Adsorption Of Nucleic Acids Research Articles

Adsorption of nucleotides on biomimetic apatite: The case of adenosine 5′ monophosphate (AMP)

This work investigates the interaction between the nucleotide adenosine 5′ monophosphate molecule (AMP) and a biomimetic nanocrystalline carbonated apatite as a model for bone mineral. The analogy of the apatite phase used in this work with biological apatite was first pointed out by complementary techniques. AMP adsorption isotherms were then investigated. Obtained data were fitted to a Sips isotherm with an exponent greater than one suggesting positive cooperativity among adsorbed molecules. The data were compared to a previous study relative to the adsorption of another nucleotide, cytidine monophosphate (CMP) onto a similar substrate, evidencing some effect of the chemical nature of the nucleic base. An enhanced adsorption was observed under acidic (pH 6) conditions as opposed to pH 7.4, which parallels the case of DNA adsorption on biomimetic apatite. An estimated standard Gibbs free energy associated to the adsorption process (ΔG°ads≅−22kJ/mol) intermediate between “physisorption” and “chemisorption” was found. The analysis of the solids after adsorption pointed to the preservation of the main characteristics of the apatite substrate but shifts or enhancements of Raman bands attributed to AMP showed the existence of chemical interactions involving both the phosphate and adenine parts of AMP. This contribution adds to the works conducted in view of better understanding the interaction of DNA/RNA and their constitutive nucleotides and the surface of biomimetic apatites. It could prove helpful in disciplines such as bone diagenesis (DNA/apatite interface in aged bones) or nanomedicine (setup of DNA- or RNA-loaded apatite systems). Also, the adsorption of nucleic acids on minerals like apatites could have played a role in the preservation of such biomolecules in the varying conditions known to exist at the origin of life on Earth, underlining the importance of dedicated adsorption studies.

A Prebiotic Chemistry Experiment on the Adsorption of Nucleic Acids Bases onto a Natural Zeolite.

There are currently few mechanisms that can explain how nucleic acid bases were synthesized, concentrated from dilute solutions, and/or protected against degradation by UV radiation or hydrolysis on the prebiotic Earth. A natural zeolite exhibited the potential to adsorb adenine, cytosine, thymine, and uracil over a range of pH, with greater adsorption of adenine and cytosine at acidic pH. Adsorption of all nucleic acid bases was decreased in artificial seawater compared to water, likely due to cation complexation. Furthermore, adsorption of adenine appeared to protect natural zeolite from thermal degradation. The C=O groups from thymine, cytosine and uracil appeared to assist the dissolution of the mineral while the NH2 group from adenine had no effect. As shown by FT-IR spectroscopy, adenine interacted with a natural zeolite through the NH2 group, and cytosine through the C=O group. A pseudo-second-order model best described the kinetics of adenine adsorption, which occurred faster in artificial seawaters.

Inverted critical adsorption of polyelectrolytes in confinement.

What are the fundamental laws for the adsorption of charged polymers onto oppositely charged surfaces, for convex, planar, and concave geometries? This question is at the heart of surface coating applications, various complex formation phenomena, as well as in the context of cellular and viral biophysics. It has been a long-standing challenge in theoretical polymer physics; for realistic systems the quantitative understanding is however often achievable only by computer simulations. In this study, we present the findings of such extensive Monte-Carlo in silico experiments for polymer-surface adsorption in confined domains. We study the inverted critical adsorption of finite-length polyelectrolytes in three fundamental geometries: planar slit, cylindrical pore, and spherical cavity. The scaling relations extracted from simulations for the critical surface charge density σc-defining the adsorption-desorption transition-are in excellent agreement with our analytical calculations based on the ground-state analysis of the Edwards equation. In particular, we confirm the magnitude and scaling of σc for the concave interfaces versus the Debye screening length 1/κ and the extent of confinement a for these three interfaces for small κa values. For large κa the critical adsorption condition approaches the known planar limit. The transition between the two regimes takes place when the radius of surface curvature or half of the slit thickness a is of the order of 1/κ. We also rationalize how σc(κ) dependence gets modified for semi-flexible versus flexible chains under external confinement. We examine the implications of the chain length for critical adsorption-the effect often hard to tackle theoretically-putting an emphasis on polymers inside attractive spherical cavities. The applications of our findings to some biological systems are discussed, for instance the adsorption of nucleic acids onto the inner surfaces of cylindrical and spherical viral capsids.

