Targeting nucleolin to obstruct vasculature feeding with an intelligent DNA nanorobot

A prototype of DNA nanorobot with the ability to transport molecular payloads was designed to target cancer cells in tissue culture. Moreover, a further step was taken to succeed in the first in vivo application of the DNA nanorobot for cancer therapy. The robot was constructed using aptamer and DNA origami to fold a 90‐nm tubular device to carry the blood coagulation protease thrombin inside, shielded from circulating platelets and plasma fibrinogen. The recognition and binding of the aptamer to its tumour‐specific target molecule triggered the robot unfolding to expose thrombin to the blood, which in turn activated coagulation at the local tumour site, resulting in tumour necrosis and inhibition of tumour growth. Since all solid‐tumour feeding vessels are virtually the same, this strategy could be effective against many types of malignant diseases.     

SSCP primer design based on single-strand DNA structure predicted by a DNA folding program.

To predict alterations in single-strand DNA mobility in non-denaturing electrophoretic gels, Zuker's RNA folding program was modified. Energy files utilized by the LRNA RNA folding algorithm were modified to emulate folding of single-strand DNA. Energy files were modified to disallow G-T base pairing. Stacking energies were corrected for DNA thermodynamics. Constraints on loop nucleotide sequences were removed. The LRNA RNA folding algorithm using the DNA fold energy files was applied to predict folding of PCR generated single-strand DNA molecules from polymorphic human ALDH2 and TPH alleles. The DNA-Fold version 1.0 program was used to design primers to create and abolish SSCP mobility shifts. Primers were made that add a 5' tag sequence or alter complementarity to an internal sequence. Differences in DNA secondary structure were assessed by SSCP analysis and compared to single-strand DNA secondary structure predictions. Results demonstrate that alterations in single-strand DNA conformation may be predicted using DNA-Fold 1.0.     

Ethidium-bromide-inhibited restriction endonucleases cleave one strand of circular DNA

We analyzed the effect of ethidium bromide (EtBr) on the cleavage of closed circular pBR322 DNA molecules by six restriction enzymes which make staggered or flush cuts (EcoRI, HindIII, BglI, PstI, HincII, PvuII). EtBr concentrations and reaction temperatures were determined at which DNA molecules with single-strand breaks were the major reaction product of digestion by all the enzymes. However, the amounts of intermediates which could be isolated differed for various enzymes. The results extend previous studies, showing that sequential cleavage of the DNA strands probably is a general property of restriction endonucleases.     

A high-throughput assay for the adenylation reaction of bacterial DNA ligase

DNA ligase catalyzes the closure of single-strand nicks in double-stranded DNA that arise during replication and recombination. Inhibition of bacterial ligase is expected to cause chromosome degradation and cell death, making it an attractive target for new antibacterials. The prototypical bacterial ligase couples the hydrolysis of NAD(+) to phosphodiester bond formation between an adjacent 3'OH and 5'-terminal phosphate of nicked duplex DNA. The first step is the reversible formation of a ligase-adenylate from the reaction between apoenzyme and NAD(+). Inhibitors that compete with NAD(+) are expected to be bacterial specific because eukaryotic DNA ligases use ATP and differ in the sequence composition of their adenylation domain. We report here a high-throughput assay that measures the adenylation reaction specifically by monitoring ligase-AMP formation via scintillation proximity technologies. Escherichia coli DNA ligase was biotinylated in vivo; after reaction with radiolabeled NAD(+), ligase-[(3)H]AMP could be captured onto the streptavidin-coated surface of the solid scintillant. The method was ideal for high-throughput screening because it required minimal manipulations and generated a robust signal with minimal scatter. Certain adenosine analogs were found to inhibit the adenylation assay and had similar potency of inhibition in a DNA ligation assay.     

Isolation of high-molecular-weight plant DNA for DNA damage quantitation: relative effects of solar 297 nm UVB and 365 nm radiation

Quantitation of UV-induced DNA damages in nanogram quantities of non-radiactive DNA from irradiated plants by gel electrophoresis requires a prompt, efficient, high-yield method of isolating DNA yielding high-molecular-weight, enzymatically digestible DNA. To meet these criteria we devised a high-yield method for isolating from plant tissue, DNA whose single-strand molecular length is greater than about 170 kb. Leaf tissue is embedded in agarose plugs, digested with Proteinase K in the presence of detergent, and treated with phenylmethylsulfonyl fluoride (PMSF). The agarose plugs are then soaked with buffer appropriate to the desired enzyme treatment. Evaluation of the DNA on neutral and alkaline gels indicates its high molecular length and low frequency of single-strand breaks. The DNA can be digested with damage-specific and other endonucleases. The method is especially suitable for DNA damage quantitation, as tissue processing is carried out immediately after harvesting (allowing DNA lesion measurement at precisely known times after irradiation), and many samples can be easily handled at once. It should also be useful for molecular analysis of large numbers of plant samples available only in small quantities. We here use this method to quantitate DNA damage induced by 297 and 365 nm radiation, and calculate the relative damaging effects of these wavebands in today's solar spectrum.     

