Incorporation Of Nucleic Acids Research Articles

Virus-like particle - mediated delivery of the RIG-I agonist M8 induces a type I interferon response and protects cells against viral infection.

Virus-Like Particles (VLPs) are nanostructures that share conformation and self-assembly properties with viruses, but lack a viral genome and therefore the infectious capacity. In this study, we produced VLPs by co-expression of VSV glycoprotein (VSV-G) and HIV structural proteins (Gag, Pol) that incorporated a strong sequence-optimized 5'ppp-RNA RIG-I agonist, termed M8. Treatment of target cells with VLPs-M8 generated an antiviral state that conferred resistance against multiple viruses. Interestingly, treatment with VLPs-M8 also elicited a therapeutic effect by inhibiting ongoing viral replication in previously infected cells. Finally, the expression of SARS-CoV-2 Spike glycoprotein on the VLP surface retargeted VLPs to ACE2 expressing cells, thus selectively blocking viral infection in permissive cells. These results highlight the potential of VLPs-M8 as a therapeutic and prophylactic vaccine platform. Overall, these observations indicate that the modification of VLP surface glycoproteins and the incorporation of nucleic acids or therapeutic drugs, will permit modulation of particle tropism, direct specific innate and adaptive immune responses in target tissues, and boost immunogenicity while minimizing off-target effects.

Extracellular Vesicle Loading Via pH-Gradient Modification.

Extracellular vesicles (EVs) have emerged as key mediators of intercellular communication and consequently have the potential to be potent therapeutic vectors. Beyond their endogenous function, EVs are also being harnessed as drug delivery vehicles with possible benefits over synthetic nanoparticle systems. Despite advances in loading exogenous molecules into extracellular vesicles, efficient incorporation of nucleic acids remains a challenge due to aggregation and degradation. In this chapter, we detail a method to load EVs with negatively charged cargo, in particular nucleic acids, by modifying the internal pH of the vesicles to be acidic. This approach demonstrates that pH modification of EVs enables efficient loading of nucleic acids with functional cargo.

Study of the incorporation of nucleic acids in chitosan-coated polystyrene nanoparticles for use as DNA carrier system

Nanotechnology is a multidisciplinary scientific field based on the development, characterization, production and application of structures, devices and systems with shape and size at the nanoscale. Polymeric systems with therapeutic purpose have been widely used since they allow a slow and gradual release of drug and allow the transport of drugs to their specific place of action. In recent years, nanoparticles have been used for DNA loading. The introduction of exogenous DNA into a cell may be applicable to fields of gene therapy, DNA vaccines and diagnosis. The development of nucleic acid loading nanoparticles, with a well characterized activity, would be very important. For this project, cationic polystyrene nanoparticles coated with chitosan was studied for a DNA carrier system. The propose is an elaboration of a dilution gradient that allows to know the pattern of incorporation of nucleic acids in the nanoparticles, permitting the development of a mathematical model that characterizes the incorporation in the different conditions studied, allowing their use in future projects. Through this, it´s found the potential of DNA saturation by this nanoparticle system, as in 29% of the incorporation mass, which reveals the capacity of DNA incorporation.

Magnolol pretreatment attenuates heat stress-induced IEC-6 cell injury.

Heat stress (HS) is an important environmental stressor that adversely influences livestock during the summer. The aim of this study was to investigate whether magnolol protects against HS-induced intestinal epithelial cell injury. An intestinal epithelial cell line (IEC-6) was subjected to HS at 42 °C, with and without magnolol pretreatment. Cell injury was detected by monitoring lactate dehydrogenase (LDH) release. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay was used to assess cell proliferation and viability, including identifying effective concentrations of magnolol. Flow cytometry confirmed G1-phase cell-cycle arrest and its alleviation by magnolol. Active DNA synthesis was measured by incorporation of nucleic acid 5-ethynyl-2'-deoxyuridine (EdU). G1-phase cell-cycle-related gene expression was assessed by real-time reverse transcription polymerase chain reaction (RT-PCR) and levels of G1-phase-related proteins by Western blotting. HS induced IEC-6 cell injury and decreased cell viability, as demonstrated by data from LDH and MTS assays, respectively. Based on a number of criteria, IEC-6 cells subjected to HS were arrested in the G1 phase of the cell cycle. Magnolol pretreatment decreased HS-induced cell injury through relief of this cell-cycle arrest. Magnolol pretreatment attenuates HS-induced injury in IEC-6 cells. Magnolol is potentially promising as a protective strategy for HS in livestock.

