an Artificial Protein Nanocage Using Alkaline Earth Metal Ions

Protein nanocages are of increasing interest for use as drug capsules, but the encapsulation and release of drug molecules at appropriate times require the reversible association and dissociation of the nanocages. One promising approach to addressing this challenge is the design of metal-dependent associating proteins. Such designed proteins typically have Cys or His residues at the protein surface for connecting the associating proteins through metal-ion coordination. However, Cys and His residues favor interactions with soft and borderline metal ions, such as Au+ and Zn2+, classified by the hard and soft acids and bases concept, restricting the types of metal ions available to drive association. Here, we show the alkaline earth (AE) metal-dependent association of the recently designed artificial protein nanocage TIP60, which is composed of 60-mer fusion proteins. The introduction of a Glu (hard base) mutation to the fusion protein (K67E mutant) prevented the formation of the 60-mer but formed the expected cage structure in the presence of Ca, Sr, or Ba ions (hard acids). Cryogenic electron microscopy (cryo-EM) analysis indicated a Ba ion at the interface of the subunits. Furthermore, we demonstrated the encapsulation and release of single-stranded DNA molecules using this system. Our results provide insights into the design of AE metal-dependent association and dissociation mechanisms for proteins.

Development and classification of RNA aptamers for therapeutic purposes: an updated review with emphasis on cancer.

Today, RNA aptamers are being considered promising theranostic tools against a wide variety of disorders. RNA aptamers can fold into complex shapes and bind to diverse nanostructures, macromolecules, cells, and viruses. It is possible to isolate RNA aptamers from a vast pool of nucleic acids via the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) method. As therapeutics, aptamers have great potential because of their ability to bind to proteins and selectively limit their activities with negligible side effects. Several RNA aptamers with potential implications in cancer diagnosis are known to confer a great affinity for single-stranded DNA molecules, long non-coding RNAs, circulating tumor cells, vascular endothelial growth factors, and tissue and sera-derived exosomes in patients with different malignancies. Furthermore, clinical investigations have revealed the efficacy of RNA aptamer-based agents in imaging modalities. This review seeks to deliver new insights into the development, classification, nanomerization, and modification of RNA aptamers, as well as their applications in cancer theranostics. The aptamers' mechanism of action and their interest to clinical trials as theranostic agents are also discussed.

Three-Component Covalent Organic Framework Nanosheets for the Detection of MicroRNAs

The development of new techniques for the detection of microRNAs (miRNAs) is highly desirable. Herein, a new crystalline three-component covalent organic framework (COF) termed EB-TAPB-TFP COF was synthesized under solvothermal conditions utilizing 1,3,5-triformylphloroglucinol, 1,3,5-tris(4-aminophenyl)benzene and ethidium bromide as monomers. Interestingly, EB-TAPB-TFP COF can be self-exfoliated into two-dimensional nanosheets (NSs) in an aqueous medium. The obtained EB-TAPB-TFP NSs exhibited a remarkable fluorescence intensity enhancement in the presence of a DNA-miRNA heteroduplex when compared to the presence of single-stranded DNA and other phosphate-based small molecules, making it promising in the detection of miRNA without tagging any fluorescent marker. Moreover, the EB-TAPB-TFP NSs can also be used as sensing material for the detection of a DNA-miRNA heteroduplex using the quartz crystal microbalance technique, which is in good agreement with the fluorescence sensing result. The exploration of COF-based sensors in this work demonstrates a new pathway for the selective detection of miRNAs.

Identification and Preliminary Characterization of a Novel Single-Stranded DNA Binding Protein of Staphylococcus aureus Phage Phi11 Expressed in Escherichia coli.