Modulation of the Pyrococcus abyssi NucS endonuclease activity by replication clamp at functional and structural levels

Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5' and 3' flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction.     

Existence of single-strand interruptions in the genomic DNA of mycobacteriophage I3

Abstract The genomic DNA of mycobacteriophage I3 harbours single-strand interruptions and is susceptible to degradation by nuclease S1, leading to random fragmentation. The native DNA serves as an efficient template for DNA polymerase I showing the presence of free 3'-OH termini which provide the priming points within the genome. The single-strand regions show no specificity for the 5'-end nucleotides flanking these interruptions and all 4 nucleotides are present with a probability equivalent to their percentage in the total DNA. The single-strand interruptions are an intrinsic character of this genome and are present irrespective of the host on which the phage is propagated.     

Chromatin Collapse during Caspase-dependent Apoptotic Cell Death Requires DNA Fragmentation Factor, 40-kDa Subunit-/Caspase-activated Deoxyribonuclease-mediated 3′-OH Single-strand DNA Breaks

Apoptotic nuclear morphology and oligonucleosomal double-strand DNA fragments (also known as DNA ladder) are considered the hallmarks of apoptotic cell death. From a classic point of view, these two processes occur concomitantly. Once activated, DNA fragmentation factor, 40-kDa subunit (DFF40)/caspase-activated DNase (CAD) endonuclease hydrolyzes the DNA into oligonucleosomal-size pieces, facilitating the chromatin package. However, the dogma that the apoptotic nuclear morphology depends on DNA fragmentation has been questioned. Here, we use different cellular models, including MEF CAD^−/− cells, to unravel the mechanism by which DFF40/CAD influences chromatin condensation and nuclear collapse during apoptosis. Upon apoptotic insult, SK-N-AS cells display caspase-dependent apoptotic nuclear alterations in the absence of internucleosomal DNA degradation. The overexpression of a wild-type form of DFF40/CAD endonuclease, but not of different catalytic-null mutants, restores the cellular ability to degrade the chromatin into oligonucleosomal-length fragments. We show that apoptotic nuclear collapse requires a 3′-OH endonucleolytic activity even though the internucleosomal DNA degradation is impaired. Moreover, alkaline unwinding electrophoresis and In Situ End-Labeling (ISEL)/In Situ Nick Translation (ISNT) assays reveal that the apoptotic DNA damage observed in the DNA ladder-deficient SK-N-AS cells is characterized by the presence of single-strand nicks/breaks. Apoptotic single-strand breaks can be impaired by DFF40/CAD knockdown, abrogating nuclear collapse and disassembly. In conclusion, the highest order of chromatin compaction observed in the later steps of caspase-dependent apoptosis relies on DFF40/CAD-mediated DNA damage by generating 3′-OH ends in single-strand rather than double-strand DNA nicks/breaks.     

Acute low-intensity microwave exposure increases DNA single-strand breaks in rat brain cells

Levels of DNA single-strand break were assayed in brain cells from rats acutely exposed to low-intensity 2450 MHz microwaves using an alkaline microgel electrophoresis method. Immediately after 2 h of exposure to pulsed (2 μs width, 500 pulses/s) microwaves, no significant effect was observed, whereas a dose rate-dependent [0.6 and 1.2 W/kg whole body specific absorption rate (SAR)] increase in DNA single-strand breaks was found in brain cells of rats at 4 h postexposure. Furthermore, in rats exposed for 2 h to continuous-wave 2450 MHz microwaves (SAR 1.2 W/kg), increases in brain cell DNA single-strand breaks were observed immediately as well as at 4 h postexposure. © 1995 Wiley-Liss, Inc.     