64Cu-ATSM therapy targets regions with activated DNA repair and enrichment of CD133+ cells in an HT-29 tumor model: Sensitization with a nucleic acid antimetabolite

64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM) is a potential theranostic agent targeting the over-reduced state under hypoxia within tumors. Recent clinical Cu-ATSM positron emission tomography studies have revealed a correlation between uptake and poor prognosis; however, the reason is unclear. Here, using a human colon carcinoma HT-29 model, we demonstrated that the intratumoral 64Cu-ATSM high-uptake regions exhibited malignant characteristics, such as upregulated DNA repair and elevated %CD133+ cancer stem-like cells. Based on this evidence, we developed a strategy to enhance the efficacy of 64Cu-ATSM internal radiotherapy (IRT) by inhibiting DNA repair with a nucleic acid (NA) antimetabolite. The results of the analyses showed upregulation of pathways related to DNA repair along with NA incorporation (bromodeoxyuridine uptake) and elevation of %CD133+ cells in 64Cu-ATSM high-uptake regions. In an in vivo64Cu-ATSM treatment study, co-administration of an NA antimetabolite and 64Cu-ATSM synergistically inhibited tumor growth, with little toxicity, and effectively reduced %CD133+ cells. 64Cu-ATSM therapy targeted malignant tumor regions with activated DNA repair and high concentrations of CD133+ cells in the HT-29 model. NA antimetabolite co-administration can be an effective approach to enhance the therapeutic effect of 64Cu-ATSM IRT.

Development of bright fluorescent quadracyclic adenine analogues: TDDFT-calculation supported rational design.

Fluorescent base analogues (FBAs) comprise a family of increasingly important molecules for the investigation of nucleic acid structure and dynamics. We recently reported the quantum chemical calculation supported development of four microenvironment sensitive analogues of the quadracyclic adenine (qA) scaffold, the qANs, with highly promising absorptive and fluorescence properties that were very well predicted by TDDFT calculations. Herein, we report on the efficient synthesis, experimental and theoretical characterization of nine novel quadracyclic adenine derivatives. The brightest derivative, 2-CNqA, displays a 13-fold increased brightness (εΦF = 4500) compared with the parent compound qA and has the additional benefit of being a virtually microenvironment-insensitive fluorophore, making it a suitable candidate for nucleic acid incorporation and use in quantitative FRET and anisotropy experiments. TDDFT calculations, conducted on the nine novel qAs a posteriori, successfully describe the relative fluorescence quantum yield and brightness of all qA derivatives. This observation suggests that the TDDFT-based rational design strategy may be employed for the development of bright fluorophores built up from a common scaffold to reduce the otherwise costly and time-consuming screening process usually required to obtain useful and bright FBAs.

Immobilized MWCNT support osteogenic cell culture

The broad use of versatile, strong, lightweight multi-walled carbon nanotubes (MWCNT) for use in biomaterial applications is tempered by ongoing debate about their safety. Recent reports suggest that factors such as their diameter and surface coating affect their function and cytotoxicity. The cell culture surfaces used in the current study were made of MWCNT immobilized in a high-density polyethylene substrate, differentiating it from most studies of MWCNT cytotoxicity. The purity, chemical functionalization, and immobilization of MWCNT were evaluated to elucidate their effect on MWCNT behavior relative to controls. While purity was found not to be significant in determining the behavior of cells on MWCNT relative to standard controls, the presence of carboxyl functional groups was generally associated with reduced cell metabolic activity, proliferation, and differentiation as measured using the MTS assay, nucleic acid incorporation, and alkaline phosphatase expression, respectively. This study demonstrates that the culture of osteogenic cells on surfaces made of nonfunctionalized and immobilized MWCNT is associated with a level of cell growth and differentiation comparable to those of standard tissue culture controls.

