Complete protection of antisense oligonucleotides against serum nuclease degradation by an avidin-biotin system.

It has been recently demonstrated that a complex of avidin, a cationic protein, and a monobiotinylated antisense oligonucleotide for the GLUT1 glucose transporter mRNA is taken up by cells in vitro and by organs in vivo via absorptive-mediated endocytosis. In the present study, a GLUT1 biotinylated oligonucleotide-avidin construct showing complete protection against serum 3'-exonuclease-mediated degradation is described. 21-mer antisense oligonucleotides complementary to nucleotides 162-182 and 161-181 of the bovine GLUT1 glucose transporter mRNA were synthesized with a 6-aminodeoxyuridine at positions 3 and 20, respectively, biotinylated with NHS- or NHS-XX-biotin to yield near 5'- or near 3'-biotinylated oligonucleotide (bio-DNA), and 5'- and 3'-end radiolabeled. Serum induced a rapid degradation of unprotected (no avidin) [5'-32P]-5'-bio-DNA (> 95% at 30 min). Avidin partially protected this construct (approximately 31% of intact 21-mer oligo remained at 1 h). Similar results were obtained with the [3'-32P]-5'-bio-DNA; however, no degradation products of varying size were observed, confirming that the degradation is mediated primarily by a 3'-exonuclease. Incubation of the [5'-32P]-3'-bio-DNA with serum showed a rapid conversion to the 20- and 19-mer forms (t1/2 approximately 13 min). Conversely, avidin totally protected this construct against the serum 3'-exonuclease. In conclusion, avidin fully protects antisense oligonucleotides biotinylated at the near 3'-terminus against serum 3'-exonuclease degradation, and this property may be useful for avidin-mediated drug delivery of oligonucleotides to tissues in vivo or to cultured cells in vitro.     

Adsorption of plasmid DNA to mineral surfaces and protection against DNase I.

The adsorption of [3H]thymidine-labeled plasmid DNA (pHC314; 2.4 kb) of different conformations to chemically pure sand was studied in a flowthrough microenvironment. The extent of adsorption was affected by the concentration and valency of cations, indicating a charge-dependent process. Bivalent cations (Mg2+, Ca2+) were 100-fold more effective than monovalent cations (Na+, K+, NH4+). Quantitative adsorption of up to 1 microgram of negatively supercoiled or linearized plasmid DNA to 0.7 g of sand was observed in the presence of 5 mM MgCl2 at pH 7. Under these conditions, more than 85% of DNA adsorbed within 60 s. Maximum adsorption was 4 micrograms of DNA to 0.7 g of sand. Supercoil molecules adsorbed slightly less than linearized or open circular plasmids. An increase of the pH from 5 to 9 decreased adsorption at 0.5 mM MgCl2 about eightfold. It is concluded that adsorption of plasmid DNA to sand depends on the neutralization of negative charges on the DNA molecules and the mineral surfaces by cations. The results are discussed on the grounds of the polyelectrolyte adsorption model. Sand-adsorbed DNA was 100 times more resistant against DNase I than was DNA free in solution. The data support the idea that plasmid DNA can enter the extracellular bacterial gene pool which is located at mineral surfaces in natural bacterial habitats.     

Adsorption of plasmid DNA to mineral surfaces and protection against DNase I.

The adsorption of [3H]thymidine-labeled plasmid DNA (pHC314; 2.4 kb) of different conformations to chemically pure sand was studied in a flowthrough microenvironment. The extent of adsorption was affected by the concentration and valency of cations, indicating a charge-dependent process. Bivalent cations (Mg2+, Ca2+) were 100-fold more effective than monovalent cations (Na+, K+, NH4+). Quantitative adsorption of up to 1 microgram of negatively supercoiled or linearized plasmid DNA to 0.7 g of sand was observed in the presence of 5 mM MgCl2 at pH 7. Under these conditions, more than 85% of DNA adsorbed within 60 s. Maximum adsorption was 4 micrograms of DNA to 0.7 g of sand. Supercoil molecules adsorbed slightly less than linearized or open circular plasmids. An increase of the pH from 5 to 9 decreased adsorption at 0.5 mM MgCl2 about eightfold. It is concluded that adsorption of plasmid DNA to sand depends on the neutralization of negative charges on the DNA molecules and the mineral surfaces by cations. The results are discussed on the grounds of the polyelectrolyte adsorption model. Sand-adsorbed DNA was 100 times more resistant against DNase I than was DNA free in solution. The data support the idea that plasmid DNA can enter the extracellular bacterial gene pool which is located at mineral surfaces in natural bacterial habitats.     