Bacteriophage Phi11 harbors a gene, gp13, encoding the putative SSB protein (GenBank accession no. NC_004615.1). SSB proteins bind to and protect the single-stranded DNA molecules from nuclease digestion and are essential for the growth and metabolic activities of the organisms encoding them. In this investigation, we have carried out the cloning, recombinant expression, and purification of rGp13 for the first time in Escherichia coli. EMSA data indicated that the purified recombinant Gp13 protein was capable of binding to single-stranded DNA. The protein exhibited maximum binding activity at 32°C. Furthermore, our bioinformatic analysis has revealed that Gp13 consists of an OB-fold, a characteristic of SSB proteins. However, the arrangement of the OB-fold is unique, being located in the C-terminal domain of Gp13. Despite the importance of SSB proteins in various metabolic processes as well as in various types of PCR, there are no reports on the purification and characterization of SSB proteins from staphylococcal bacteriophages. We expect that the purification and characterization of recombinant Gp13 will help us gain a better insight into its biological activity and make it available in large quantities for molecular biology work.

Genome-wide identification, phylogenetic, and expression analysis under abiotic stress conditions of Whirly (WHY) gene family in Medicago sativa L.

The WHY family is a group of plant-specific transcription factors, that can bind to single-stranded DNA molecules and play a variety of functions in plant nuclei and organelles, participating in the regulation of plant leaf senescence. It has been identified and analyzed in many species, however, the systematic identification and analysis of the WHY genes family have not yet been reported in alfalfa (Medicago sativa L.). Therefore, to explore the function of alfalfa the WHY genes, and 10 MsWHY genes were identified and further characterized their evolutionary relationship and expression patterns by analyzing the recently published genome of alfalfa. Comprehensive analysis of the chromosome location, physicochemical properties of the protein, evolutionary relationship, conserved motifs, and responses to abiotic stresses of the WHY gene family in alfalfa using bioinformatics methods. The results showed that 10 MsWHY genes were distributed on 10 chromosomes, and collinearity analysis showed that many MsWHYs might be derived from segmental duplications, and these genes are under purifying selection. Based on phylogenetic analyses, the WHY gene family of alfalfa can be divided into four subfamilies: I-IV subfamily, and approximately all the WHY genes within the same subfamily share similar gene structures. The 10 MsWHY gene family members contained 10 motifs, of which motif 2 and motif 4 are the conserved motifs shared by these genes. Furthermore, the analysis of cis-regulatory elements indicated that regulatory elements related to transcription, cell cycle, development, hormone, and stress response are abundant in the promoter sequence of the MsWHY genes. Real-time quantitative PCR demonstrated that MsWHYs gene expression is induced by drought, salt, and methyl jasmonate. The present study serves as a basic foundation for future functional studies on the alfalfa WHY family.

Systematic bio-fabrication of aptamers and their applications in engineering biology.

Aptamers are single-stranded DNA or RNA molecules that have high affinity and selectivity to bind to specific targets. Compared to antibodies, aptamers are easy to in vitro synthesize with low cost, and exhibit excellent thermal stability and programmability. With these features, aptamers have been widely used in biology and medicine-related fields. In the meantime, a variety of systematic evolution of ligands by exponential enrichment (SELEX) technologies have been developed to screen aptamers for various targets. According to the characteristics of targets, customizing appropriate SELEX technology and post-SELEX optimization helps to obtain ideal aptamers with high affinity and specificity. In this review, we first summarize the latest research on the systematic bio-fabrication of aptamers, including various SELEX technologies, post-SELEX optimization, and aptamer modification technology. These procedures not only help to gain the aptamer sequences but also provide insights into the relationship between structure and function of the aptamers. The latter provides a new perspective for the systems bio-fabrication of aptamers. Furthermore, on this basis, we review the applications of aptamers, particularly in the fields of engineering biology, including industrial biotechnology, medical and health engineering, and environmental and food safety monitoring. And the encountered challenges and prospects are discussed, providing an outlook for the future development of aptamers.

Heavy Metal Ions Trigger a Fluorescent Quenching in DNA-Organic Semiconductor Hybrid Assemblies.