Equitoxic Doses of 5-Azacytidine and 5-Aza-2′Deoxycytidine Induce Diverse Immediate and Overlapping Heritable Changes in the Transcriptome

BackgroundThe hypomethylating agent 5-Azacytidine (5-Aza-CR) is the first drug to prolong overall survival in patients with myelodysplastic syndrome (MDS). Surprisingly, the deoxyribonucleoside analog 5-Aza-2′deoxycytidine (5-Aza-CdR) did not have a similar effect on survival in a large clinical trial. Both drugs are thought to exert their effects after incorporation into DNA by covalent binding of DNA methyltransferase (DNMT). While 5-Aza-CdR is incorporated into only DNA, 5-Aza-CR is also incorporated into RNA. Here, we have analyzed whether this difference in nucleic acid incorporation may influence the capacities of these drugs to regulate the expression of mRNA and microRNAs (miRNA), which may potentially affect the activities of the drugs in patients.Methodology/Principal FindingsA hematopoietic (HL-60; acute myeloid leukemia) and a solid (T24; transitional cell carcinoma) cancer cell line were treated with equitoxic doses of 5-Aza-CR and 5-Aza-CdR for 24 hrs, and the immediate (day 2) and lasting (day 8) effects on RNA expression examined. There was considerable overlap between the RNAs heritably upregulated by both drugs on day 8 but more RNAs were stably induced by the deoxy analog. Both drugs strongly induced expression of cancer testis antigens. On day 2 more RNAs were downregulated by 5-Aza-CR, particularly at higher doses. A remarkable downregulation of miRNAs and a significant upregulation of tRNA synthetases and other genes involved in amino acid metabolism was observed in T24 cells.Conclusions/SignificanceOverall, this suggests that significant differences exist in the immediate action of the two drugs, however the dominant pattern of the lasting, and possible heritable changes, is overlapping.

New insights into the pharmacology and cytotoxicity of gemcitabine and 2′,2′-difluorodeoxyuridine

In a clinical study with oral gemcitabine (2',2'-difluorodeoxycytidine, dFdC), 2',2'-difluorodeoxyuridine (dFdU) was extensively formed and accumulated after multiple oral dosing. Here, we have investigated the in vitro cytotoxicity, cellular uptake, efflux, biotransformation, and nucleic acid incorporation of dFdC and dFdU. Short-term and long-term cytotoxicity assays were used to assess the cytotoxicity of dFdC and dFdU in human hepatocellular carcinoma HepG2, human lung carcinoma A549, and Madin-Darby canine kidney cell lines transfected with the human concentrative or equilibrative nucleoside transporter 1 (hCNT1 or hENT1), or empty vector. Radiolabeled dFdC and dFdU were used to determine cellular uptake, efflux, biotransformation, and incorporation into DNA and RNA. The compounds dFdC, dFdU, and their phosphorylated metabolites were quantified by high-performance liquid chromatography with UV and radioisotope detection. dFdU monophosphate, diphosphate, and triphosphate (dFdU-TP) were formed from dFdC and dFdU. dFdU-TP was incorporated into DNA and RNA. The area under the intracellular concentration-time curve of dFdC-TP and dFdU-TP and their extent of incorporation into DNA and RNA inversely correlated with the IC(50) of dFdC and dFdU, respectively. The cellular uptake and cytotoxicity of dFdU were significantly enhanced by hCNT1. dFdU inhibited cell cycle progression and its cytotoxicity significantly increased with longer duration of exposure. dFdU is taken up into cells with high affinity by hCNT1 and phosphorylated to its dFdU-TP metabolite. dFdU-TP is incorporated into DNA and RNA, which correlated with dFdU cytotoxicity. These data provide strong evidence that dFdU can significantly contribute to the cytotoxicity of dFdC.

Effects of oxaliplatin and CPT-11 on cytotoxicity and nucleic acid incorporation of the fluoropyrimidines.

