Stabilization and structural changes of 2D DNA origami by enzymatic ligation

The low thermal stability of DNA nanostructures is the major drawback in their practical applications. Most of the DNA nanotubes/tiles and the DNA origami structures melt below 60°C due to the presence of discontinuities in the phosphate backbone (i.e., nicks) of the staple strands. In molecular biology, enzymatic ligation is commonly used to seal the nicks in the duplex DNA. However, in DNA nanotechnology, the ligation procedures are neither optimized for the DNA origami nor routinely applied to link the nicks in it. Here, we report a detailed analysis and optimization of the conditions for the enzymatic ligation of the staple strands in four types of 2D square lattice DNA origami. Our results indicated that the ligation takes overnight, efficient at 37°C rather than the usual 16°C or room temperature, and typically requires much higher concentration of T4 DNA ligase. Under the optimized conditions, up to 10 staples ligation with a maximum ligation efficiency of 55% was achieved. Also, the ligation is found to increase the thermal stability of the origami as low as 5°C to as high as 20°C, depending on the structure. Further, our studies indicated that the ligation of the staple strands influences the globular structure/planarity of the DNA origami, and the origami is more compact when the staples are ligated. The globular structure of the native and ligated origami was also found to be altered dynamically and progressively upon ethidium bromide intercalation in a concentration-dependent manner.     

CRISPR‐Cas9‐mediated efficient directed mutagenesis and RAD51‐dependent and RAD51‐independent gene targeting in the moss Physcomitrella patens

The ability to address the CRISPR‐Cas9 nuclease complex to any target DNA using customizable single‐guide RNAs has now permitted genome engineering in many species. Here, we report its first successful use in a nonvascular plant, the moss Physcomitrella patens. Single‐guide RNAs (sgRNAs) were designed to target an endogenous reporter gene, PpAPT, whose inactivation confers resistance to 2‐fluoroadenine. Transformation of moss protoplasts with these sgRNAs and the Cas9 coding sequence from Streptococcus pyogenes triggered mutagenesis at the PpAPT target in about 2% of the regenerated plants. Mainly, deletions were observed, most of them resulting from alternative end‐joining (alt‐EJ)‐driven repair. We further demonstrate that, in the presence of a donor DNA sharing sequence homology with the PpAPT gene, most transgene integration events occur by homology‐driven repair (HDR) at the target locus but also that Cas9‐induced double‐strand breaks are repaired with almost equal frequencies by mutagenic illegitimate recombination. Finally, we establish that a significant fraction of HDR‐mediated gene targeting events (30%) is still possible in the absence of PpRAD51 protein, indicating that CRISPR‐induced HDR is only partially mediated by the classical homologous recombination pathway.     

Glucose metabolism down‐regulates the uptake of 6‐(N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino)‐2‐deoxyglucose (6‐NBDG) mediated by glucose transporter 1 isoform (GLUT1): theory and simulations using the symmetric four‐state carrier model

The non‐metabolizable fluorescent glucose analogue 6‐(N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)amino)‐2‐deoxyglucose (6‐NBDG) is increasingly used to study cellular transport of glucose. Intracellular accumulation of exogenously applied 6‐NBDG is assumed to reflect concurrent gradient‐driven glucose uptake by glucose transporters (GLUTs). Here, theoretical considerations are provided that put this assumption into question. In particular, depending on the microscopic parameters of the carrier proteins, theory proves that changes in glucose transport can be accompanied by opposite changes in flow of 6‐NBDG. Simulations were carried out applying the symmetric four‐state carrier model on the GLUT1 isoform, which is the only isoform whose kinetic parameters are presently available. Results show that cellular 6‐NBDG uptake decreases with increasing rate of glucose utilization under core‐model conditions, supported by literature, namely where the transporter is assumed to work in regime of slow reorientation of the free‐carrier compared with the ligand–carrier complex. To observe an increase of 6‐NBDG uptake with increasing rate of glucose utilization, and thus interpret 6‐NBDG increase as surrogate of glucose uptake, the transporter must be assumed to operate in regime of slow ligand–carrier binding, a condition that is currently not supported by literature. Our findings suggest that the interpretation of data obtained with NBDG derivatives is presently ambiguous and should be cautious because the underlying transport kinetics are not adequately established.     