The significance of DNA is no longer limited to its role as a biological information carrier; as a natural polymer, it also become in the field of materials. Single-stranded DNA (ssDNA) molecules with specific sequences can form a G-quadruplex or hairpin-shaped conformation with specific heavy metal ions through coordination bonds. In this study, ssDNA molecules of the four sequences were prepared into hybrid assemblies with one of the famous display materials, the tris-(8-hydroxyquinoline)aluminum (Alq3) semiconductor. Based on these hybrid assemblies, heavy metal ions, namely Pb2+, Hg2+, Cd2+ and As3+, were detected individually at the ppb level. Apart from this, in practical application, many samples containing heavy metal ions are digested with acid. By introducing MES buffer solution, the influence of acidity on the fluorescent signal of Alq3 was excluded. This strategy showed promising results in the practical application of detecting heavy metal ions in shrub branches and leaves.

Patterns of Genetic Diversity among Alphasatellites Infecting Gossypium Species.

Alphasatellites are small single-stranded circular DNA molecules associated with geminiviruses and nanoviruses. In this study, a meta-analysis of known alphasatellites isolated from the genus Gossypium (cotton) over the last two decades was performed. The phylogenetic and pairwise sequence identity analysis suggested that cotton-infecting begomoviruses were associated with at least 12 different alphasatellites globally. Three out of twelve alphasatellite were associated with cotton leaf curl geminiviruses but were not isolated from cotton plants. The cotton leaf curl Multan alphasatellite, which was initially isolated from cotton, has now been reported in several plant species, including monocot plants such as sugarcane. Our recombination analysis suggested that four alphasatellites, namely cotton leaf curl Lucknow alphasatellites, cotton leaf curl Multan alphasatellites, Ageratum yellow vein Indian alphasatellites and Ageratum enation alphasatellites, evolved through recombination. Additionally, high genetic variability was detected among the cotton-infecting alphasatellites at the genome level. The nucleotide substitution rate for the replication protein of alphasatellites (alpha-Rep) was estimated to be relatively high (~1.56 × 10−3). However, unlike other begomoviruses and satellites, the first codon position of alpha-Rep rapidly changed compared to the second and third codon positions. This study highlights the biodiversity and recombination of alphasatellites associated with the leaf curl diseases of cotton crops.

An Overview of Chili Leaf Curl Disease: Molecular Mechanisms, Impact, Challenges, and Disease Management Strategies in Indian Subcontinent

Leaf curl disease in a chili plant is caused mainly by Chili leaf curl virus (ChiLCV) (Family: Geminiviridae, Genus: Begomovirus). ChiLCV shows a widespread occurrence in most of the chili (Capsicum spp.) growing regions. ChiLCV has a limited host range and infects tomatoes (Solanum lycopersicum), potatoes (S. tuberosum), and amaranth (Amaranthus tricolor). The virus genome is a monopartite circular single-stranded DNA molecule of 2.7 kb and associated with α and β-satellites of 1.3 and 1.4 kb, respectively. The virus genome is encapsulated in distinct twinned icosahedral particles of around 18–30 nm in size and transmitted by Bemisia tabaci (Family: Aleyrodidae, Order: Hemiptera). Recently, bipartite begomovirus has been found to be associated with leaf curl disease. The leaf curl disease has a widespread distribution in the major equatorial regions viz., Australia, Asia, Africa, Europe, and America. Besides the PCR, qPCR, and LAMP-based detection systems, recently, localized surface-plasmon-resonance (LPSR) based optical platform is used for ChiLCV detection in a 20–40 μl of sample volume using aluminum nanoparticles. Management of ChiLCV is more challenging due to the vector-borne nature of the virus, therefore integrated disease management strategies need to be followed to contain the spread and heavy crop loss. CRISPR/Cas-mediated virus resistance has gained importance in disease management of DNA and RNA viruses due to certain advantages over the conventional approaches. Therefore, CRISPR/Cas system-mediated resistance needs to be explored in chili against ChiLCV.

Distribution Behavior of Single-Stranded DNA Molecules in an Amino-Group-Functionalized Silica Microparticle.

