• 972 Views
  • 371 Downloads
Research Article
The Open Access Journal of Science and Technology
Vol. 5 (2017), Article ID 101190, 18 pages
doi:10.11131/2017/101190

Adsorption and Elution of Nucleic Acids: Mesoporous Materials and Methods

Baochuan Lin1, Brandy J. Johnson1, Brian J. Melde1, Genevieve M. Haas2, Miles K. J. McConner3 and Jenna R. Taft4

1Center for Bio/Molecular Science & Engineering, US Naval Research Laboratory, Washington, DC, USA 20375

2Department of Biology, Northern Michigan University, Marquette, MI, USA 49855, at NRL § summers 2012 and 2013

3Department of Biology, Norfolk State University, Norfolk, VA, USA 23504, at NRL 1 summer 2014

4Formerly NOVA Research Incorporated, Alexandria, VA, USA 22308, currently Department of Chemistry, University of Vermont, Burlington, VT, USA 05405

Received 30 November 2015; Accepted 19 January 2017

Editors: Jung-Hua Kuo, Zhengguo Xiao, and Chunyi Zhi

Copyright © 2017 Baochuan Lin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Physical protection of nucleic acids from the environment for improved stability through encapsulation or adsorption has been reported using various materials, including liposomes, metal particles, mesoporous silica nanoparticles, and polymers. In an extension of that approach, our previous study demonstrated the potential of mesoporous silica nanoparticles with and without covalently attached stabilizing reagents, such as sugar and bovine serum albumin, for improving the stability of RNA. The goal of the current study is to evaluate the potential of silicate sorbents bearing chemical functionalities for stabilization of nucleic acid targets. Materials offering charged groups, metal chelating sites, and π-bonding sites are considered. Adsorption and elution of RNA, DNA, and single stranded DNA (ssDNA) are evaluated as is subsequent elution of the bound target. A sorbent functionalized with primary amine groups showed promising results for RNA and ssDNA stabilization. The impact of the sorbents on long term viability the targets is also evaluated. Storage of adsorbed targets at room temperature and 37C over a period of 200 d indicates the potential for stabilization of RNA and ssDNA using several of the functionalities. None of the sorbents improved the stability of DNA either under room temperature or 37C storage.

1. Introduction

Successful advancements in molecular diagnostics related to a wide range of fields, including medical, biological, environmental, forensics, and food safety, drive the need for preservation of nucleic acid integrity during sample collection, transportation, processing, and storage. The most common method for maintaining nucleic acid integrity is freezing at low temperature (−20C or −80C). This approach is not practical for routine specimen processing, storage, or shipping related to austere field conditions. Furthermore, the costs associated with maintaining large sets of samples under the necessary conditions over long periods of time can be prohibitive [1,2,3]. To address these issues, several technologies have been developed for the stabilization and storage of nucleic acids at room temperature. These technologies are primarily based on three principles. The first is anhydrobiosis, the dehydration process used by some organisms to survive extreme conditions [4,5]. Anhydrobiosis based methods are commonly used for DNA and include spray drying, spray-freeze-drying, air drying, and lyophilization with or without additives (i.e. trehalose) for preservation [6,7,8,9,10,11,12]. One study also indicated that anhydrobiosis worked well for RNA preservation [13]. While in the dry state, matrix components form a thermo-stable barrier around the DNA protecting the sample from damage and degradation. DNA can be recovered by rehydration as the matrix will completely dissolve [1,14]. The second approach to stabilization is to use chemicals or proteins to bind nucleic acids; this changes the characteristics of interactions with the nucleic acids to provide stability. Several chemicals and compounds have been reported to preserve nucleic acids at room temperature from periods of weeks to months. DNA-binding protein from starved cells (Dps) and poly(A) binding protein (Pab1p) were reported to stabilize DNA and mRNA, respectively [3,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30]. Commercial products, such as RNAlater and Trizol (ThermoFisher Scientific, Waltham, MA, USA), are based on this approach and have been documented to stabilize nucleic acids at room temperature for long periods of time [16,26,31,32,33,34].

Physically protecting nucleic acids from the environment, through encapsulation or adsorption onto a solid support, is the third of the stabilization principles and has emerged for the delivery of gene therapeutics. Materials, such as liposomes, metal particles, mesoporous silica nanoparticles, polymers, potato starch, silk fibron and surfactants, have been developed with these applications in mind [35,36,37,38,39,40,41,42,43,44,45,46,47]. In a previous study, we demonstrated the potential of RNA adsorption onto mesoporous silica nanoparticles (MSNs) with and without additional stabilizing reagents. The MSNs provided enhanced stability for extended periods allowing the adsorbed RNA to be eluted using simple buffers and employed directly for downstream molecular diagnostic assays. While promising, the RNA recovery rates from these MSNs were not ideal (less than 25%) [48]. The goal of the ongoing effort is to control interactions with the nucleic acids that result in degradation. Some of these interactions are restricted through adsorption or encapsulation; for example, the access of enzymes and microorganisms as well as the mobility of the nucleic acids. Other features of interest for the sorbents are altering solvent interactions, providing reducing sites and chelating groups, and inhibiting nuclease activity. While the previous study focused on common sugars and bovine serum albumin as stabilizing agents, the study presented here uses chemical functionalities incorporated into the sorbents to provide the potential for addressing other aspects of nucleic acid degradation. Binding and elution of RNA, DNA, and single stranded DNA (ssDNA) are evaluated. Elution methods are compared and the impact of the sorbents on long term sample viability is assessed.

2. Experimental Section

1,2-bis(trimethoxysilyl)ethane 96% (BTE), mesitylene (1,3,5-trimethylbenzene or TMB), 2-(N-morpholino)ethanesulfonic acid sodium salt (MES), cetyltrimethylammonium bromide (CTAB), PAMAM-25% C12 dendrimer (ethylenediamine core, generation 4.0 solution 10 wt. % in methanol), zinc acetate, copper chloride, chitosan (low molecular weight), N(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), tetraethyl orthosilicate (TEOS), tetramethyl orthosilicate 98% (TMOS), PluronicⓇP123 (P123), nitric acid 70%, and hydrochloric acid 37% were obtained from Sigma-Aldrich (St. Louis, MO, USA). Bis(trimethoxysilylethyl) benzene (DEB, mixture of 1,4 and 1,3 isomers), 3- aminopropyltrimethoxysilane (APS), phenyltrimethoxysilane (PTS), N-(2-aminoethyl)-3- aminopropyltrimethoxysilane (EDA), 3-isocyanatopropyltriethoxysilane (ICS), N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride (TSPMC, 50% in methanol), and N-tri- methoxysilylpropyl-N,N,N-tri-n-butylammonium chloride (TSPBC, 50% in methanol) were obtained from Gelest, Inc (Morrisville, PA, USA). Ethanol (200 proof) was obtained from the Warner-Graham Company (Cockeysville, MS, USA). Water was deionized to 18.2 MΩ-cm by a Millipore Milli Q UV-Plus water purification system.