Boronate affinity nanoparticles for nucleoside separation

Boronate affinity systems have been recently used for the specific isolation of cis-diol group carrying biomolecules such as glycoproteins, nucleosides, carbohydrates. Nanosized materials have been extremely used for the biotechnological purposes due to their unique properties and their high surface areas. The objective of this presented work was to develop a new boronate affinity system for the nucleoside adsorption. For this purpose, poly(HEMA) nanoparticles were synthesized by using surfactant free emulsion polymerization technique and then functionalized with phenylboronic acid. Synthesized nanoparticles were characterized with FTIR, SEM, and Zeta size analysis. Nucleic acid adsorption experiments were repeated for different medium pH values, for various nucleosides concentrations, for different temperatures and ionic strengths, in order to determine the optimum adsorption conditions. In the light of these studies, it can be concluded that this boronate ligand carrying nanoparticles were very valuable for the separation of nucleosides.

Improved yield of high molecular weight DNA coincides with increased microbial diversity access from iron oxide cemented sub-surface clay environments.

Despite over three decades of progress, extraction of high molecular weight (HMW) DNA from high clay soils or iron oxide cemented clay has remained challenging. HMW DNA is desirable for next generation sequencing as it yields the most comprehensive coverage. Several DNA extraction procedures were compared from samples that exhibit strong nucleic acid adsorption. pH manipulation or use of alternative ion solutions offered no improvement in nucleic acid recovery. Lysis by liquid N2 grinding in concentrated guanidine followed by concentrated sodium phosphate extraction supported HMW DNA recovery from clays high in iron oxides. DNA recovered using 1 M sodium phosphate buffer (PB) as a competitive desorptive wash was 15.22±2.33 µg DNA/g clay, with most DNA consisting of >20 Kb fragments, compared to 2.46±0.25 µg DNA/g clay with the Powerlyzer system (MoBio). Increasing PB concentration in the lysis reagent coincided with increasing DNA fragment length during initial extraction. Rarefaction plots of 16S rRNA (V1–V3 region) pyrosequencing from A-horizon and clay soils showed an ∼80% and ∼400% larger accessed diversity compared to the Powerlyzer soil DNA system, respectively. The observed diversity from the Firmicutes showed the strongest increase with >3-fold more operational taxonomic units (OTU) recovered.

Nano-graphene oxide as a novel platform for monitoring the effect of LNA modification on nucleic acid interactions

Graphene oxide has gained significant attention due to its exceptional physical properties at biological interfaces. It has extraordinary quenching, fast adsorption and desorption properties that are suitable for detection of molecular interactions in nucleic acids. Here we studied the interaction between locked nucleic acid (LNA) modified oligonucleotides and its complementary miR-10b DNA analog. We demonstrate that LNA modification does not alter the hybridization yield, despite a slight difference in the rate, however it does increase the duplex stability dramatically. The noncovalent nucleic acid-graphene oxide complex maintained the stability between 25 and 90 °C in the absence of oligonucleotide-induced desorption. The melting temperatures of duplexes with or without LNA base modification were determined due to remarkable fluorescence quenching and fast oligonucleotide adsorption with graphene oxide. The difference in melting temperatures was used to control the release of surface adsorbed nucleic acids at 70 °C. Finally, a mutation in the oligonucleotide sequence is detected by the complementary oligonucleotides on the graphene oxide surface. Due to its extraordinary physical properties, graphene oxide represents a remarkable platform for studying nucleic acid interactions and serves as a promising material for biomedical applications.