Intramuscular plasmid DNA electrotransfer: biodistribution and degradation

We have studied radiolabelled plasmid DNA biodistribution and degradation in the muscle at different times after injection, with or without electrotransfer using previously defined conditions. Radiolabelled plasmid progressively left the muscle and was degraded as soon as 5 min after plasmid injection, with or without electrotransfer. Autoradiography showed that the major part of injected radioactivity was detected in the interfibrilar space of a large proportion of the muscle. Large zones of accumulation of radioactivity, which seems to be contained in some fibres (more than 20 microm), were identified as soon as 5 min after electrotransfer. Such structures were never observed on slices of non-electrotransferred muscles. However, these structures were not frequent and probably lesional. The surprising fact is that despite the amount of intact plasmid having been greatly reduced between 5 min and 3 h after injection, the level of transfection remains unchanged whether electric pulses were delivered 20 s or 3 h after injection. Such a behavior was similarly observed when injecting 0.3, 3 or 30 microg of plasmid DNA. Moreover, the transfection level was correlated to the amount of plasmid DNA injected. These results suggest that as soon as it is injected, plasmid DNA is proportionally partitioned between at least two compartments. While a major part of plasmid DNA is rapidly cleared and degraded, the electrotransferable pool of plasmid DNA represents a very small part of the amount injected and belongs to another compartment where it is protected from endogenous DNAses.     

Dimethyl fumarate protection against collagen II degradation

Degradation of collagen type II caused by pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) is one of the major pathological characteristics of osteoarthritis (OA). Dimethyl fumarate (DMF) is a medication approved by the US Food and Drug Administration (FDA) as an oral multiple sclerosis (MS) therapy. In this study, we found that DMF ameliorated collagen type II degradation by inhibiting the expression of MMP-1, MMP-3, and MMP-13 caused by TNF-α. Mechanistically, DMF attenuated MMPs expression by suppressing JAK/STAT3 pathway. These findings imply that DMF treatment might be a potential therapeutic strategy for chondroprotective therapy.     

Structure- and sequence-specificity of ozone degradation of supercoiled plasmid DNA.

Ozone-reactive sites on the nucleobase moieties in supercoiled pBR322 DNA were investigated by using sequencing procedures. Ozonolysis in the absence of salt resulted in degradation of thymine residues in the A + T rich region located at 3100-3400bp. In the presence of salt, such as NaCl or MgCl2, a conformational change of plasmid DNA was induced. Subsequently the thymine and guanine residues in the loop of the cruciform located at 3120bp and 3220bp were degraded. In addition, central thymine residues present in sequences GTA, GTT and ATA were also degraded.     

Protamine sulfate protects exogenous DNA against nuclease degradation but is unable to improve the efficiency of bovine sperm mediated transgenesis

The present study investigated whether protamine sulfate can be used to improve the efficiency of bovine sperm mediated transgenesis (SMT) by protecting the plasmid pCX-EGFP against nuclease activity. A high proportion (31%) of bovine spermatozoa transfected with the plasmid pCX-EGFP maintain their motility. Using an in vitro assay, protamine sulfate protected the plasmid against degradation by DNase I. However, upon transfecting spermatozoa, the plasmid remained intact regardless of whether it was complexed to protamine sulfate. When in vitro fertilisation (IVF) was undertaken using transfected sperm, 14.6 and 10.2% of blastocysts derived from pCX-EGFP only and pCX-EGFP-protamine transfected sperm, respectively, were PCR positive for the plasmid. In conclusion, using spermatozoa transfected with either pCX-EGFP or pCX-EGFP-protamine complexes, produced PCR positive blastocysts after SMT. However, the use of protamine sulfate does not improve the efficiency of SMT suggesting that factors other than nuclease activity could be limiting.     