Kinetic mechanism of human DNA ligase I reveals magnesium-dependent changes in the rate-limiting step that compromise ligation efficiency

DNA ligase I (LIG1) catalyzes the ligation of single-strand breaks to complete DNA replication and repair. The energy of ATP is used to form a new phosphodiester bond in DNA via a reaction mechanism that involves three distinct chemical steps: enzyme adenylylation, adenylyl transfer to DNA, and nick sealing. We used steady state and pre-steady state kinetics to characterize the minimal mechanism for DNA ligation catalyzed by human LIG1. The ATP dependence of the reaction indicates that LIG1 requires multiple Mg(2+) ions for catalysis and that an essential Mg(2+) ion binds more tightly to ATP than to the enzyme. Further dissection of the magnesium ion dependence of individual reaction steps revealed that the affinity for Mg(2+) changes along the reaction coordinate. At saturating concentrations of ATP and Mg(2+) ions, the three chemical steps occur at similar rates, and the efficiency of ligation is high. However, under conditions of limiting Mg(2+), the nick-sealing step becomes rate-limiting, and the adenylylated DNA intermediate is prematurely released into solution. Subsequent adenylylation of enzyme prevents rebinding to the adenylylated DNA intermediate comprising an Achilles' heel of LIG1. These ligase-generated 5'-adenylylated nicks constitute persistent breaks that are a threat to genomic stability if they are not repaired. The kinetic and thermodynamic framework that we have determined for LIG1 provides a starting point for understanding the mechanism and specificity of mammalian DNA ligases.     

Designing a Bio-responsive Robot from DNA Origami

Nucleic acids are astonishingly versatile. In addition to their natural role as storage medium for biological information¹, they can be utilized in parallel computing^2,3 , recognize and bind molecular or cellular targets^4,5 , catalyze chemical reactions^6,7 , and generate calculated responses in a biological system^8,9. Importantly, nucleic acids can be programmed to self-assemble into 2D and 3D structures^10-12, enabling the integration of all these remarkable features in a single robot linking the sensing of biological cues to a preset response in order to exert a desired effect.Creating shapes from nucleic acids was first proposed by Seeman¹³, and several variations on this theme have since been realized using various techniques^11,12,14,15 . However, the most significant is perhaps the one proposed by Rothemund, termed scaffolded DNA origami¹⁶. In this technique, the folding of a long (>7,000 bases) single-stranded DNA ''scaffold'' is directed to a desired shape by hundreds of short complementary strands termed ''staples''. Folding is carried out by temperature annealing ramp. This technique was successfully demonstrated in the creation of a diverse array of 2D shapes with remarkable precision and robustness. DNA origami was later extended to 3D as well^17,18 .The current paper will focus on the caDNAno 2.0 software¹⁹ developed by Douglas and colleagues. caDNAno is a robust, user-friendly CAD tool enabling the design of 2D and 3D DNA origami shapes with versatile features. The design process relies on a systematic and accurate abstraction scheme for DNA structures, making it relatively straightforward and efficient.In this paper we demonstrate the design of a DNA origami nanorobot that has been recently described²⁰. This robot is ''robotic'' in the sense that it links sensing to actuation, in order to perform a task. We explain how various sensing schemes can be integrated into the structure, and how this can be relayed to a desired effect. Finally we use Cando²¹ to simulate the mechanical properties of the designed shape. The concept we discuss can be adapted to multiple tasks and settings.     

"Run-off" synthesis and application of defined single-stranded DNA hybridization probes.

A simple and efficient method for synthesizing radioactively labeled single-stranded DNA hybridization probes with Thermus aquaticus (Taq) DNA polymerase is described. This is done in a "run-off" polymerization with repeated cycles of denaturation, annealing, and extension. It leads to high yields of a single-stranded DNA of defined length (up to 5000 nt), which is labeled to a high specific activity (1.3 x 10(8) cpm/micrograms DNA). These hybridization probes are equally sensitive as nick-translated DNA probes, but strand specific. This was tested by slot blot hybridization with in vitro-transcribed target RNAs and by Northern blotting. The use of single-stranded DNA hybridization probes combines the benefits of DNA stability and single-strand RNA probes.     

Normal response of Fanconi's anemia cells to high concentrations of O2 as determined by alkaline elution

In this investigation, normal and Fanconi's anemia fibroblasts were exposed to high concentrations of oxygen and the effects of this treatment on DNA were analyzed by alkaline elution. No DNA single-strand breaks were detected in either cell type with up to 20 h incubation in high (50–95%) concentrations of O₂. No evidence of DNA damage by O₂ could be detected with an endonuclease preparation from Micrococcus luteus. Cells which have been treated with various DNA-damaging agents in the presence of the polymerase inhibitor cytosine arabinoside have been shown to accumulate DNA single-strand breaks during DNA excision repair. When cells were treated with the polymerase inhibitor in 50 or 95% O₂, a low level of DNA single-strand breaks accumulated in both cell types. However, no significant differences in the frequency of DNA single-strand breaks were detected between normal and Fanconi's anemia cells after exposure to high O₂.     