Optimization of influencing factors of nucleic acid adsorption onto silica-coated magnetic particles: Application to viral nucleic acid extraction from serum

We present a detailed study of nucleic acid adsorption onto silica-coated magnetic particles in the presence of guanidinium thiocyanate, and extraction of nucleic acid from two important transfusion-transmitted viruses using these particles. Silica-coated magnetic particles were prepared by encapsulating Fe3O4 nanoparticles with tetraethylorthosilicate (TEOS) hydrolysis. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS) and vibrating sample magnetometer (VSM) were used for particle characterization. The results indicate that silica-coated magnetic particles are spheroid with a narrow hydrodynamic size distribution of about 500nm. VSM data indicates that these particles display paramagnetic behavior with saturation magnetization of about 30emu/g. The adsorption capacities were evaluated with DNA from salmon sperm and RNA of Escherichia coli strain JM109 in the presence of guanidinium thiocyanate. The maximum of adsorption is up to 10.6mg DNA or 7.7mg RNA per 1g of silica-coated magnetic particles with 4M guanidinium thiocyanate (GTC) at pH 5.5 without adding ethanol. The influencing factors were analyzed in term of the adsorption of nucleic acids onto silica-coated magnetic particles. The adsorption capacity in acidic condition is found to be larger than that in alkaline condition and increases with adding equivalent volume of ethanol. A simple method was therefore established to extract nucleic acids of two important transfusion-transmitted viruses from serum and compared with the commercial kits. The results indicate that the extraction method based on silica-coated magnetic particles can be adapted to rapidly and facilely isolate viral nucleic acid for diagnosis of viral infection from serum within 30min, irrespective of genome compositions of virus.

Improving Griffith's protocol for co-extraction of microbial DNA and RNA in adsorptive soils

Quantification of microbial gene expression is increasingly being used to study key functions in soil microbial communities, yet major limitations still exist for efficient extraction of nucleic acids, especially RNA for transcript analysis, from this complex matrix. We present an improved extraction protocol that was optimized by: i) including an adsorption-site competitor prior to cell lysis to decrease adsorption of nucleic acids to soil particles, and ii) optimizing the PEG concentration used for nucleic acid precipitation. The extraction efficiency was determined using quantitative real-time PCR on both the RNA (after conversion to cDNA) and the DNA fraction of the extracts. Non-adsorptive soils were characterized by low clay content and/or high phosphate content, whereas adsorptive soils had clay contents above 20% and/or a strong presence of divalent Ca2+ in combination with high pH. Modifications to the co-extraction protocol improved nucleic acid extraction efficiency from all adsorptive soils and were successfully validated by DGGE analysis of the indigenous community based on 16S rRNA gene and transcripts in soils representing low biomass and/or high clay content. This new approach reveals a robust co-extraction protocol for a range of molecular analysis of diverse soil environments.

Bovine Serum Albumin-Based Probe Carrier Platform for Electrochemical DNA Biosensing

A bovine serum albumin (BSA)-monolayer-based probe carrier platform is shown to improve the performance of a conventional thiolated single-stranded DNA probe self-assembled-monolayer-based electrochemical DNA hybridization biosensor. A detection limit of 0.5 fM can be obtained in a very reproducible manner (relative standard deviation <5%), along with high specificity. The performance of the biosensor can be attributed primarily to the enhanced spatial positioning range and accessibility of the probes on the BSA-based platform. Furthermore, the novel biosensor shows high resistance to nonspecific adsorption of nucleic acid and protein and can be directly employed in detection in biological fluids. These advantages give this simple developed methodology great promise for a wide range of nucleic acid testing.

RNA and DNA Association to Zwitterionic and Charged Monolayers at the Air–Liquid Interface