2.1. Materials synthesis

Table 1 provides a summary of the sorbent materials utilized and their morphological characteristics. In general, synthesis involves establishment of morphology-directing surfactant micelles in solution followed by condensation of precursors around those micelles. The surfactant is then removed from the material leaving a porous structure to which further modification can be made. Below, specific syntheses for the various materials are detailed.

BTE- and DEB-based materials. Synthesis of the BTE- and DEB-based sorbents was based on a previously described approach [49,50,51] and began with dissolving TMB and P123 (1.9 g) in 0.1 M HNO3 with stirring at 60C. The stirring solution was cooled to room temperature and the silane mixture was added drop-wise. The reaction mixture was stirred until homogeneous and transferred to a culture tube which was sealed tightly and heated at 60C overnight (∼18 h). The tube was unsealed and the white gel was heated at 60C for 2 d and then 80C for 2 d. P123 was extracted by refluxing the monolith three times in ethanol for at least 12 h. A powdered product was collected by suction filtration, rinsed with ethanol and water, and dried at 100C. For MM5, 0.3 g TMB was used with 6 g 0.1 M HNO3. The total mol Si used was 7.84 mmol with 50:50 BTE:DEB. In the case of P10 and P5, 0.55 g TMB was used with 7.5 g 0.1 M HNO3. The silane mixture consisted of 15.7 mmol total Si with either 50:40:10 (P10) or 50:45:5 (P5) BTE:DEB:PTS. For N5, the protocol utilized 0.3 g TMB with 9.5 g 0.1 M HNO3 and the silane mixture was 100% BTE [49]. Following synthesis, amine groups were grafted on to the materials by adding sorbent (1 g) to 200 mL of toluene with 1 g APS [52]. This mixture was refluxed for 24 h after which the grafted product was collected by vacuum filtration, washed with toluene then ethanol, and dried at 110C. The DEN sorbent is a variation of this material. Following the amine functionalization protocol, isocyanate groups were incorporated using the ICS precursor [52]. This sorbent (1 g) was then placed in 50 mL MES buffer (100 mM, pH 5.5) with 1.3 g PAMAM dendrimer (10 wt. % in methanol) and mixed on a rotisserie mixer overnight at room temperature. The sorbent was collected by vacuum filtration, washed with methanol, and dried at 110C.

Sorbents with alkylammonium groups. The alkylammonium group bearing materials were synthesized based on a previously published approach [50,51,53]. For synthesis of the HX sorbent, 4.0 g of Pluronic P123 and 0.85 g of TMB were dissolved in 12.0 g of 1.0 M HNO3 with magnetic stirring and heating at 60C. The stirring mixture was allowed to cool to room temperature and 5.15 g of TMOS was added drop-wise. The mixture was stirred until homogeneous, transferred to a culture tube, sealed tightly, and heated at 60C overnight (≥18 h). The white monolith was dried in the unsealed tube at 60C for approximately 5 days before calcination (ambient atmosphere, temperature ramped 1C/min to 650C and held for 5 h) to remove surfactant. Materials were dried at 110C prior to grafting with alkylammonium silanes which was accomplished by adding sorbent (1 g) to 100 mL of toluene followed by addition of 2 mmol of both TSPMC and TSPBC. This mixture was refluxed for 24 h after which the grafted product was collected by vacuum filtration, washed with toluene then ethanol, and dried at 110C. The CF2M2B sorbent was synthesized identically with the exception of the TMB included which was 3.10 g.

Metal functionalized sorbents. EDA materials were synthesized using an adapted protocol [54,55,56] in which BTE (3.2 g) was dissolved in 0.01 M HCl (4 g). P123 (0.65 g) was added to the mixture and allowed to fully dissolve. The metal chelating group, EDA (0.11 g) was then added with either zinc chloride (0.04 g) or copper chloride (0.04 g) and a vacuum was pulled on the solutions for 24 h. The tube was sealed and placed in an oven at 100C for 0.5 h followed by 60C for 24 h. Sorbents were refluxed twice in acidified ethanol to remove the surfactant and soaked overnight in an ammonium hydroxide solution. After rinsing, metals were reincorporated through refluxing in a 0.1 M solution of either copper chloride or zinc acetate.

Chitosan functionalized sorbent. The reactor for this synthesis consisted of a 1000 mL PTFE jar in a water bath maintained at 80C. Cetyltrimethylammonium bromide (1.0 g) and 1.0 N NaOH (6.0 mL) were dissolved in 475 mL of H2O with magnetic stirring [57,58]. Mesitylene (6.0 mL) was added, and the solution was stirred for 3 h. Tetraethyl orthosilicate (TEOS; 5 mL) was added, and the mixture was stirred; white precipitate formed quickly. After 2 h, the precipitate was collected on filter paper by gravity filtration. When dry, the material was refluxed in 160 mL of ethanol with 9 mL of hydrochloric acid (37%) for 1 d to extract surfactant. The extracted product was collected by centrifugation, and washed with ethanol followed by water (3 times). The sorbent was dried at 110C prior to functionalization. To incorporate chitosan, a mixture of 1 g chitosan and 100 mL of 1 vol% acetic acid was prepared and filtered to remove insoluble matter. The sorbent was magnetically stirred in 50 mL of chitosan solution at room temperature for 1 d. The functionalized material (ChTS) was collected by centrifugation and washed with H2O three times before drying at 70C.

2.2. Characterization methods

Nitrogen sorption experiments were conducted using a Micromeritics ASAP 2010 at 77 K with DataMaster v. 4.03 E control and analysis software. Samples were degassed to 1 μm Hg at 100C prior to analysis. Standard methods were used for calculation of material characteristics. The Brunauer-Emmett-Teller (BET) method was used to determine surface area; Barrett-Joyner-Halenda (BJH) method was used to calculate pore size from the adsorption branch of the isotherm; the single point method was used calculate pore volume at relative pressure (P/P0) 0.97.

Powder X-ray diffraction patterns were collected at room temperature using CuKα radiation from a Brüker MICROSTAR-H X-ray generator operated at 40 kV and 20 mA equipped with a 5 m Radian collimator, and a Brüker Platinum-135 CCD area detector. A custom fabricated beamstop was mounted on the detector to allow data collection to approximately 0.4 2θ (approximately 210 Å) with a sample to detector distance of 30 cm. After unwarping the images, the XRD2 plug-in was used to integrate the diffraction patterns from 0.3 to 8.4 2θ.