Co-administration of a plasmid DNA encoding IL-15 improves long-term protection of a genetic vaccine against Trypanosoma cruzi

Background

Immunization of mice with the Trypanosoma cruzi trans-sialidase (TS) gene using plasmid DNA, adenoviral vector, and CpG-adjuvanted protein delivery has proven highly immunogenic and provides protection against acute lethal challenge. However, long-term protection induced by TS DNA vaccines has not been reported. The goal of the present work was to test whether the co-administration of a plasmid encoding IL-15 (pIL-15) could improve the duration of protection achieved through genetic vaccination with plasmid encoding TS (pTS) alone.

Methodology

We immunized BALB/c mice with pTS in the presence or absence of pIL-15 and studied immune responses [with TS-specific IFN-γ ELISPOT, serum IgG ELISAs, intracellular cytokine staining (IFN-γ, TNF-α, and IL-2), tetramer staining, and CFSE dilution assays] and protection against lethal systemic challenge at 1 to 6 months post vaccination. Mice receiving pTS alone developed robust TS-specific IFN-γ responses and survived a lethal challenge given within the first 3 months following immunization. The addition of pIL-15 to pTS vaccination did not significantly alter T cell responses or protection during this early post-vaccination period. However, mice vaccinated with both pTS and pIL-15 challenged 6 months post-vaccination were significantly more protected against lethal T. cruzi challenges than mice vaccinated with pTS alone (P<0.05). Improved protection correlated with significantly higher numbers of TS-specific IFN-γ producing total and CD8⁺ T cells detected>6 months post immunization. Also, these TS-specific T cells were better able to expand after in vitro re-stimulation.

Conclusion

Addition of pIL-15 during genetic vaccination greatly improved long-term T cell survival, memory T cell expansion, and long-term protection against the important human parasite, T. cruzi.

Author Summary

Over 11 million individuals are infected with Trypanosoma cruzi, the causative agent of Chagas disease, which kills >50,000 people annually. Although recent vector control efforts and increased use and effectiveness of chemotherapeutic drugs including benznidazole have reduced infection rates and mortality, a safe, effective vaccine is needed. Vaccination with the T. cruzi trans-sialidase (TS) has been used effectively in mice to reduce mortality and chronic disease, however, the establishment of vaccine-induced long-term protective immunity remains elusive. Co-immunization strategies utilizing immune regulators such as interleukin-12 (IL-12) and interleukin-15 (IL-15) can be used to enhance antigen-specific T cell responses and prolong protective immunity. In the present report, we show that genetic vaccination of BALB/c mice with plasmid DNA encoding both TS and IL-15 compared with plasmid DNA encoding TS alone significantly enhanced CD4⁺ and CD8⁺ T cell responses including increased TNF-α, IFN-γ, and IL-2 production, and long-term protection against lethal systemic parasite challenge.     

DNA replication and degradation in mammalian tissue I. Changes in DNA polymerase and nuclease during rat liver regeneration

The incorporation of [³H]thymidine into rat liver DNA, DNA polymerase activity, and deoxyribonuclease activity have been compared at various times following partial hepatectomy of the rat.

• 1.1. DNA polymerase activity increases when [³H]thymidine incorporation in vivo increased and remained elevated for at least 60 h following partial hepatectomy, despite waves of DNA synthesis. Fluctuations in nuclease activity were directly opposite to the peaks of [³H]thymidine incorporation at different times during regeneration. It is suggested that deoxyribonuclease plays an important role in the control of DNA synthesis.
• 2.2. Rat liver polymerase had a preference for native DNA primer whereas deoxyribonuclease in the same preparation acted preferentially on denatured DNA. It is suggested that the apparent difference in polymerase primer preference is due to nuclease activity associated with the pH 5.0 protein fraction.

     

The combination of DNA methylation and H1 histone binding inhibits the action of a restriction nuclease on plasmid DNA.