The objective of this work is to establish under which conditions short RNA molecules (similar to miRNA) associate with zwitterionic phospholipids and how this differs from the association with cationic surfactants. We study how the base pairing (i.e., single stranded versus double stranded nucleic acids) and the length of the nucleic acid and the charge of the lipid/surfactant monolayer affect the association behavior. For this purpose, we study the adsorption of nucleic acids to monolayers composed of dipalmitoyl phosphatidylcholine (DPPC) or dioctadecyl-dimethyl-ammoniumbromide (DODAB) using the surface film balance, neutron reflectometry, and fluorescence microscopy. The monolayer studies with the surface film balance suggested that short single-stranded ssRNA associates with liquid expanded zwitterionic phospholipid monolayers, whereas less or no association is detected for double-stranded dsRNA and dsDNA. In order to quantify the interaction and to determine the location of the nucleic acid in the lipid/surfactant monolayer we performed neutron reflectometry measurements. It was shown that ssRNA adsorbs to and penetrates the liquid expanded monolayers, whereas there is no penetration of nucleic acids into the liquid condensed monolayer. No adsorption was detected for dsDNA to zwitterionic monolayers. On the basis of these results, we propose that the association is driven by the hydrophobic interactions between the exposed hydrophobic bases of the ssRNA and the hydrocarbon chains of the phospholipids. The addition of ssRNA also influences domain formation in the DPPC monolayer, leading to fractal-like interconnected domains. The experimental results are discussed in terms of the implication for biological processes and new leads for applications in medicine and biotechnology.

Sensitive life detection strategies for low-biomass environments: optimizing extraction of nucleic acids adsorbing to terrestrial and Mars analogue minerals

The adsorption of nucleic acids to mineral matrixes can result in low extraction yields and negatively influences molecular microbial ecology studies, in particular for low-biomass environments on Earth and Mars. We determined the recovery of nucleic acids from a range of minerals relevant to Earth and Mars. Clay minerals, but also other silicates and nonsilicates, showed very low recovery (< 1%). Consequently, optimization of DNA extraction was directed towards clays. The high temperatures and acidic conditions used in some methods to dissolve mineral matrices proved to destruct DNA. The most efficient method comprised a high phosphate solution (P/EtOH; 1 M phosphate, 15% ethanol buffer at pH 8) introduced at the cell-lysing step in DNA extraction, to promote chemical competition with DNA for adsorption sites. This solution increased DNA yield from clay samples spiked with known quantities of cells up to nearly 100-fold. DNA recovery was also enhanced from several mineral samples retrieved from an aquifer, while maintaining reproducible DGGE profiles. DGGE profiles were obtained for a clay sample for which no profile could be generated with the standard DNA isolation protocol. Mineralogy influenced microbial community composition. The method also proved suitable for the recovery of low molecular weight DNA (< 1.5 kb).

Freeze-dried cationic calcium phosphatenanorods as versatile carriers of nucleic acids (DNA, siRNA)

Functionalized calcium phosphate (hydroxyapatite) nanorods were freeze-dried in the presence of the cryoprotectant trehalose, giving a storable and easily redispersible system which can adsorb nucleic acids for transfection and gene silencing. The nanorods were first surface-functionalized with a layer of polyethyleneimine (PEI), purified by ultracentrifugation and redispersion, and freeze-dried in the presence of trehalose. The nanorods can be easily redispersed in water. The concentration-dependent adsorption of nucleic acids (DNA and siRNA) onto the surface of the redispersed cationic nanorods was measured by dynamic light scattering (particle diameter and zeta potential). The transfection efficiency on epithelial cells (HeLa) and osteoblasts (MG-63) was systematically determined for increasing amounts of added nucleic acid, up to a charge reversal by the anionic nucleic acids. Both transfection and gene silencing efficiency increased with increasing amount of nucleic acid, but went through minimum around the point of zero charge where the particles agglomerated. The application of an additional outer layer of polyethyleneimine around the hydroxyapatite/PEI/nucleic acid-nanoparticles reversed the charge back to positive, resulting in even higher transfection rates with almost complete cell viability.

Magnetic silica spheres with large nanopores for nucleic acid adsorption and cellular uptake