2.3. Control nucleic acids

Triosephosphate isomerase (TIM, accession no. AF247559) of Arabidopsis thaliana was chosen as control RNA. TIM RNA transcripts were generated as previously described from pSP64poly(A)-TIM linearized with EcoRI and in vitro transcribed from the SP6 promoter using the MEGAscript high-yield transciption kit (formerly Life Technologies, Carlsbad, CA, USA; now ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer's recommended protocol [58]. A. thaliana NAC1 (accession no. AF198054) was chosen as control DNA and a 1059 nucleotide segment of the gene was PCR out of cDNAs of A. thaliana and cloned into TOPO4.0 vector (Life Technologies, Carlsbad, CA). The plasmid containing the NAC1 gene was used as a template for PCR with M13 primers, then digested with restriction enzymes PstI and XbaI (New England BioLabs, Inc., Ipswich, MA, USA). This was then cloned into pSP64 polyA Vector (Promega Corporation, Madison, WI, USA) digested with the same enzymes to generate pSP64poly(A)-NAC. NAC1 DNA was amplified by PCR with SP6 and M13R primers, and the PCR products were purified using a QIAquick PCR purification kit (QIAGEN, Valencia, CA, USA).

Single strand NAC1 DNA (ssNAC1) was generated based on the protocol developed by Tang et al. [59] with slight modification. Briefly, ssNAC1 was PCR amplified in 50 μL reaction volume containing 1x Green GoTaqⓇ reaction buffer (Promega Corporation, Madison, WI, USA), 3 mM MgCl2, 200 μM each of dNTPs, 20 nM each of SP6 and M13R primers, 375 nM of NAC1 forward primer, 1 unit of GoTaqⓇ DNA polymerase (Promega Corporation, Madison, WI, USA), and 0.01 ng of pSP64poly(A)-NAC DNA. Reactions were performed with initial denaturation at 95C, 3 min, followed by 20 cycles of 95C, 20 sec; 52C, 30 sec; and 72C, 60 sec; and 20 cycles 95C, 20 sec; 58C, 30 sec; and 72C, 60 sec with final extension at 72C, 5 min. The ssNAC1 PCR products were confirmed using 1.5% TAE agarose gel, then purified using DNA clean & concentratorTM-5 (Zymo Research, Irvine, CA, USA) according to manufacturer's recommended protocol for ssDNA purification.

2.4. Nucleic acid adsorption and elution

Adsorption of nucleic acids (NAs) by the porous sorbents was performed as previously described [58]. Briefly, samples were vortexed, placed on an agitator, and incubated for 20 min. Following incubation, samples were centrifuged at 1,000 g for 10 min and supernatants were separated from the precipitated sorbents. Bound target was calculated by comparison of supernatant content as determined by PCR to that determined for the control solution. Generation of isotherms and fits of the NA binding data were completed using PSI-plot v. 9.5 (Poly Software International, Inc., Pearl River, NY, USA).

To wash sorbents prior to nucleic acid adsorption, 0.3 mg of sorbent in 300 μL nuclease-free water was centrifuged at 1,000 g for 10 min. The supernatant was removed, and the sorbent was resuspended in 300 μL of 10 mM Tris/1% Triton X-100 followed by incubation for 15 min at room temperature with agitation. The sorbents were again centrifuged at 1,000 g for 10 min, and the supernatant was aspirated and discarded. The sorbents were then resuspended in 330 μL of 10 mM Tris/1% Triton X-100 for use in adsorption experiments as described above.

NA elution was performed using 20 to 100 μL of different buffers at various temperatures as indicated in the text and figure captions. Sorbents with encapsulated NA were mixed with elution buffer and vortexed briefly, then incubated at the indicated temperature for 10 min. After incubation, the samples were centrifuged at 1,000 g for 10 min; the supernatants were used for quantitative real-time reverse transcription-PCR (qRT-PCR) or real-time PCR (qPCR). NA content was determined based on a comparison of the results of PCR analysis to that of control solutions.

For stability testing, supernatants were separated from the precipitated sorbents, and the sorbents were left to dry at room temperature overnight. Control target samples were stored as prepared for adsorption experiments (in solution). To collect a time point, elution was completed as described above. NAs recovered at various time points were compared to recovery of NAs from the same sorbent on day 1 of the experiment.

2.5. Real-time RT-PCR or PCR (qRT-PCR or qPCR)

The recovery rates of TIM RNA were quantified using qRT-PCR as previously described [58]. The recovery rates of NAC1 and ssNAC1 were quantified using qPCR. qPCR was performed using iQ SYBRⓇ Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) with primers NAC1-225F (5'-ATCGACCACCTCTTGTCCTG-3') and NAC1-377R (5'-CCGTTGCTCGGTTAGTTCTC-3'). Eluted NA (1 μL) was used as template for 25 μL PCR reactions using MyiQ Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). A serial dilution of 10x NAC1 DNA (1–105 fg/μL) was used as standard curve. The qPCR reactions were performed using the following conditions: 95C for 3 min, followed by 30–35 cycles of 10 sec of denaturing at 95C, 10 sec of annealing at 56C, 10 sec of extension at 72C.

3. Results and Discussion

The sorbents utilized for this effort offer a range of morphological and chemical functionalities (Table 1). Pore diameters for the materials range from 43 to 223 Å while the BET surface areas range from 140 to 1,000 m2/g. Surface area, together with morphological organization, can impact the total binding sites available to an application. As the nucleic acid targets considered here are large molecules, average pore diameters and related factors, such as diffusion, may also contribute to the binding characteristics. While these factors cannot be ignored in overall sorbent optimization, this study did not focus on the impact of morphology; rather, the emphasis is given to the potential for the chemical composition of the materials in stabilizing the targets against typical degradation processes.

T1

Table 1: Material characteristics.

The chemical composition of the sorbents considered covers a wide range of possible activities (Figure 1). Diethylbenzene-bridged materials and those functionalized with pendent phenyl groups offer a somewhat hydrophobic environment as well as a high concentration of π-bonds (MM5, P5, P10). The hydroxyl groups of these types of silicate materials tend to be acidic; incorporation of primary amine groups offers basic sites (N5). The dendrimer modification (DEN) provides a greater number of basic sites at greater distance from the surface and increased hydrophobicity in the sorbent. The alkylammonium functionalities offer cationic groups in two different material morphologies with relatively disordered (CF2M2B) and ordered (HX2M2B) mesopore structures. Chitosan (ChTS) offers antimicrobial activity as well as the potential for multiple and complex cationic interactions with nucleic acids. The materials with ethylenediamine pendent groups (CuEDA and ZnEDA) offer sites for metal ion chelation. The presence of cations is known to impact the secondary structure of DNA; the presence of copper has been shown to decrease DNA melting temperatures while zinc causes an increase [60].