To investigate the potentials of DNA methylation and H1 histone in regulating the action of DNA binding proteins, well ordered complexes were formed by slow salt gradient dialysis of mixtures of H1 histone with either methylated or nonmethylated DNA. The sites methylated in the plasmids were CCGG. Methylation of cytosine in this site protects the DNA against HpaII endonuclease but not against MspI. However, when the methylated DNA was complexed to H1, it was protected against MspI. The protection was only effective for a subset of the MspI restriction sites. The protection of DNA afforded by the combination of H1 binding and DNA methylation did not apply to EcoRI, PstI, or BamHI sites and so did not seem to be due to aggregation of the DNA by H1 histone. Gel retardation assays indicated that the affinity of H1 for methylated DNA was not detectably different from its affinity for nonmethylated DNA. Probably methylated DNA when bound to H1 is in a conformation that is resistant to MspI endonuclease. Such conformational changes induced by DNA methylation and H1 binding might affect the action of other DNA binding proteins, perhaps in chromatin as well as in H1.DNA complexes.     

Nitroxide-mediated protection against X-ray- and neocarzinostatin-induced DNA damage.

The stable free radical Tempol (4-hydroxy-2,2,6,6-tetramethyl-piperidinyloxy) has been shown to protect against X-ray-induced cytotoxicity and hydrogen peroxide- or xanthine oxidase-induced cytotoxicity and mutagenicity. The ability of Tempol to protect against X-ray- or neocarzinostatin (NCS)-induced mutagenicity or DNA double-strand breaks (dsb) was studied in Chinese hamster cells. Tempol (50 mM) provided a protection factor of 2.7 against X-ray-induced mutagenicity in Chinese hamster ovary (CHO) AS52 cells, with a protection factor against cytotoxicity of 3.5. Using the field inversion gel electrophoresis technique of measuring DNA dsb, 50 mM Tempol provides a threefold reduction in DNA damage at an X-ray dose of 40 Gy. For NCS-induced damage, Tempol increased survival from 9% to 80% at 60 ng/mL NCS and reduced mutation induction by a factor of approximately 3. DNA dsb were reduced by a factor of approximately 7 at 500 ng/mL NCS. Tempol is representative of a class of stable nitroxide free radical compounds that have superoxide dismutase-mimetic activity, can oxidize metal ions such as ferrous iron that are complexed to DNA, and may also detoxify radiation-induced organoperoxide radicals by competitive scvenging. The NCS chromophore is reduced by sulfhydryls to an active form. Electron spin resonance (ESR) spectroscopy shows that 2-mercaptoethanol-activated NCS reacts with Tempol 3.5 times faster than does unactivated NCS. Thus, Tempol appears to inactivate the NCS chromophore before a substantial amount of DNA damage occurs.     

Site-directed protection of RNA during nuclease digestion

We report the use of oligodeoxynucleotides to block the nucleolytic hydrolysis of single-stranded regions of RNA. Using complementary oligomers, the hydrolysis of the CCA terminus of methionine initiator tRNA could be prevented. This method can be useful in the production of specific single-stranded fragments of RNA, which are necessary in recombinant RNA technology.     

Protection against radiation-induced degradation of DNA bases by polyamines

Polyamines have been reported to protect DNA against the formation of radiation-induced strand breaks and crosslinks to proteins. The present study was aimed at investigating the protective effect of spermine, spermidine and putrescine against the degradation of DNA bases upon exposure to gamma rays in aerated aqueous solution. The yield of 8-oxo-7,8-dihydroguanine and 5-hydroxycytosine was found to decrease for concentrations of spermine and spermidine greater than 0.1 mM. A protection factor of 10 was observed for a concentration of 1 mM of the latter two polyamines. Putrescine afforded a lower protection. In addition, the formation yield of a series of radiation-induced degradation products of the purine and pyrimidine bases was determined within DNA in the presence or absence of spermine. The protection factor was within the same range for all the lesions measured. The latter observation ruled out the possibility of degradation of DNA by radiation-induced polyamine peroxyl radicals. This was confirmed by studies involving radiolysis of DMSO and decomposition of 2,2'-azobis(2-methyl-propionamidine) as sources of alkylperoxyl radicals. Therefore, it is likely that the polyamine-mediated protection against the radiation-induced degradation of DNA bases is due to the compaction of the DNA structure and the reduction in the accessibility of DNA to .OH rather than by scavenging .OH in the bulk solution or in the vicinity of the DNA.     