Template assisted fabrication of magnetic silica nanospheres with large nanopores (MSNLP) and their adsorption and delivery of nucleic acids are reported in this paper. Silica spheres with controlled particle diameter (∼400 nm) and large nanopore size (13–24 nm) are prepared by using Brij56 as a template of mesopore, enabling incorporation of magnetic nanocrystals into the particles under mild neutral synthesis conditions. High resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), and field-dependent magnetisation measurements confirm that the magnetic nanocrystals have been encapsulated into the silica spheres. The saturation magnetisation values of the resulted magnetic-silica nanocomposites are tunable by adjusting the amount of Fe 3O 4 magnetic nanocrystals used in the synthesis process. The nitrogen sorption analysis reveals that mesopores with large pore size exist in the silica matrix. After functionalisation of the silica surface with poly-( l-lysine) (PLL), the nanoparticles show strong adsorption capacity (q m ranging from 10 to 22.5 μg/mg) for CpG DNA. We have further demonstrated successful delivery of miRNA into rat proximal tubular epithelial cells, facilitated by efficient cellular uptake of the nanocomposites. This work provides a convenient strategy to prepare MSNLP which can offer a versatile platform for biological applications such as simultaneous drug delivery and magnetic resonance imagining under external magnetic field.

Adsorption of nucleic acids on collodion films

Adsorption of nucleic acids on collodion films from solutions of different ionic strengths was studied, and the adsorption equilibrium constants were determined. The primary adsorption of DNA samples on collodion films was examined depending on various factors. A comparative analysis of different techniques (vacuum drying, UV irradiation) employed for immobilization of DNA samples on collodion films was carried out.

Adsorption of Nucleic Acid Components on Rutile (TiO2) Surfaces

Nucleic acids, the storage molecules of genetic information, are composed of repeating polymers of ribonucleotides (in RNA) or deoxyribonucleotides (in DNA), which are themselves composed of a phosphate moiety, a sugar moiety, and a nitrogenous base. The interactions between these components and mineral surfaces are important because there is a tremendous flux of nucleic acids in the environment due to cell death and horizontal gene transfer. The adsorption of mono-, oligo-, and polynucleotides and their components on mineral surfaces may have been important for the origin of life. We have studied here interactions of nucleic acid components with rutile (TiO(2)), a mineral common in many terrestrial crustal rocks. Our results suggest roles for several nucleic acid functional groups (including sugar hydroxyl groups, the phosphate group, and extracyclic functional groups on the bases) in binding, in agreement with results obtained from studies of other minerals. In contrast with recent studies of nucleotide adsorption on ZnO, aluminum oxides, and hematite, our results suggest a different preferred orientation for the monomers on rutile surfaces. The conformations of the molecules bound to rutile surfaces appear to favor specific interactions, which in turn may allow identification of the most favorable mineral surfaces for nucleic acid adsorption.

Nucleic Acids in Protein Samples Interfere with Phosphopeptide Identification by Immobilized-Metal-Ion Affinity Chromatography and Mass Spectrometry

Immobilized-metal-ion affinity chromatography (IMAC) is used extensively for phosphopeptide enrichment in phosphoproteomics. However, the effect of nucleic acids in protein samples on phosphopeptide enrichment by IMAC has not yet been well clarified. In this study, we demonstrate that IMAC beads possess a strong adsorption of nucleic acids, especially single-stranded or single-stranded-region-containing nucleic acids, leading to approximately 50% loss of phosphopeptides during the process of IMAC enrichment. Therefore, nucleic acids must be removed from protein samples prior to IMAC. Acetonitrile (ACN) precipitation, a simple and efficient procedure, was established to remove nucleic acids from the protein samples. We showed that ACN precipitation approximately doubled the phosphopeptide number identified by IMAC and mass spectrometry, indicating that nucleic acid removal significantly improves the identification of phosphopeptides.

Biochemical applications of surface-enhanced infrared absorption spectroscopy

An overview is presented on the application of surface-enhanced infrared absorption (SEIRA) spectroscopy to biochemical problems. Use of SEIRA results in high surface sensitivity by enhancing the signal of the adsorbed molecule by approximately two orders of magnitude and has the potential to enable new studies, from fundamental aspects to applied sciences. This report surveys studies of DNA and nucleic acid adsorption to gold surfaces, development of immunoassays, electron transfer between metal electrodes and proteins, and protein–protein interactions. Because signal enhancement in SEIRA uses surface properties of the nano-structured metal, the biomaterial must be tethered to the metal without hampering its functionality. Because many biochemical reactions proceed vectorially, their functionality depends on proper orientation of the biomaterial. Thus, surface-modification techniques are addressed that enable control of the proper orientation of proteins on the metal surface.FigureSurface enhanced infrared absorption spectroscopy (SEIRAS) on the studies of tethered protein monolayer (cytochrome c oxidase and cytochrome c) on gold substrate (left), and its potential induced surface enhanced infrared difference absorption (SEIDA) spectrum