F1
Figure 1: Precursors groups utilized in synthesis of sorbents.
3.1. Target adsorption and elution

The materials were screened by generating a data set consisting of binding data for RNA and DNA using two target concentrations (Figure 2a). The goal was to identify the types of functional characteristics that provided significant adsorption of the two targets, so that those materials could be evaluated fully. The results of this screening were unexpected; some materials bound high percentages of the DNA or RNA while binding little to none of the other target (see items identified by asterisks). Similar data for the binding of ssDNA was collected to determine if the single versus double stranded nature of the targets was a significant contributor to these differences and to provide an additional point of comparison between the materials. The results of ssDNA binding were consistent with neither the DNA nor RNA binding (Figure 2a) indicating that the single or double stranded nature of the targets was not the primary contribution to the difference in binding efficiency of the materials.

F2
Figure 2: NA targets bound from solutions consisting of 30 μg sorbent with 300 ng of TIM RNA (red); 3 ng NAC1 DNA (blue); or 30 ng NAC1 ssDNA (black). Sorbents were used without pre-washing (a) and following the described wash steps (b). Error bars indicate standard deviation across six measurements. Data with * indicate those for which distinct differences in target binding was noted..

The Langmuir-Freundlich (LF) binding isotherm is a generalized form of the phenomenological Langmuir model often applied to solid sorbents. It allows for calculation of an effective affinity constant for the target (k), the saturated loading capacity of the sorbent (qs), and the site heterogeneity (n) within the sorbent based on the free ([L], ng) and bound target (q, ng/μg) [58,61,62]. Here, the constant α divided by the mass (m) yields the more typically utilized saturation capacity (qs) for the model.

q = α m k [ l ] n 1 + k [ l ] n

In order to better understand the interactions between the NA targets and the sorbents, isotherms for each of the materials with each of the three targets were generated. Table 2 provides a summary of the resulting parameters and fit statistics. In the case of these materials, site heterogeneity was found to be one (n = 1) for all targets; this result was also observed for a previous RNA binding study [58]. Figure 3a presents data from RNA adsorption experiments and the calculated binding isotherms for N5. As in past studies of this type, adsorption of target using varied target and sorbent concentrations provides sampling across a larger region of the binding space and allows for generation of fit parameters with greater confidence [48].

T2

Table 2: Langmuir isotherm parameters for RNA, DNA, and ssDNA binding by unwashed sorbents.

F3
Figure 3: Binding Isotherms. RNA bound by the N5 sorbent without pre-washing (a) and following the described wash step (b). Here, sorbent masses of 2 (black circle), 8 (red square), 13 (blue triangle), 18 (green diamond), 23 (purple hexagon), and 40 μg (orange star) were utilized for capture of RNA from a 240 μl solution containing ∼300 ng TIM RNA target.

The parameters obtained indicate the maximum target that can be bound (α) and the rate at which that limit will be approached (k). For example, the RNA saturated loading limit for HX2M2B is greater than that of N5, but, at low free RNA concentrations, N5 will bind more target than HX2M2B (k = 0.0467 ng−1 versus 0.00818 ng−1). N5 provided the greatest DNA saturated loading limit, while HX2M2B provided the greatest limit for ssDNA. MM5, P5, and P10 showed moderate to low total binding and affinity for all three targets. DEN performed moderately for RNA and ssDNA and bound by far the greatest amount of DNA. CuEDA and ZnEDA performed moderately for DNA and ssDNA, but CuEDA provided the greatest RNA saturated loading limit. Elution of bound target from the sorbent materials was also evaluated. Initial attempts at recovering RNA using EB buffer (10 mM Tris-Cl, pH 8.5) at 50C (total volume 20 μL) provided minimal return from these materials. NEB buffer (20 mM Tris-Cl, pH7.5, 1 mM EDTA) provided the best performance in our previous study [58], but did not offer target recovery from these materials. Variations on temperature, volume, incubation period, and detergent concentrations were considered and tested as was the inclusion of solvent and sodium chloride [63,64]. Other studies have indicated the impact of buffer pH on the elution efficiency related to silicate materials [65,66,67]; however, varying pH (5.7 to 8.0) did not have an impact on RNA recovery. It has been argued that nucleic acid interactions with silicate materials are via amine and carboxyl groups [68]. Methods used to displace these interactions as well as those used to displace RNA from negatively charged membranes [69] were considered and tested without improvement. Finally, various nucleic acid washing solutions and hybridization buffers were evaluated without success (less than 1% of target recovered). The solutions and condition variations evaluated are summarized in Table 3.

T3

Table 3: Elution solutions and conditions evaluated.

Given the failure of this wide range of elution solutions, the possibility that the nucleic acids were destroyed upon interaction with the sorbents was considered; however, given previous work in this area and the wide range of sorbent variations involved, these types of destructive interactions are unlikely to be observed for all of the materials under consideration. Based on previous experience and other materials used for nucleic acid hybridization, a pre-wash step was evaluated for the sorbents. This type of prehybridization step has been used in Northern and Southern blotting technologies to reduce nonspecific binding of nucleic acids [70]. Here, the procedure involved incubating the sorbent in 10 mM Tris with 1% Triton X-100 for 15 min at room temperature prior to NA adsorption. Figure 2b provides single point data on target binding by the washed N5, CuEDA, HX2M2B, CF2M2B, DEN and ChTS. Other sorbents considered for this study bound less than 5% of all three targets. This pre-conditioning step strongly impacted the binding behavior of the sorbents and led to less error in the resulting fits (Table 2; Figure 3b). ZnEDA offered lower saturated loading capacities than CuEDA prior to washing and likely lost binding capacity upon interaction with the Triton X-100 as observed for CuEDA. This surfactant would also be expected to interact with the surfaces of the MM5, P5, and P10 sorbents given their somewhat hydrophobic nature and the available π-interaction sites. Additional experiments using other prehybridization solutions, such as 2X SSC (0.3 M sodium chloride with 30 mM trisodium citrate at pH 7) with 0.1% SDS or 6X SSPE (900 mM NaCl with 60 mM sodium phosphate and 6 mM ethylenediaminetetraacetic acid) with 0.1% SDS, could be considered for use with these sorbents.

F4
Figure 4: Elution. RNA eluted from washed sorbent materials reported as a percentage of the target initially adsorbed: N5 (red); HX2M2B (blue); DEN (black); CuEDA (green). All elution solutions utilized 100 μL at 50C for 20 min: RNA (a), DNA (b), and ssDNA (c).