In this study, we investigated the distribution behavior of single-stranded DNA molecules with 20 bases in silica particles (particle size: ∼30 μm) using confocal fluorescence microspectroscopy. The distribution kinetics was investigated under various conditions, such as the type of base (adenine, thymine, guanine, and cytosine), pore size of the particle (30 and 50 nm), and salt concentration (100, 200, and 500 mM), which changed the distribution behavior. At high salt concentrations, we observed sigmoidal kinetic behavior, which does not occur in the general distribution of small organic molecules but is often observed in protein aggregation and nuclear growth. An analytical model based on DNA aggregation explained the sigmoidal distribution behavior well, and this model also worked well when the number of DNA molecules involved in DNA aggregation was greater than two. The intraparticle diffusion of DNA molecules was analyzed using the pore and surface diffusion model. As a result, the intraparticle diffusion of DNA aggregates mainly occurs according to surface diffusion, and the surface diffusion coefficient has the same value ((2.4-6.7) × 10-9 cm2 s-1) independent of the pore size and type of base.

Diffusion mechanisms of DNA in agarose gels: NMR studies and Monte Carlo simulations.

We report on the diffusion mechanism of short, single-stranded DNA molecules with up to 100 nucleobases in agarose gels with concentrations of up to 2.0% with the aim to characterize the DNA-agarose interaction. The diffusion coefficients were measured directly, i.e., without any model assumptions, by pulsed field gradient nuclear magnetic resonance (PFG-NMR). We find that the diffusion coefficient decreases, as expected, with an increase in both DNA strand length and gel concentration. In addition, we performed Monte Carlo simulations of particle diffusion in a model network of polymer chains, considering our experimental conditions. Together, the Monte Carlo simulations and the PFG-NMR results show that the decrease in diffusion coefficients in the presence of the agarose gel is due to a temporary adhesion of the DNA molecules to the surface of gel fibers. The average adhesion time to a given gel fiber increases with the length of the DNA strands but is independent of the number of gel fibers. The corresponding magnitude of the binding enthalpies of DNA strands to gel fibers indicates that a mixture of van der Waals interactions and hydrogen bonding contributes to the decreased diffusion of DNA in agarose gels.

Methylene Blue functionalized carbon nanodots combined with different shape gold nanostructures for sensitive and selective SARS-CoV-2 sensing

The development of DNA-sensing platforms based on new synthetized Methylene Blue functionalized carbon nanodots combined with different shape gold nanostructures (AuNs), as a new pathway to develop a selective and sensitive methodology for SARS-CoV-2 detection is presented. A mixture of gold nanoparticles and gold nanotriangles have been synthetized to modify disposable electrodes that act as an enhanced nanostructured electrochemical surface for DNA probe immobilization. On the other hand, modified carbon nanodots prepared a la carte to contain Methylene Blue (MB-CDs) are used as electrochemical indicators of the hybridization event. These MB-CDs, due to their structure, are able to interact differently with double and single-stranded DNA molecules. Based on this strategy, target sequences of the SARS-CoV-2 virus have been detected in a straightforward way and rapidly with a detection limit of 2.00 aM. Moreover, this platform allows the detection of the SARS-CoV-2 sequence in the presence of other viruses, and also a single nucleotide polymorphism (SNPs). The developed approach has been tested directly on RNA obtained from nasopharyngeal samples from COVID-19 patients, avoiding any amplification process. The results agree well with those obtained by RT-qPCR or reverse transcription quantitative polymerase chain reaction technique.

Rh Blood Group D Antigen Genotyping Using a Portable Nanopore-based Sequencing Device: Proof of Principle.