Tunable 2D diffusion of DNA nanostructures on lipid membranes

DNA nanotechnology facilitates the synthesis of biomimetic models for studying biological systems. This work uses lipid bilayers as platforms for two-dimensional single-particle tracking of the dynamics of DNA nanostructures. Three different DNA origami structures adhere to the membrane through hybridization with cholesterol-modified strands. Their two-dimensional diffusion coefficient is modulated by changing the concentration of monovalent and divalent salts and the number of anchors. In addition, the diffusion coefficient is tuned by targeting cholesterol-modified anchor strands with strand-displacement reactions. We demonstrate a responsive system with changing diffusivity by selectively displacing membrane-bound anchor strands. We also show the programmed release of origami structures from the lipid membranes.     

cGAS is activated by DNA in a length‐dependent manner

Cytosolic DNA stimulates innate immune responses, including type I interferons (IFN), which have antiviral and immunomodulatory activities. Cyclic GMP‐AMP synthase (cGAS) recognizes cytoplasmic DNA and signals via STING to induce IFN production. Despite the importance of DNA in innate immunity, the nature of the DNA that stimulates IFN production is not well described. Using low DNA concentrations, we show that dsDNA induces IFN in a length‐dependent manner. This is observed over a wide length‐span of DNA, ranging from the minimal stimulatory length to several kilobases, and is fully dependent on cGAS irrespective of DNA length. Importantly, in vitro studies reveal that long DNA activates recombinant human cGAS more efficiently than short DNA, showing that length‐dependent DNA recognition is an intrinsic property of cGAS independent of accessory proteins. Collectively, this work identifies long DNA as the molecular entity stimulating the cGAS pathway upon cytosolic DNA challenge such as viral infections.     

Inhibition of mutant BRAF splice variant signaling by next‐generation,selective RAF inhibitors

Vemurafenib and dabrafenib block MEK‐ERK1/2 signaling and cause tumor regression in the majority of advanced‐stage BRAF^V600E melanoma patients; however, acquired resistance and paradoxical signaling have driven efforts for more potent and selective RAF inhibitors. Next‐generation RAF inhibitors, such as PLX7904 (PB04), effectively inhibit RAF signaling in BRAF^V600E melanoma cells without paradoxical effects in wild‐type cells. Furthermore, PLX7904 blocks the growth of vemurafenib‐resistant BRAF^V600E cells that express mutant NRAS. Acquired resistance to vemurafenib and dabrafenib is also frequently driven by expression of mutation BRAF splice variants; thus, we tested the effects of PLX7904 and its clinical analog, PLX8394 (PB03), in BRAF^V600E splice variant‐mediated vemurafenib‐resistant cells. We show that paradox‐breaker RAF inhibitors potently block MEK‐ERK1/2 signaling, G1/S cell cycle events, survival and growth of vemurafenib/PLX4720‐resistant cells harboring distinct BRAF^V600E splice variants. These data support the further investigation of paradox‐breaker RAF inhibitors as a second‐line treatment option for patients failing on vemurafenib or dabrafenib.     

Direct sequencing of haplotypes from diploid individuals through a modified emulsion PCR‐based single‐molecule sequencing approach

While standard DNA‐sequencing approaches readily yield genotypic sequence data, haplotype information is often of greater utility for population genetic analyses. However, obtaining individual haplotype sequences can be costly and time‐consuming and sometimes requires statistical reconstruction approaches that are subject to bias and error. Advancements have recently been made in determining individual chromosomal sequences in large‐scale genomic studies, yet few options exist for obtaining this information from large numbers of highly polymorphic individuals in a cost‐effective manner. As a solution, we developed a simple PCR‐based method for obtaining sequence information from individual DNA strands using standard laboratory equipment. The method employs a water‐in‐oil emulsion to separate the PCR mixture into thousands of individual microreactors. PCR within these small vesicles results in amplification from only a single starting DNA template molecule and thus a single haplotype. We improved upon previous approaches by including SYBR Green I and a melted agarose solution in the PCR, allowing easy identification and separation of individually amplified DNA molecules. We demonstrate the use of this method on a highly polymorphic estuarine population of the copepod Eurytemora affinis for which current molecular and computational methods for haplotype determination have been inadequate.     