Modeling of nucleic acid adsorption on 3D prisms in microchannels

Understanding nucleic acid adsorption in microchannels is critical to improve the efficiency of purifying and extracting nucleic acid (NA) from sample solutions by microfluidic technologies. Using a microchannel with 3D prismatic silica elements on the wall can dramatically increase the surface area-to-volume ratio, and hence facilitate the nucleic acid adsorption on the wall. In this study a theoretical model for modeling adsorption in a microchannel with a designed 3D surface structure was developed, and five dimensionless numbers were found to be the key parameters in the adsorption process. Extensive numerical simulations were conducted. Two flow modes, the electroosmotic flow (EOF) and pressure-driven flow (PDF), were investigated for their effect on the adsorption. It was found that the EOF is more desirable than PDF. The 3D prismatic elements can increases the NA molecule adsorption not only by providing more surface areas, but also by the induced pressure resisting the central bulk electroosmotic flow. Finally, the effects of adsorption kinetic parameters (i.e., the kinetic association/dissociation constants, the diffusion coefficient, the total site density, the loading concentration, and the channel height), on the adsorption process were discussed in detail.

Water‐elutability of nucleic acids from metal‐chelate affinity adsorbents: enhancement by control of surface charge density

Immobilized metal affinity chromatography (IMAC) is widely used for purification of proteins, especially "hexahistidine-tagged" recombinant proteins. We previously demonstrated the application of IMAC to selective capture of nucleic acids, including RNA, selectively-denatured genomic DNA, and PCR primers through interactions with purine bases exposed in single-stranded regions. We also found that the binding affinity of nucleic acids for IMAC adsorbents can be increased several-fold by addition of 20 volume% of neutral additives such as ethanol or DMSO. In the present work, it is demonstrated that bound nucleic acids can be effectively eluted with water instead of the usual imidazole-containing competitive eluants, when the surface density of negative charges is enhanced by operation at alkaline pH, or by deliberate metal-underloading of the anionic chelating ligands. With enhanced negative surface charge density, nucleic acid adsorption can be made strongly dependent on the presence of adsorption-promoting additives and/or repulsion-shielding salts, and removal of these induces elution. Complete water-elutability is demonstrated for baker's yeast RNA bound to 10% Cu(II)- underloaded IDA Chelating Sepharose in a binding buffer of 20 mM HEPES, 240 mM NaCl, pH 7. Water elutability will significantly enhance the utility of IMAC in nucleic acid separations.

Neutral additives enhance the metal-chelate affinity adsorption of nucleic acids: Role of water activity

Immobilized metal-chelate affinity chromatography has been widely used in the purification of proteins, and we have recently found that it can also be applied to purification of nucleic acids through interactions involving exposed bases, especially purines. Here we report that the inclusion of moderate quantities of neutral solutes in the buffer substantially enhances the binding affinity of nucleic acids for immobilized metal-chelate affinity adsorbents. Addition of 20% (v/v) of solutes such as ethanol, methanol, isopropanol, n-propanol, and dimethyl sulfoxide enhances the initial affinity of binding of total yeast RNA by 4.4-, 3.8-, 3.7-, 3.0-, and 2.8-fold, respectively for Cu(II)–iminodiacetic acid (IDA) agarose adsorbent, and the weaker adsorption by Cu(II)–nitrilotriacetic acid (NTA) agarose was even more strongly enhanced. The adsorption affinities of the smaller oligodeoxynucleotide molecules A 20, G 20, C 20 and T 20 also increase with the addition of ethanol, suggesting that the effect is not significantly mediated by conformational changes. Binding enhancement generally correlates with reduction of water activity by the various solutes, as predicted by several models of solution thermodynamics, consistent with an entropic contribution by displacement of waters from the metal-chelate. Interestingly, the enhancement was not seen with the proteins bovine serum albumin and lysozyme.