The potential for elution of RNA was evaluated using the pre-conditioned materials, and variations on the elution solution were again considered (Table 3). As shown in Figure 4a, RNA recovery was significantly increased from less than 1% in the unwashed sorbents to between 20 and 80% using 100 mM Tris with 0.1% SDS for HX2M2B, N5, and CuEDA. Unfortunately, prewashing did not significantly improve recovery from DEN; a wide range of elution solutions were again considered with no recovery greater than 2% (Table 3). It is unclear if this is a result of continuing strong interactions between DEN and the NA targets or target degradation. Recovery of DNA and ssDNA was also improved following the pre-conditioning step with a small amount of DNA recovered from even the DEN sorbent.

3.2. NA stability

In order to evaluate the potential of the sorbents to enhance the stability of stored NA targets, a large batch sample for each target adsorbed onto each material was prepared. The sample was then divided into aliquots, the supernatants were separated from the precipitated sorbents, and the sorbents were left to dry at room temperature overnight. A control sample consisting of the target only in solution was retained for comparison. The sorbents were sampled following storage either at room temperature or at 37C. No special protection from light or control of humidity was employed. Storage at 37C is intended to simulate temperatures relevant to those expected at the high end of operational conditions. Over the course of the experiments, room temperature varied between 18 and 23C while relative humidity ranged from 42 to 61%. Figure 5a presents the results of RNA storage over a period of 200 days at room temperature. The recovered RNA is normalized to the amount recovered on day one of the experiment. Over this period, RNA eluted from N5 gradually decreases to 20% of the day one recovery while that from HX2M2B decreases to 5%. Recovery from DEN, significantly lower on day one, decreases to <10% by day 80. RNA in the control sample drops to <10% by day 29. At 37C, recovery of RNA from all three materials was increased as compared to that from the control sample (Figure 6a). More than 20% was recovered from N5 through day 140, from HX2M2B through day 80, and from DEN through day 50. These results indicate that the three sorbents offer improvements in RNA stability both at room temperature and at 37C.

F5
Figure 5: Stability of NA targets. NA targets recovered from N5 (red square), HX2M2B (blue triangle), and DEN (green diamond) following storage at room temperature. Data for similarly stored target only in solution (circle) is provided for comparison: RNA (a), DNA (b), and ssDNA (c).

The decrease in DNA recovered from N5 and HX2M2B at room temperature and at 37C was similar to the decrease in the DNA content of the control sample (Figure 5b and Figure 6b). For ssDNA, on the other hand, while the control sample at room temperature dropped below 20% of the original content on day 121, recovery from N5 remained above 20% beyond day 170. When stored at 37C, however, the decrease in ssDNA recovered from N5 was similar to that of the control sample. While the decrease in ssDNA recovered from HX2M2B at room temperature was similar to the decrease in the ssDNA content of the control sample, HX2M2B showed slightly improved recovery of ssDNA over the first 20 days at 37C. Results with DEN showed more rapidly decreasing DNA and ssDNA content than that observed for the control samples.

F6
Figure 6: Stability of NA targets at 37C. NA targets recovered from N5 (red square), HX2M2B (blue triangle), and DEN (green diamond) following storage at 37C. Data for similarly stored target only in solution (circle) is provided for comparison: RNA (a), DNA (b), and ssDNA (c).

4. Conclusions

The study presented here evaluated the potential of mesoporous sorbents bearing chemical functionalities for stabilization of nucleic acid targets. The idea was to access the activities of common stabilization reagents within these sorbents while making separation of the NAs from stabilizing compounds simple. This would allow for stabilization of the NAs during storage and transportation while providing a system that does not interfere with further (down-stream) analysis of the targets. While many of the porous materials were found to remove RNA, DNA, and ssDNA from solution, recovery of the targets from the sorbents proved challenging. The desire to avoid downstream contamination prevents the use of harsher elution conditions. The necessary pre-conditioning step using a Triton X-100 wash prevented the types of interactions desired with several of the surfaces considered. It is likely that π-interactions contributed to the noted reductions in binding for DEB based sorbents. Other commonly used prehybridization washing solutions, such as 2X SSC with 0.1% SDS or 6X SSPE with 0.1% SDS, could offer substitutions that reduce interaction with the π-bonds of the sorbents and are currently being evaluated.

Of the materials that were suitable for use with the pre-conditioning wash step, the sorbent functionalized with primary amine groups (N5) showed promising results for RNA and ssDNA stabilization. The performance of the sorbent at room temperature was approximately equivalent to the performance of our previously reported trehalose sorbent at 4C [58]. N5 showed significant stabilization of RNA even when stored at 37C. The HX2M2B sorbent also showed stabilization of RNA and ssDNA, but recovery from this sorbent declined more quickly than that of the N5 sorbent. None of the sorbents improved the stability of DNA either under room temperature or 37C storage. Based on the results of these studies, we are currently evaluating the potential for combinations of functional groups within the sorbent structure. Specifically, combinations of sugars (trehalose, glucosamine) and the functional groups considered here within sorbent morphologies similar to those described for this study.

Competing Interests

The authors declare that they have no competing interests.

Acknowledgments

This research was sponsored by the U.S. Office of Naval Research through Naval Research Laboratory base funds (69-6595). Participation of M. McConner through Office of Naval Research (ONR) sponsored Historically Black Colleges and Universities/Minority Institutions (HBCU/MI) summer internship program. Participation of G. Haas through Naval Research Laboratory Student Temporary Employment Program (STEP). We applied the SDC approach (“sequence-determines-credit”) for determining the sequence of authors [71]. The views expressed here are those of the authors and do not represent those of the U.S. Navy, the U.S. Department of Defense, or the U.S. Government.