Nanopore sequencing is direct sequencing of a single-stranded DNA molecule using biological pores. A portable nanopore-based sequencing device from Oxford Nanopore Technologies (MinION) depends on driving a DNA molecule through nanopores embedded in a membrane using a voltage. Changes in current are then measured by a sensor, thousands of times per second and translated to nucleobases. Genomic DNA (gDNA) samples (n = 13) were tested for Rh blood group D antigen (RHD) gene zygosity using droplet digital PCR. The RHD gene was amplified in 6 overlapping amplicons using long-range PCR. Amplicons were purified, and the sequencing library was prepared following the 1D Native barcoding gDNA protocol. Sequencing was carried out with 1D flow cells R9 version. Data analysis included basecalling, aligning to the RHD reference sequence, and calling variants. Variants detected were compared to the results acquired previously by the Ion Personal Genome Machine (Ion PGM). Up to 500× sequence coverage across the RHD gene allowed accurate variant calling. Exonic changes in the RHD gene allowed RHD allele determination for all samples sequenced except 1 RHD homozygous sample, where 2 heterozygous RHD variant alleles are suspected. There were 3 known variant RHD alleles (RHD*01W.02, RHD*11, and RHD*15) and 6 novel RHD variant alleles, as previously seen in Ion PGM sequencing data for these samples. MinION was effective in blood group genotyping, provided enough sequencing data to achieve high coverage of the RHD gene, and enabled confident calling of variants and RHD allele determination.

Magnetoresistive effect in a quantum heterostructure with helical spacer: interplay between helicity and external electric field

Giant magnetoresistive effect in a multi-layered structure not only depends on the properties of magnetic systems, it also strongly depends on the type of non-magnetic spacer that is clamped between magnetic layers. In this work, we critically investigate the role of a helical spacer in presence of a transverse electric field. Two kinds of helical geometries, possessing short-range (SRH) and long-range hopping (LRH) of electrons, are taken into account mimicking single-stranded DNA and protein molecules respectively. Sandwiching the magnetic–non-magnetic–magnetic quantum heterostructure between source and drain contact electrodes, we investigate the properties of giant magnetoresistance (GMR) following the Green’s function formalism within a tight-binding framework. The interplay between SRHs and LRHs of electrons provides several nontrivial signatures in GMR, especially in the presence of transverse electric field, as it makes the system a deterministic disordered one, similar to the well-known Aubry–Andre–Harper from. The famous gapped nature of energy band structure in presence of cosine modulation leads to high degree of magnetoresistance at multiple Fermi energies, compared to the traditional spacers. The magnetoresistive effect can be monitored selectively by adjusting the electric field strength and its direction. Comparing the results between the SRH and LRH cases, we find that the later one is more superior. Finally, to make the system more realistic we include the effect of dephasing. Our analysis may provide some fundamental aspects of designing electronic and spintronic devices based on magnetoresistive effect.

Distinct mechanisms for genomic attachment of the 5' and 3' ends of Agrobacterium T-DNA in plants.

Agrobacterium tumefaciens, a pathogenic bacterium capable of transforming plants through horizontal gene transfer, is nowadays the preferred vector for plant genetic engineering. The vehicle for transfer is the T-strand, a single-stranded DNA molecule bound by the bacterial protein VirD2, which guides the T-DNA into the plant's nucleus where it integrates. How VirD2 is removed from T-DNA, and which mechanism acts to attach the liberated end to the plant genome is currently unknown. Here, using newly developed technology that yields hundreds of T-DNA integrations in somatic tissue of Arabidopsis thaliana, we uncover two redundant mechanisms for the genomic capture of the T-DNA 5' end. Different from capture of the 3' end of the T-DNA, which is the exclusive action of polymerase theta-mediated end joining (TMEJ), 5' attachment is accomplished either by TMEJ or by canonical non-homologous end joining (cNHEJ). We further find that TMEJ needs MRE11, whereas cNHEJ requires TDP2 to remove the 5' end-blocking protein VirD2. As a consequence, T-DNA integration is severely impaired in plants deficient for both MRE11 and TDP2 (or other cNHEJ factors). In support of MRE11 and cNHEJ specifically acting on the 5' end, we demonstrate rescue of the integration defect of double-deficient plants by using T-DNAs that are capable of forming telomeres upon 3' capture. Our study provides a mechanistic model for how Agrobacterium exploits the plant's own DNA repair machineries to transform it.