Next‐generation DNA barcoding: using next‐generation sequencing to enhance and accelerate DNA barcode capture from single specimens

DNA barcoding is an efficient method to identify specimens and to detect undescribed/cryptic species. Sanger sequencing of individual specimens is the standard approach in generating large‐scale DNA barcode libraries and identifying unknowns. However, the Sanger sequencing technology is, in some respects, inferior to next‐generation sequencers, which are capable of producing millions of sequence reads simultaneously. Additionally, direct Sanger sequencing of DNA barcode amplicons, as practiced in most DNA barcoding procedures, is hampered by the need for relatively high‐target amplicon yield, coamplification of nuclear mitochondrial pseudogenes, confusion with sequences from intracellular endosymbiotic bacteria (e.g. Wolbachia) and instances of intraindividual variability (i.e. heteroplasmy). Any of these situations can lead to failed Sanger sequencing attempts or ambiguity of the generated DNA barcodes. Here, we demonstrate the potential application of next‐generation sequencing platforms for parallel acquisition of DNA barcode sequences from hundreds of specimens simultaneously. To facilitate retrieval of sequences obtained from individual specimens, we tag individual specimens during PCR amplification using unique 10‐mer oligonucleotides attached to DNA barcoding PCR primers. We employ 454 pyrosequencing to recover full‐length DNA barcodes of 190 specimens using 12.5% capacity of a 454 sequencing run (i.e. two lanes of a 16 lane run). We obtained an average of 143 sequence reads for each individual specimen. The sequences produced are full‐length DNA barcodes for all but one of the included specimens. In a subset of samples, we also detected Wolbachia, nontarget species, and heteroplasmic sequences. Next‐generation sequencing is of great value because of its protocol simplicity, greatly reduced cost per barcode read, faster throughout and added information content.     

Transcript profiling of jasmonate‐elicited Taxus cells reveals a β‐phenylalanine‐CoA ligase

Plant cell cultures constitute eco‐friendly biotechnological platforms for the production of plant secondary metabolites with pharmacological activities, as well as a suitable system for extending our knowledge of secondary metabolism. Despite the high added value of taxol and the importance of taxanes as anticancer compounds, several aspects of their biosynthesis remain unknown. In this work, a genomewide expression analysis of jasmonate‐elicited Taxus baccata cell cultures by complementary DNA‐amplified fragment length polymorphism (cDNA‐AFLP) indicated a correlation between an extensive elicitor‐induced genetic reprogramming and increased taxane production in the targeted cultures. Subsequent in silico analysis allowed us to identify 15 genes with a jasmonate‐induced differential expression as putative candidates for genes encoding enzymes involved in five unknown steps of taxane biosynthesis. Among them, the TB768 gene showed a strong homology, including a very similar predicted 3D structure, with other genes previously reported to encode acyl‐CoA ligases, thus suggesting a role in the formation of the taxol lateral chain. Functional analysis confirmed that the TB768 gene encodes an acyl‐CoA ligase that localizes to the cytoplasm and is able to convert β‐phenylalanine, as well as coumaric acid, into their respective derivative CoA esters. β‐phenylalanyl‐CoA is attached to baccatin III in one of the last steps of the taxol biosynthetic pathway. The identification of this gene will contribute to the establishment of sustainable taxol production systems through metabolic engineering or synthetic biology approaches.     

Geometrical principles of homomeric β‐barrels and β‐helices: Application to modeling amyloid protofilaments

Examples of homomeric β‐helices and β‐barrels have recently emerged. Here we generalize the theory for the shear number in β‐barrels to encompass β‐helices and homomeric structures. We introduce the concept of the “β‐strip,” the set of parallel or antiparallel neighboring strands, from which the whole helix can be generated giving it n‐fold rotational symmetry. In this context, the shear number is interpreted as the sum around the helix of the fixed register shift between neighboring identical β‐strips. Using this approach, we have derived relationships between helical width, pitch, angle between strand direction and helical axis, mass per length, register shift, and number of strands. The validity and unifying power of the method is demonstrated with known structures including α‐hemolysin, T4 phage spike, cylindrin, and the HET‐s(218‐289) prion. From reported dimensions measured by X‐ray fiber diffraction on amyloid fibrils, the relationships can be used to predict the register shift and the number of strands within amyloid protofilaments. This was used to construct models of transthyretin and Alzheimer β(40) amyloid protofilaments that comprise a single strip of in‐register β‐strands folded into a “β‐strip helix.” Results suggest both stabilization of an individual β‐strip helix and growth by addition of further β‐strip helices can involve the same pair of sequence segments associating with β‐sheet hydrogen bonding at the same register shift. This process would be aided by a repeat sequence. Hence, understanding how the register shift (as the distance between repeat sequences) relates to helical dimensions will be useful for nanotube design.     