References

  1. C. Frippiat, S. Zorbo, D. Leonard et al., “Evaluation of novel forensic DNA storage methodologies,” Forensic Science International: Genetics, vol. 5, no. 5, pp. 386–392, 2011.
  2. M. Shabihkhani, G. M. Lucey, B. Wei et al., “The procurement, storage, and quality assurance of frozen blood and tissue biospecimens in pathology, biorepository, and biobank settings,” Clinical Biochemistry, vol. 47, no. 4-5, pp. 258–266, 2014.
  3. J. A. Blow, C. N. Mores, J. Dyer, and D. J. Dohm, “Viral nucleic acid stabilization by RNA extraction reagent,” Journal of Virological Methods, vol. 150, no. 1-2, pp. 41–44, 2008.
  4. A. H. García, “Anhydrobiosis in bacteria: From physiology to applications,” Journal of Biosciences, vol. 36, no. 5, pp. 939–950, 2011.
  5. W. Block, R. I. Lewis Smith, and A. D. Kennedy, “Strategies of survival and resource exploitation in the Antarctic fellfield ecosystem,” Biol Rev Camb Philos Soc, vol. 84, pp. 449–484, 2009.
  6. D. Clermont, S. Santoni, S. Saker, M. Gomard, E. Gardais, and C. Bizet, “Assessment of DNA encapsulation, a new room-temperature DNA storage method,” Biopreservation and Biobanking, vol. 12, no. 3, pp. 176–183, 2014.
  7. J. Bonnet, M. Colotte, D. Coudy et al., “Chain and conformation stability of solid-state DNA: Implications for room temperature storage,” Nucleic Acids Research, vol. 38, no. 5, Article ID gkp1060, pp. 1531–1546, 2009.
  8. C. Brus, E. Kleemann, A. Aigner, F. Czubayko, and T. Kissel, “Stabilization of oligonucleotide-polyethylenimine complexes by freeze-drying: Physicochemical and biological characterization,” Journal of Controlled Release, vol. 95, no. 1, pp. 119–131, 2004.
  9. J.-H. S. Kuo and R. Hwang, “Preparation of DNA dry powder for non-viral gene delivery by spray-freeze drying: Effect of protective agents (polyethyleneimine and sugars) on the stability of DNA,” Journal of Pharmacy and Pharmacology, vol. 56, no. 1, pp. 27–33, 2004.
  10. B. Li, S. Li, Y. Tan et al., “Lyophilization of cationic lipid-protamine-DNA (LPD) complexes,” Journal of Pharmaceutical Sciences, vol. 89, no. 3, pp. 355–364, 2000.
  11. D. Dimitrellou, P. Kandylis, T. Petrović et al., “Survival of spray dried microencapsulated Lactobacillus casei ATCC 393 in simulated gastrointestinal conditions and fermented milk,” LWT - Food Science and Technology, vol. 71, pp. 169–174, 2016.
  12. A. H. Garcia, 2015, Induced anhydrobiosis Powerful method for preservation of industrial microorganisms,.
  13. G. E. Hernandez, T. S. Mondala, and S. R. Head, “Assessing a novel room-temperature RNA storage medium for compatibility in microarray gene expression analysis,” BioTechniques, vol. 47, no. 2, pp. 667–670, 2009.
  14. E. Wan, M. Akana, J. Pons et al., “Green technologies for room temperature nucleic acid storage,” Current Issues in Molecular Biology, vol. 12, no. 3, pp. 135–142, 2010.
  15. G. Seutin, B. N. White, and P. T. Boag, “Preservation of Avian Blood and Tissue Samples for DNA Analyses,” Can J Zool, vol. 69, pp. 82–90, 1991.
  16. C. L. Michaud and D. R. Foran, “Simplified field preservation of tissues for subsequent DNA analyses,” Journal of Forensic Sciences, vol. 56, no. 4, pp. 846–852, 2011.
  17. A. J. Rissanen, E. Kurhela, T. Aho, T. Oittinen, and M. Tiirola, “Storage of environmental samples for guaranteeing nucleic acid yields for molecular microbiological studies,” Applied Microbiology and Biotechnology, vol. 88, no. 4, pp. 977–984, 2010.
  18. P. Vonhippel and K. Wong, NEUTRAL SALTS: THE GENERALITY OF THEIR EFFECTS ON THE STABILITY OF MACROMOLECULAR CONFORMATIONS. Science, vol. 145, pp. 577–580, 1964.
  19. A. Allen-Hall and D. McNevin, “Human tissue preservation for disaster victim identification (DVI) in tropical climates,” Forensic Science International: Genetics, vol. 6, no. 5, pp. 653–657, 2012.
  20. R. M. Tuttle, J. K. Waselenko, P. Yosseffi, N. Weigand, and R. K. Martin, “Preservation of nucleic acids for polymerase chain reaction after prolonged storage at room temperature,” Diagnostic Molecular Pathology, vol. 7, no. 6, pp. 302–309, 1998.
  21. C. A. Whittier, W. Horne, B. Slenning, M. Loomis, and M. K. Stoskopf, “Comparison of storage methods for reverse-transcriptase PCR amplification of rotavirus RNA from gorilla (Gorilla g. gorilla) fecal samples,” Journal of Virological Methods, vol. 116, no. 1, pp. 11–17, 2004.
  22. C. J. Fregeau, H. Vanstone, S. Borys et al., “AmpFlSTR® profiler plus™ and AmpFlSTR® COfiler™ analysis of tissues stored in GenoFix™, a new tissue preservation solution for mass disaster DNA identification,” Journal of Forensic Sciences, vol. 46, no. 5, pp. 1180–1190, 2001.
  23. V. Vincek, M. Nassiri, M. Nadji, and A. R. Morales, “A Tissue Fixative that Protects Macromolecules (DNA, RNA, and Protein) and Histomorphology in Clinical Samples,” Laboratory Investigation, vol. 83, no. 10, pp. 1427–1435, 2003.
  24. T. E. Andrisin, L. M. Humma, and J. A. Johnson, “Collection of genomic DNA by the noninvasive mouthwash method for use in pharmacogenetic studies,” Pharmacotherapy, vol. 22, no. 8, pp. 954–960, 2002.
  25. A. E. Krafft, K. L. Russell, A. W. Hawksworth et al., “Evaluation of PCR testing of ethanol-fixed nasal swab specimens as an augmented surveillance strategy for influenza virus and adenovirus identification,” Journal of Clinical Microbiology, vol. 43, no. 4, pp. 1768–1775, 2005.
  26. D. S. Bachoon, F. Chen, and R. E. Hodson, “RNA recovery and detection of mRNA by RT-PCR from preserved prokaryotic samples,” FEMS Microbiology Letters, vol. 201, no. 2, pp. 127–132, 2001.
  27. Y. Terui, M. Ohnuma, K. Hiraga, E. Kawashima, and T. Oshima, “Stabilization of nucleic acids by unusual polyamines produced by an extreme thermophile, Thermus thermophilus,” Biochemical Journal, vol. 388, no. 2, pp. 427–433, 2005.
  28. C. Tribioli and T. Lufkin, “Long-term room temperature storage of high-quality embryonic stem cell genomic DNA extracted with a simple and rapid procedure,” Journal of Biomolecular Techniques, vol. 17, no. 4, pp. 249–251, 2006.
  29. S. G. Wolf, D. Frenkiel, T. Arad, S. E. Finkeil, R. Kolter, and A. Minsky, “DNA protection by stress-induced biocrystallization,” Nature, vol. 400, no. 6739, pp. 83–85, 1999.
  30. J. M. Coller, N. K. Gray, and M. P. Wickens, “mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation,” Genes and Development, vol. 12, no. 20, pp. 3226–3235, 1998.
  31. S. D. Blacksell, S. Khounsy, and H. A. Westbury, “The effect of sample degradation and RNA stabilization on classical swine fever virus RT-PCR and ELISA methods,” Journal of Virological Methods, vol. 118, no. 1, pp. 33–37, 2004.
  32. S. R. Florell, C. M. Coffin, J. A. Holden et al., “Preservation of RNA for functional genomic studies: A multidisciplinary tumor bank protocol,” Modern Pathology, vol. 14, no. 2, pp. 116–128, 2001.
  33. W. Ma, M. Wang, Z.-Q. Wang et al., “Effect of long-term storage in TRIzol on microarray-based gene expression profiling,” Cancer Epidemiology Biomarkers and Prevention, vol. 19, no. 10, pp. 2445–2452, 2010.
  34. A. Sorensen, E. Rahman, C. Canela, D. Gangitano, and S. Hughes-Stamm, “Preservation and rapid purification of DNA from decomposing human tissue samples,” Forensic Sci. Int.-Genet, vol. 25, pp. 182–190, 2016.
  35. L. Brannon-Peppas, B. Ghosn, K. Roy, and K. Cornetta, “Encapsulation of nucleic acids and opportunities for cancer treatment,” Pharmaceutical Research, vol. 24, no. 4, pp. 618–627, 2007.
  36. R. T. Chacko, J. Ventura, J. Zhuang, and S. Thayumanavan, “Polymer nanogels: A versatile nanoscopic drug delivery platform,” Advanced Drug Delivery Reviews, vol. 64, no. 9, pp. 836–851, 2012.