Search for apoptotic nucleases in yeast: role of Tat-D nuclease in apoptotic DNA degradation

DNA fragmentation/degradation is an important step for apoptosis. However, in unicellular organisms such as yeast, this process has rarely been investigated. In the current study, we revealed eight apoptotic nuclease candidates in Saccharyomyces cerevisiae, analogous to the Caenorhabditis elegans apoptotic nucleases. One of them is Tat-D. Sequence comparison indicates that Tat-D is conserved across kingdoms, implicating that it is evolutionarily and functionally indispensable. In order to better understand the biochemical and biological functions of Tat-D, we have overexpressed, purified, and characterized the S. cerevisiae Tat-D (scTat-D). Our biochemical assays revealed that scTat-D is an endo-/exonuclease. It incises the double-stranded DNA without obvious specificity via its endonuclease activity and excises the DNA from the 3'- to 5'-end by its exonuclease activity. The enzyme activities are metal-dependent with Mg(2+) as an optimal metal ion and an optimal pH around 5. We have also identified three amino acid residues, His(185), Asp(325), and Glu(327), important for its catalysis. In addition, our study demonstrated that knock-out of TAT-D in S. cerevisiae increases the TUNEL-positive cells and cell survival in response to hydrogen hyperoxide treatment, whereas overexpression of Tat-D facilitates cell death. These results suggest a role of Tat-D in yeast apoptosis.     

Sequence-specific protection of plasmid DNA from restriction endonuclease hydrolysis by pyrrole-imidazole-cyclopropapyrroloindole conjugates

The pyrrole-imidazole (Py-Im) triamide-cyclopropa pyrroloindole (CPI) conjugates ImPyImLDu86 (7) and ImImPyLDu86 (14) were synthesized and their alkylating activities and inhibitory effects on DNA hydrolysis by restriction endonucleases were examined. Sequencing gel analysis demonstrated that conjugates 7 and 14 specifically alkylated DNA at 5'-CGCGCG-3' and 5'-PyGGCCPu-3', respectively. Agarose gel electrophoresis indicated that incubation of a supercoiled plasmid, pSPORT I (4109 bp), with conjugate 7 effectively inhibited its hydrolysis by BssHII (5'-G_CGCGC-3'), whereas conjugate 14 had no effect on this hydrolysis. These results suggest that conjugate 7 sequence-specifically inhibits the hydrolysis of DNA by BssHII. Sequence-specific alkylation by the Py-Im triamide-CPI conjugates was further confirmed by inhibition of the Eco52I (5'-C_GGCCG-3') hydrolysis of conjugate 14-treated pQBI PGK (5387 bp). In clear contrast, hydrolysis of pQB1 PGK by DraI (3'-TTT_AAA-3') was not inhibited by 5 micro M conjugate 14. That ImImPy did not inhibit the hydrolysis of pQB1 PGK indicates that covalent bond formation is necessary for inhibition. A similar experiment, using linear pQBI PGK, achieved the same extent of protection of the DNA with approximately half the concentration of conjugate 14 as was required to protect supercoiled DNA from hydrolysis.     

Similar ultraviolet action spectra for the protection by glycerol of transforming DNA against single-strand breaks and inactivation of biological activity

An action spectrum for the protection of purified DNA by glycerol against the induction of single-strand breaks in the DNA by ultraviolet (uv) light is described. Protection was not observed below 300 nm, was maximal between 334 and 365 nm, and decreased at 405 nm. This spectrum closely matched the spectrum for the protection by glycerol against the inactivation of biological transforming activity by near uv, described previously. Also, deviations from the reciprocity rule are similar for inactivation of transforming activity and for induction of DNA breaks by 365-nm radiation. That is, the deviations for the two end points are quantitatively the same, such that high fluence rates are less effective than low fluence rates.     