Pulsed-field gel electrophoresis analysis of higher-order chromatin structures of Zea mays. Highly methylated DNA in the 50 kb chromatin structure

We have investigated the presence of higher-order chromatin structures in different maize tissues. Taking advantage of the pulsed-field gel electrophoresis technique to analyse large DNA fragments from intact nuclei and cells, we have determined the size distribution of the high-molecular-weight DNA fragments obtained from chromatin degradation by endogenous nucleases in isolated nuclei. Chromatin digestion leads to the appearance of stable DNA fragments of about 50 kb in all the tissues examined, suggesting the folding of DNA in higher-order chromatin domain structures. It has been reported that such chromatin domains are formed by loops of the 30 nm fibres anchored to the nuclear matrix by a complex set of proteins, including DNA topoisomerase II. Treatment of maize protoplasts with the calcium ionophore A23187 and the antitumour drug VM-26, which specifically inhibit the religation of the cleaved DNA in the topoisomerase II reaction, also produces the 50 kb structure. Analysis of the DNA contained in the 50 kb chromatin structure shows a higher degree of methylation than in bulk maize chromosomal DNA. The role of methylated DNA in the chromatin folding is discussed.     

Experimental Evidence for Slipped Loop DNA,a Novel Folding Type for Polynucleotide Chain

Abstract

DNA regions with short direct repeats (5–7bp) with a spacer in between, when under super-helical stress, are known to become susceptible to single-strand specific nuclease S1. This is in accord with formation of two shifted loops protruding from the opposite chains. Such type of folding could have been additionally stabilized by base pairing between the complementary parts of the loops that explains existence of the protected from SI moieties of the loops. To test this possibility we designed and synthesized an oligonucleotide of 56 bases, so that it forms a hairpin with a stem which fails to acquire a traditional helix due to a special sequence but may favor the formation of the proposed Slipped Loop Structure (SLS). The oligonucleotide folding was studied by a chemical modification method at one nucleotide level resolution. Three zones, protected from the used probes were found: the one that forms the stem, and the others that are located within the two by-loops in those moieties which have the base pairing potential. Proceeding from the data obtained and stereochemical analysis a 3-D scheme for the SLS form of DNA is suggested.     

Herpes simplex virus type 1 single-strand DNA binding protein ICP8 enhances the nuclease activity of the UL12 alkaline nuclease by increasing its processivity

UL12 is a 5'- to 3'-exonuclease encoded by herpes simplex virus type 1 (HSV-1) which degrades single- and double-stranded DNA. UL12 and the single-strand DNA binding protein ICP8 mediate a strand exchange reaction. We found that ICP8 inhibited UL12 digestion of single-stranded DNA but stimulated digestion of double-stranded DNA threefold. The stimulatory effect of ICP8 was independent of a strand exchange reaction; furthermore, the effect was specific to ICP8, as it could not be reproduced by Escherichia coli single-stranded DNA binding protein. The effect of ICP8 on the rate of UL12 double-stranded DNA digestion is attributable to an increase in processivity in the presence of ICP8.     

P1 nuclease cleavage is dependent on length of the mismatches in DNA

P1 nuclease is one of the most extensively used single-strand DNA specific nucleases in molecular biology. In modern biology, it is used as an enzymatic probe to detect altered DNA conformations. It is well documented that P1 cleaves single-stranded nucleic acids and single-stranded DNA regions. The fact that P1 can act under a wide range of conditions, including physiological pH and temperature make it the most commonly used enzymatic probe in DNA structural studies. Surprisingly, to this date, there is no study to characterize the influence of length of mismatches on P1 sensitivity. Using a series of radioactively labeled oligomeric DNA substrates-containing mismatches, we find that P1 nuclease cleavage is dependent on the length of mismatches. P1 does not cleave DNA when there is a single-base mismatch. P1 cleavage efficiency is optimum when mismatch length is 3 nt or more. Changing the position of the mismatches also does not make any difference in cleavage efficacy. These novel findings on P1 properties have implications for its use in DNA structure and DNA repair studies.     

Comment: a biological guide for electromagnetic safety: the stress response

Questions of safety of electromagnetic (EM) fields should be based on relevant biological properties, i.e., specific cellular reactions to potentially harmful stimuli. The stress response is a well documented protective reaction of plant and animal cells to a variety of environmental threats, and it is stimulated by both extremely low frequency (ELF) and radio frequency (RF) EM fields. It involves activation of DNA to initiate synthesis of stress proteins. Thermal and non-thermal stimuli affect different segments of DNA and utilize different biochemical pathways. However, both ELF and RF stimulate the same non-thermal pathway. Since the same biochemical reactions are stimulated in different frequency ranges with very different specific absorption rates (SARs), SAR level is not a valid basis for safety standards. Studies of EM field interactions with DNA and with model systems provide insight into a plausible mechanism that can be effective in ELF and RF ranges.