  37. J.-H. Ryu, R. T. Chacko, S. Jiwpanich, S. Bickerton, R. P. Babu, and S. Thayumanavan, “Self-cross-linked polymer nanogels: A versatile nanoscopic drug delivery platform,” Journal of the American Chemical Society, vol. 132, no. 48, pp. 17227–17235, 2010.
  38. A. V. Kabanov and S. V. Vinogradov, “Nanogels as pharmaceutical carriers: finite networks of infinite capabilities. Angew Chem Int,” Engl, vol. 48, pp. 5418–5429, 2009.
  39. K. Khosravi-Darani, A. Pardakhty, H. Honarpisheh, V. S. N. M. Rao, and M. R. Mozafari, “The role of high-resolution imaging in the evaluation of nanosystems for bioactive encapsulation and targeted nanotherapy,” Micron, vol. 38, no. 8, pp. 804–818, 2007.
  40. C. S. Lengsfeld, M. C. Manning, and T. W. Randolph, “Encapsulating DNA within biodegradable polymeric micorparticles,” Current Pharmaceutical Biotechnology, vol. 3, no. 3, pp. 227–235, 2002.
  41. W. Li and F. C. Szoka Jr., “Lipid-based nanoparticles for nucleic acid delivery,” Pharmaceutical Research, vol. 24, no. 3, pp. 438–449, 2007.
  42. M. L. Tan, P. F. M. Choong, and C. R. Dass, “Cancer, chitosan nanopartides and catalytic nucleic acids,” Journal of Pharmacy and Pharmacology, vol. 61, no. 1, pp. 3–12, 2009.
  43. L. Xu and T. Anchordoquy, “Drug delivery trends in clinical trials and translational medicine: Challenges and opportunities in the delivery of nucleic acid-based therapeutics,” Journal of Pharmaceutical Sciences, vol. 100, no. 1, pp. 38–52, 2011.
  44. K. D. Clark, M. Sorensen, O. Nacham, and J. L. Anderson, “Preservation of DNA in nuclease-rich samples using magnetic ionic liquids,” RSC Advances, vol. 6, no. 46, pp. 39846–39851, 2016.
  45. J.-M. Cayuela, C. Mauté, A.-L. Fabre et al., “A novel method for room temperature distribution and conservation of RNA and DNA reference materials for guaranteeing performance of molecular diagnostics in onco-hematology: A GBMHM study,” Clinical Biochemistry, vol. 48, no. 15, pp. 982–987, 2015.
  46. X. Liu, Q. Li, X. Wang et al., “Evaluation of DNA/RNAshells for room temperature nucleic acids storage,” Biopreservation and Biobanking, vol. 13, no. 1, pp. 49–55, 2015.
  47. R. Muller, F. Betsou, M. G. Barnes et al., “Int Soc Biol Environm, R. Preservation of Biospecimens at Ambient Temperature: Special Focus on Nucleic Acids and Opportunities for the Biobanking Community,” Biopreserv. Biobank, vol. 14, pp. 89–98, 2016.
  48. B. J. Johnson, A. P. Malanoski, I. A. Leska et al., “Adsorption of organophosphates from solution by porous organosilicates: Capillary phase-separation,” Microporous and Mesoporous Materials, vol. 195, pp. 154–160, 2014.
  49. B. J. Melde, B. J. Johnson, M. A. Dinderman, and J. R. Deschamps, “Macroporous periodic mesoporous organosilicas with diethylbenzene bridging groups,” Microporous and Mesoporous Materials, vol. 130, no. 1-3, pp. 180–188, 2010.
  50. K. Nakanishi, T. Amatani, S. Yano, and T. Kodaira, “Multiscale templating of siloxane gels via polymerization-induced phase separation,” Chemistry of Materials, vol. 20, no. 3, pp. 1108–1115, 2008.
  51. T. Amatani, K. Nakanishi, K. Hirao, and T. Kodaira, “Monolithic periodic mesoporous silica with well-defined macropores,” Chemistry of Materials, vol. 17, no. 8, pp. 2114–2119, 2005.
  52. B. J. Johnson, B. J. Melde, G. W. Peterson, B. J. Schindler, and P. Jones, “Functionalized organosilicate materials for irritant gas removal,” Chemical Engineering Science, vol. 68, no. 1, pp. 376–382, 2012.
  53. B. J. Johnson, I. A. Leska, B. J. Melde et al., “Extraction of perchlorate using porous organosilicate materials,” Materials, vol. 6, no. 4, pp. 1403–1419, 2013.
  54. M. C. Burleigh, S. Dai, E. W. Hagaman, and J. S. Lin, “Imprinted polysilsesquioxanes for the enhanced recognition of metal ions,” Chemistry of Materials, vol. 13, no. 8, pp. 2537–2546, 2001.
  55. H. Zhu, D. J. Jones, J. Zajac, R. Dutartre, M. Rhomari, and J. Rozière, “Synthesis of periodic large mesoporous organosilicas and functionalization by incorporation of ligands into the framework wall,” Chemistry of Materials, vol. 14, no. 12, pp. 4886–4894, 2002.
  56. B. J. White, B. J. Melde, M. H. Moore, and G. W. Peterson, “Functionalized organosilicate sorbents for air purification,” in NRL/FR/6920--13-10, pp. 6920–13, US Naval Research, Laboratory: Washington DC, 12/23/2013, 2013.
  57. I. I. Slowing, B. G. Trewyn, and V. S.-Y. Lin, “Mesoporous silica nanoparticles for intracellular delivery of membrane-impermeable proteins,” Journal of the American Chemical Society, vol. 129, no. 28, pp. 8845–8849, 2007.
  58. B. J. Johnson, B. J. Melde, M. A. Dinderman, and B. Lin, “Stabilization of RNA through Absorption by Functionalized Mesoporous Silicate Nanospheres,” PLoS ONE, vol. 7, no. 11, Article ID e50356, 2012.
  59. X. Tang, S. L. Morris, J. J. Langone, and L. E. Bockstahler, “Simple and effective method for generating single-stranded DNA targets and probes,” BioTechniques, vol. 40, no. 6, pp. 759–762, 2006.
  60. H. Venner and C. Zimmer, “Studies on nucleic acids. VIII. Changes in the stability of DNA secondary structure by interaction with divalent metal ions,” Biopolymers, vol. 4, no. 3, pp. 321–335, 1966.
  61. R. J. Umpleby II, S. C. Baxter, M. Bode, J. K. Berch Jr., R. N. Shah, and K. D. Shimizu, “Application of the Freundlich adsorption isotherm in the characterization of molecularly imprinted polymers,” Analytica Chimica Acta, vol. 435, no. 1, pp. 35–42, 2001.
  62. H. Kim and G. Guiochon, “Comparison of the thermodynamic properties of particulate and monolithic columns of molecularly imprinted copolymers,” Analytical Chemistry, vol. 77, no. 1, pp. 93–102, 2005.
  63. M. C. Morán, M. G. Miguel, and B. Lindman, “Surfactant-DNA gel particles: Formation and release characteristics,” Biomacromolecules, vol. 8, no. 12, pp. 3886–3892, 2007.
  64. X. Li, J. X. Zhang, and H. C. Gu, “Study on the Adsorption Mechanism,” Langmuir : the ACS journal of surfaces and colloids, vol. 28, pp. 2827–2834, 2012.
  65. A. Masotti, F. Bordi, G. Ortaggi, F. Marino, and C. Palocci, “A novel method to obtain chitosan/DNA nanospheres and a study of their release properties,” Nanotechnology, vol. 19, no. 5, Article ID 055302, 2008.
  66. S. Wu, X. Huang, and X. Du, “Glucose- and pH-responsive controlled release of cargo from protein-gated carbohydrate-functionalized mesoporous silica nanocontainers,” Angewandte Chemie - International Edition, vol. 52, no. 21, pp. 5580–5584, 2013.
  67. Y. Zhao, B. G. Trewyn, I. I. Slowing, and V. S.-Y. Lin, “Mesoporous silica nanoparticle-based double drug delivery system for glucose-responsive controlled release of insulin and cyclic AMP,” Journal of the American Chemical Society, vol. 131, no. 24, pp. 8398–8400, 2009.
  68. F. Pu, Z. Liu, J. Ren, and X. Qu, “Nucleic acid-mesoporous silica nanoparticle conjugates for keypad lock security operation,” Chemical Communications, vol. 49, no. 23, pp. 2305–2307, 2013.
  69. A. de Abreu Corrêa and M. P. Miagostovich, “Optimization of an Adsorption-Elution Method with a Negatively Charged Membrane to Recover Norovirus from Lettuce,” Food and Environmental Virology, vol. 5, no. 3, pp. 144–149, 2013.
  70. J. Sambrook, E. F. Fritsch, and T. Maniatis, “Molecular cloning: A laboratory manual,” in Molecular cloning: A laboratory manual, 2nd. edition, Ed., vol. 2, p. p, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 2nd edition, 1989.
  71. T. Tscharntke, M. E. Hochberg, T. A. Rand, V. H. Resh, and J. Krauss, “Author sequence and credit for contributions in multiauthored publications.,” PLoS biology, vol. 5, no. 1, p. e18, 2007.
Research Article
The Open Access Journal of Science and Technology
Vol. 5 (2017), Article ID 101190, 18 pages
doi:10.11131/2017/101190