The addition of oxaliplatin or CPT-11 to 5-FU has become common practice in the treatment of colorectal cancer. It is not known, however, which fluoropyrimidine drug (5-FU, FUdR, or FUR) will produce superior cytotoxicity when combined with either oxaliplatin or CPT-11. The purpose of the study was to determine the effects of oxaliplatin and CPT-11 on cytotoxicity and nucleic acid incorporation of all three fluoropyrimidines. HT-29 cells were exposed for 2 h to IC(10), IC(30), and IC(70) of oxaliplatin and CPT-11. Subsequently, cells were exposed for 24 h to IC(10), IC(30), and IC(70) of 5-FU, FUdR, and FUR. Cytotoxicity was measured by the MTT assay. Nucleic acid incorporation of [(3)H]fluoropyrimidine was then compared in the presence and absence of oxaliplatin or CPT-11 pretreatment. Synergistic cytotoxicity was displayed when IC(30) of oxaliplatin or CPT-11 was combined with IC(10) and IC(30) of the fluoropyrimidines. One fluoropyrimidine did not achieve superior cytotoxicity over the others. After pretreatment with oxaliplatin or CPT-11, cytotoxic antagonism was observed as the concentration of a fluoropyrimidine increased up to IC(70). The increasing cytotoxic antagonism correlated with decreases in fluoropyrimidine nucleic acid incorporation. The most significant incorporation difference existed within the 5-FU treated group. No single fluoropyrimidine is more cytotoxically effective over the others when combined with oxaliplatin or CPT-11. Correlation of cytotoxic antagonism to the inhibition of fluoropyrimidine nucleic acid incorporation implies difficulties in drug transport and/or metabolism only after oxaliplatin or CPT-11 pretreatment.

Towards proto-cells: "primitive" lipid vesicles encapsulating giant DNA and its histone complex.

An important prebiotic event was probably the incorporation of nucleic acids by lipid vesicles. This process was studied with artificial model cells and fluorescent probes. Pictures 1–4 represent a time series of fluorescence microscopic images of T4 DNA molecules (166 kb) incorporated into giant vesicles of 5 μm diameter. The entrapped DNA molecules are in a coiled state.

Fas/Apo [Apoptosis]-1 and Associated Proteins in the Differentiating Cerebral Cortex: Induction of Caspase-Dependent Cell Death and Activation of NF-κB

The developing cerebral cortex undergoes a period of substantial cell death. The present studies examine the role of the suicide receptor Fas/Apo[apoptosis]-1 in cerebral cortical development. Fas mRNA and protein are transiently expressed in subsets of cells within the developing rat cerebral cortex during the peak period of apoptosis. Fas-immunoreactive cells were localized in close proximity to Fas ligand (FasL)-expressing cells. The Fas-associated signaling protein receptor interacting protein (RIP) was expressed by some Fas-expressing cells, whereas Fas-associated death domain (FADD) was undetectable in the early postnatal cerebral cortex. FLICE-inhibitory protein (FLIP), an inhibitor of Fas activation, was also expressed in the postnatal cerebral cortex. Fas expression was more ubiquitous in embryonic cortical neuroblasts in dissociated culture compared to in situ within the developing brain, suggesting that the environmental milieu partly suppresses Fas expression at this developmental stage. Furthermore, FADD, RIP, and FLIP were also expressed by subsets of dissociated cortical neuroblasts in culture. Fas activation by ligand (FasL) or anti-Fas antibody induced caspase-dependent cell death in primary embryonic cortical neuroblast cultures. The activation of Fas was also accompanied by a rapid downregulation of Fas receptor expression, non-cell cycle-related incorporation of nucleic acids and nuclear translocation of the RelA/p65 subunit of the transcription factor NF-kappaB. Together, these data suggest that adult cortical cell number may be established, in part, by an active process of receptor-mediated cell suicide, initiated in situ by killer (FasL-expressing) cells and that Fas may have functions in addition to suicide in the developing brain.

Detection of DNA hybridization using self‐assembled bilayer lipid membranes (BLMs)

AbstractThe present work reports the use of self assembled bilayer lipid membranes supported on the surface of a metal (s‐BLMs) as transducers for the direct electrochemical monitoring of DNA hybridization. Characterized oligomers based on single stranded deoxyribonucleic acid (ssDNA) thymidylic acid icosanucleotides that were terminated with C16 (dT20‐C16) and deoxyadenylic acid icosanucleotides (dA20) were used for the hybridization procedure at the lipid membrane surface. The decrease of the ion conductivity observed during the hybridization procedure is indicative of the formation of the duplex, which may subsequently be excluded from the membrane. The incorporation of nucleic acids into the lipid matrix was investigated by differential scanning calorimetric (DSC) experiments. The present simple electrochemical biosensor for monitoring of DNA hybridization was regenerable for multiple cycles of application. This approach provides low detection limits for DNA (a few hundreds of fmol), fast response times (on the order of a few minutes), mild conditions of hybridization and capability of analyzing small volumes of sample.