Development of a vaccine against murine cytomegalovirus (MCMV), consisting of plasmid DNA and formalin-inactivated MCMV, that provides long-term, complete protection against viral replication

We previously demonstrated that immunization of mice with plasmid DNAs (pDNAs) expressing the murine cytomegalovirus (MCMV) genes IE1-pp89 and M84 provided synergistic protection against sublethal viral challenge, while immunization with plasmids expressing putative virion proteins provided no or inconsistent protection. In this report, we sought to augment protection by increasing the breadth of the immune response. We identified another MCMV gene (m04 encoding gp34) that provided strong and consistent protection against viral replication in the spleen. We also found that immunization with a DNA pool containing 10 MCMV genes that individually were nonprotective elicited reproducible protection against low to intermediate doses of challenge virus. Moreover, inclusion of these plasmids into a mixture with gp34, pp89, and M84 DNAs provided even greater protection than did coimmunization with pp89 and M84. The highest level of protection was achieved by immunization of mice with the pool of 13 pDNAs, followed by formalin-inactivated MCMV (FI-MCMV). Immunization with FI-MCMV elicited neutralizing antibodies against salivary gland-derived MCMV, and of greatest importance, mice immunized with both the combined pDNA pool and FI-MCMV had undetectable levels of virus in the spleen and salivary glands after challenge. Intracellular cytokine staining of splenocytes from pDNA- and FI-MCMV-immunized mice showed that pDNA immunization elicited high levels of pp89- and M83-specific CD8(+) T cells, whereas both pDNA and FI-MCMV immunizations generated strong CD8(+)-T-cell responses against virion-associated antigens. Taken together, these results show that immunization with pDNA and inactivated virus provides strong antibody and cell-mediated immunity against CMV infection.     

Chemical and biological consequences of the radioactive decay of iodine-125 in plasmid DNA

Doubly labeled [U-14C, 5-125I]iododeoxycytidine (IdC) triphosphate was synthesized and incorporated enzymatically into defined positions of the plasmid pBR322. After storage under various conditions, the stable end products were analyzed using radio-GC, radio-HPLC, and electron microscopy. In addition, solutions of 14C-IdC-labeled DNA containing Na125I as an internal radiation source were studied to investigate the influence of internal radiolysis. Transmutation of the covalently bound 125I leads to complete destruction of the labeled nucleotide, giving rise to 14CO2 and 14CO as major products. Fragmentation of the pyrimidine base is independent of solvent and DNA configuration. Internal radiolysis caused by Na125I leads to only minor damage. Electron microscopy studies reveal that decay-induced double strand breaks (dsb) occur both at the site of decay and in areas as far as hundreds of base pairs apart from that site. Number and distribution of the breaks is strongly dependent on solvent and DNA configuration. A direct correlation exists between the extent of fragmentation of the nucleotide and the mean number of dsb.     

Immunization of mice with plasmid DNA coding for NcGRA7 or NcsHSP33 confers partial protection against vertical transmission of Neospora caninum

The purpose of the present study was to use direct plasmid deoxyribonucleic acid (DNA) injection to identify specific antigens that confer protection against congenital transfer of Neospora caninum. Inbred BALB/c mice were vaccinated before pregnancy with a recombinant plasmid containing sequences encoding N. caninum antigen NcGRA7 or NcsHSP33. The mice were challenged with N. caninum tachyzoites at 10-12 days of gestation. Whereas 100% of pups born from dams immunized with control plasmid contained detectable levels of N. caninum DNA in a Neospora-specific polymerase chain reaction assay, only 46% of pups from pCMVi-NcGRA7-immunized mice and 53% of pCMVi-NcsHSP33-immunized mice were N. caninum positive, and none of the mice immunized with tachyzoite extract contained N. caninum DNA. Thus, immunization of mice with plasmid DNA expressing N. caninum antigens conferred partial protection against congenital neosporosis.