Targeting human telomeric G‐quadruplex DNA with antitumour natural alkaloid aristololactam‐β‐D‐glucoside and its comparison with daunomycin

Study on anticancer agents that act via stabilization of telomeric G‐quadruplex DNA has emerged as novel and exciting field for anticancer drug discovery. The interaction of carbohydrate containing anticancer alkaloid aristololactam‐β‐D‐glucoside (ADG) with human telomeric G‐quadruplex DNA sequence was characterized by different biophysical techniques. The binding parameters were compared with daunomycin (DAN), a well‐known chemotherapeutic drug. The Scatchard binding isotherms revealed noncooperative binding for both with the binding affinity values of (1.01 ± 0.05) × 10⁶ and (1.78 ± 0.18) × 10⁶ M⁻¹ for ADG and DAN, respectively. Circular dichroism, ferrocyanide quenching study, anisotropy study, thiazole orange displacement, optical melting, differential scanning calorimetry study, and molecular docking study suggest significant stacking and stabilizing efficiency of ADG with comparison to DAN. The energetics of the interaction for ADG and DAN revealed that both reactions were predominantly entropy driven. Negative heat capacity values were obtained from the temperature dependence of the enthalpy change. The standard molar Gibbs energy change exhibited only marginal alterations with temperature suggesting the occurrence of enthalpy‐entropy compensation. These findings indicate that ADG can act as a stabilizer of telomeric G‐quadruplex DNA and thereby can be considered as a potential telomerase inhibitor.     

Longer wings for faster springs – wing length relates to spring phenology in a long‐distance migrant across its range

In migratory birds, morphological adaptations for efficient migratory flight often oppose morphological adaptations for efficient behavior during resident periods. This includes adaptations in wing shape for either flying long distances or foraging in the vegetation and in climate‐driven variation of body size. In addition, the timing of migratory flights and particularly the timely arrival at local breeding sites is crucial because fitness prospects depend on site‐specific phenology. Thus, adaptations for efficient long‐distance flights might be also related to conditions at destination areas. For an obligatory long‐distance migrant, the common nightingale, we verified that wing length as the aerodynamically important trait, but not structural body size increased from the western to the eastern parts of the species range. In contrast with expectation from aerodynamic theory, however, wing length did not increase with increasing migration distances. Instead, wing length was associated with the phenology at breeding destinations, namely the speed of local spring green‐up. We argue that longer wings are beneficial for adjusting migration speed to local conditions for birds breeding in habitats with fast spring green‐up and thus short optimal arrival periods. We suggest that the speed of spring green‐up at breeding sites is a fundamental variable determining the timing of migration that fine tune phenotypes in migrants across their range.     

Probing tethered targets of a single biomolecular complex with atomic force microscopy

DNA origami shows tremendous promise as templates for the assembly of nano‐components and detection of molecular recognition events. So far, the method of choice for evaluating these structures has been atomic force microscopy (AFM), a powerful tool for imaging nanoscale objects. In most cases, tethered targets on DNA origami have proven to be highly effective samples for investigation. Still, while maximal assembly of the nanostructures might benefit from the greatest flexibility in the tether, AFM imaging requires a sufficient stability of the adsorbed components. The balance between the tether flexibility and sample stability is a major, poorly understood, concern in such studies. Here, we investigated the dependence of the tethering length on molecular capture events monitored by AFM. In our experiments, single biotin molecules were attached to DNA origami templates with various linker lengths of thymidine nucleotides, and their interaction with streptavidin was observed with AFM. Our results show that the streptavidin‐biotin complexes are easily detected with short tethered lengths, and that their morphological features clearly change with the tethering length. We identify the functionally useful tether lengths for these investigations, which are also expected to prove useful in the construction and further application of DNA origami in bio‐nanotechnology studies. Copyright © 2013 John Wiley & Sons, Ltd.     