Uptake, Transport and Metabolism of Exogenous Nucleosides in Intestinal Epithelial Cell Cultures

The uptake and transport of nucleosides (adenosine, cytidine, guanosine, thymidine, uridine and inosine) were examined in intestinal cell lines (Caco-2 and IEC-6). Well-differentiated Caco-2 cells took up significantly more uridine and thymidine than less-differentiated cells over 24 h. These differences were reflected in the resulting trichloroacetic acid-soluble (nucleotide, nucleoside and nucleobase) pool sizes rather than in differences in nucleic acid incorporation. IEC-6 cells have smaller soluble pools than Caco-2 cells, and the uptake of nucleosides over 60 min reflected this difference. Caco-2 cells transported significantly more thymidine, cytidine, guanosine, adenosine and inosine into acid-soluble pools than IEC-6 cells. Caco-2 cells were grown on filters to examine the transcellular transport of nucleosides and to examine the chemical form in which nucleosides appeared at the basolateral aspect of the cell monolayer. The pattern of transport varied for individual nucleosides. The transport of cytidine and guanosine was significantly greater in the apical-to-basolateral direction than in the opposite direction over 2 h. No purine nucleoside was transported intact across the Caco-2 cell monolayer in either direction, with the majority of the transported material appearing as nucleobases. Nucleobases also represented the main metabolites of pyrimidines transported. However, some cytidine and uridine were detectable, but each constituted < 20% of transported material. Nucleosides are therefore transported by enterocytes but metabolized during transport.

Use of radioactive ethanolamine incorporation into phospholipids to assess in vitro antimalarial activity by the semiautomated microdilution technique.

Phospholipid biosynthetic activity is intense in the erythrocytic stage of Plasmodium falciparum because of the parasite's own enzymatic machinery. The incorporation of various labeled phospholipid precursors in comparison with the incorporation of nucleic acid and protein precursors was tested to evaluate P. falciparum growth in vitro. These precursors, namely, [3H]ethanolamine, [3H]hypoxanthine, [3H]palmitate, [14C]serine, [3H]choline, [3H]inositol, and [3H]isoleucine, were all accurate indicators of parasite growth. However, because of its high level of incorporation, [3H]ethanolamine proved to be the best tool for assessing parasite viability. When culture parameters were carefully controlled, [3H]ethanolamine incorporation into phospholipids was proportional to pulse time, precursor concentration, and initial parasitemia and was sensitive to the number of uninfected erythrocytes (hematocrit). It can be used for a wide range of infected erythrocytes, from 2 x 10(4) to 5 x 10(5). The use of [3H]ethanolamine for in vitro antimalarial drug screening is a good alternative to the method of Desjardins et al. (R. E. Desjardins, C. J. Canfield, J. D. Haynes, and J. D. Chulay, Antimicrob. Agents. Chemother. 16:710-718, 1979). The major advantage is that the culture medium can be supplemented with hypoxanthine, which results in better parasite growth. [3H]ethanolamine is also an ideal tool when compounds that interfere with DNA and/or RNA metabolism are to be investigated for their effect on Plasmodium growth.

Experimental Evaluation of Conversion Factors for the [ 3 H]Thymidine Incorporation Assay of Bacterial Secondary Productivity

The relationship between bacterial growth and incorporation of [methyl-H]thymidine in oligotrophic lake water cultures was investigated. Prescreening, dilution, and addition of organic and inorganic nutrients were treatments used to prevent bacterivory and stimulate bacterial growth. Growth in unmanipulated samples was estimated through separate measurements of grazing losses. Both bacterial number and biovolume growth responses were measured, and incorporation of [H]thymidine in both total macromolecules and nucleic acids was assayed. The treatments had significant effects on conversion factors used to relate thymidine incorporation to bacterial growth. Cell number-based factors ranged from 1.1 x 10 to 38 x 10 cells mol of total thymidine incorporation and varied with treatment up to 10-fold for the same initial bacterial assemblage. In contrast, cell biovolume-based conversion factors were similar for two treatment groups across a 16-fold range of [H]thymidine incorporation rates: 5.54 x 10 mum mol of total thymidine incorporation and 15.2 x 10 mum mol of nucleic acid incorporation. Much of the variation in cell number-based conversion factors was related to changes in apparent mean cell volume of produced bacteria. Phosphorus addition stimulated [H]thymidine incorporation more than it increased bacterial growth, which resulted in low conversion factors.