Adsorption and Elution of Nucleic Acids: Mesoporous Materials and Methods

Baochuan Lin1, Brandy J. Johnson1, Brian J. Melde1, Genevieve M. Haas2, Miles K. J. McConner3 and Jenna R. Taft4

1Center for Bio/Molecular Science & Engineering, US Naval Research Laboratory, Washington, DC, USA 20375

2Department of Biology, Northern Michigan University, Marquette, MI, USA 49855, at NRL § summers 2012 and 2013

3Department of Biology, Norfolk State University, Norfolk, VA, USA 23504, at NRL 1 summer 2014

4Formerly NOVA Research Incorporated, Alexandria, VA, USA 22308, currently Department of Chemistry, University of Vermont, Burlington, VT, USA 05405

Received 30 November 2015; Accepted 19 January 2017

Editors: Jung-Hua Kuo, Zhengguo Xiao, and Chunyi Zhi

Copyright © 2017 Baochuan Lin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Physical protection of nucleic acids from the environment for improved stability through encapsulation or adsorption has been reported using various materials, including liposomes, metal particles, mesoporous silica nanoparticles, and polymers. In an extension of that approach, our previous study demonstrated the potential of mesoporous silica nanoparticles with and without covalently attached stabilizing reagents, such as sugar and bovine serum albumin, for improving the stability of RNA. The goal of the current study is to evaluate the potential of silicate sorbents bearing chemical functionalities for stabilization of nucleic acid targets. Materials offering charged groups, metal chelating sites, and π-bonding sites are considered. Adsorption and elution of RNA, DNA, and single stranded DNA (ssDNA) are evaluated as is subsequent elution of the bound target. A sorbent functionalized with primary amine groups showed promising results for RNA and ssDNA stabilization. The impact of the sorbents on long term viability the targets is also evaluated. Storage of adsorbed targets at room temperature and 37C over a period of 200 d indicates the potential for stabilization of RNA and ssDNA using several of the functionalities. None of the sorbents improved the stability of DNA either under room temperature or 37C storage.