Arabidopsis ZDP DNA 3′‐phosphatase and ARP endonuclease function in 8‐oxoG repair initiated by FPG and OGG1 DNA glycosylases

Oxidation of guanine in DNA generates 7,8‐dihydro‐8‐oxoguanine (8‐oxoG), an ubiquitous lesion with mutagenic properties. 8‐oxoG is primarily removed by DNA glycosylases distributed in two families, typified by bacterial Fpg proteins and eukaryotic Ogg1 proteins. Interestingly, plants possess both Fpg and Ogg1 homologs but their relative contributions to 8‐oxoG repair remain uncertain. In this work we used Arabidopsis cell‐free extracts to monitor 8‐oxoG repair in wild‐type and mutant plants. We found that both FPG and OGG1 catalyze excision of 8‐oxoG in Arabidopsis cell extracts by a DNA glycosylase/lyase mechanism, and generate repair intermediates with blocked 3′‐termini. An increase in oxidative damage is detected in both nuclear and mitochondrial DNA from double fpg ogg1 mutants, but not in single mutants, which suggests that a single deficiency in one of these DNA glycosylases may be compensated by the other. We also found that the DNA 3′‐phosphatase ZDP (zinc finger DNA 3′‐phosphoesterase) and the AP(apurinic/apyirmidinic) endonuclease ARP(apurinic endonuclease redox protein) are required in the 8‐oxoG repair pathway to process the 3′‐blocking ends generated by FPG and OGG1. Furthermore, deficiencies in ZDP and/or ARP decrease germination ability after seed deteriorating conditions. Altogether, our results suggest that Arabidopsis cells use both FPG and OGG1 to repair 8‐oxoG in a pathway that requires ZDP and ARP in downstream steps.     

Che‐1‐induced inhibition of mTOR pathway enables stress‐induced autophagy

Mammalian target of rapamycin (mTOR) is a key protein kinase that regulates cell growth, metabolism, and autophagy to maintain cellular homeostasis. Its activity is inhibited by adverse conditions, including nutrient limitation, hypoxia, and DNA damage. In this study, we demonstrate that Che‐1, a RNA polymerase II‐binding protein activated by the DNA damage response, inhibits mTOR activity in response to stress conditions. We found that, under stress, Che‐1 induces the expression of two important mTOR inhibitors, Redd1 and Deptor, and that this activity is required for sustaining stress‐induced autophagy. Strikingly, Che‐1 expression correlates with the progression of multiple myeloma and is required for cell growth and survival, a malignancy characterized by high autophagy response.     

‘Last In–First Out’: seasonal variations of non‐structural carbohydrates,glucose‐6‐phosphate and ATP in tubers of two Arum species

• Knowledge on the metabolism of polysaccharide reserves in wild species is still scarce. In natural sites we collected tubers of Arum italicum Mill. and A. maculatum L. – two geophytes with different apparent phenological timing, ecology and chorology – during five stages of the annual cycle in order to understand patterns of reserve accumulation and degradation.
• Both the entire tuber and its proximal and distal to shoot portion were utilised. Pools of non‐structural carbohydrates (glucose, sucrose and starch), glucose‐6‐phosphate and ATP were analysed as important markers of carbohydrate metabolism.
• In both species, starch and glucose content of the whole tuber significantly increased from sprouting to the maturation/senescence stages, whereas sucrose showed an opposite trend; ATP and glucose‐6‐phosphate were almost stable and dropped only at the end of the annual cycle. Considering the two different portions of the tuber, both ATP and glucose‐6‐phosphate concentrations were higher in proximity to the shoot in all seasonal stages, except the flowering stage.
• Our findings suggest that seasonal carbon partitioning in the underground organ is driven by phenology and occurs independently of seasonal climate conditions. Moreover, our results show that starch degradation, sustained by elevated ATP and glucose‐6‐phosphate pools, starts in the peripheral, proximal‐to‐shoot portion of the tuber, consuming starch accumulated in the previous season, as a ‘Last In–First Out’ mechanism of carbohydrate storage.