Incorporation of nucleic acid and protein precursors in enriched populations of proliferating normal and leukemic cells

Nucleic acid and protein synthesis was determined in immature myeloid cells isolated from seven normal bone marrow samples and compared to blasts from nine patients with full-blown acute non-lymphocytic leukemia. Samples obtained from normal bone marrow contained a mean of 82% immature myeloid cells with 29.9% S-phase cells. These cells incorporated [ 3H]thymidine with a mean of 17,500 counts/min [ 3H]uridine with a mean of 24,000 counts/min and [ 3H]leucine with a mean of 2100 counts/min per 0.2 × 10 6 cells. Leukemic bone marrow cells could be separated in fractions with different proliferative activities. Leukemic samples with a mean of 3.6% S-phase cells incorporated [ 3H]thymidine with a mean of 1100 counts/min, [ 3H]uridine with a mean of 15,700 counts/min and [ 3H]leucine with a mean of 2600 counts/min per 0.2 × 10 6 cells. For leukemic samples with a mean of 30.6% S-phase cells these values were: [ 3H]thymidine 22,200 counts/min, [ 3H]uridine 49,700 counts/min and [ 3H]leucine 6700 counts/min per 0.2 × 10 6 cells. The incorporation studies were carried out for the first time in normal and leukemic cells with a comparable proliferative activity. Non-lymphocytic leukemic blastic cells showed a two-fold increase in RNA synthesis and a three-fold increase in protein synthesis compared to enriched samples of normal early myeloid cells with the same proliferative activity.

258) - Effect of 60-F product from streptococcus hemolyticus (Su-strain) on incorporation of nucleic acid and protein precursors into Ehrlich carcinoma cells in vitro

258) - Effect of 60-F product from streptococcus hemolyticus (Su-strain) on incorporation of nucleic acid and protein precursors into Ehrlich carcinoma cells in vitro

Nucleases activity in different segments of the human digestive tube compared to the incidence of carcinomas (histochemical study).

The alkaline and acid DNase and RNase activity was histochemically investigated in biopsies from the human digestive tube. Activity of these enzymes in the mucosal epithelium in different segments of the digestive tube was compared to the statistical incidence of malignant tumors deriving from this tissue (carcinomas). It was found that the alkaline and acid nucleases activity was very intense in small intestine precisely in this segment where the incidence of carcinomas was low, whereas the low activity of these enzymes in the stomach and large intestine corresponded to the high incidence of carcinomas. This observation confirmed our previously elaborated hypothesis, according to which the low activity of nucleases in normal tissues appeared to be a predisposing factor for malignant transformation. It could be also supposed that the nucleases constitute some kind of double barrier mechanism protecting the genetical stability of the cell against foreign nucleic acid incorporation or production; alkaline nucleases being an extracellular and acid nucleases an intracellular barrier.

An autoradiographic comparison of the effects of enucleation and actinomycin D on the incorporation of nucleic acid and protein precursors by Acetabularia chloroplasts

Surgical enucleation of the giant, unicellular alga ( Acetabularia mediterranea) eliminates the genetic material of the nucleus, while treatment with actinomycin D is presumed to prevent the transcription of the genetic material within the chloroplasts as well as that of the nucleus. An autoradiographic comparison of the effects of both treatments on the incorporation of radioactive thymidine, uridine and leucine by the chloroplasts gave the following results. 1. 1. Enucleation does not prevent the incorporation of thymidine into the DNA of the chloroplasts. 2. 2. Enucleation has little effect on the incorporation of uridine into the RNA of the chloroplasts while actinomycin abolished this incorporation in both normal and enucleate plants. 3. 3. Enucleation has only a minor effect on the incorporation of leucine by the chloroplasts while actinomycin greatly reduced such incorporation in normal and enucleate plants. These results are compatible with the hypothesis of almost complete autonomy of chloroplasts in replicating their DNA and in the production of most, if not all, plastid RNA and protein species from this DNA template.