Graphene Oxide as a Bifunctional Material toward Superior RNA

Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Graphene Oxide as a Bifunctional Material toward Superior RNA Protection and Extraction Yuhui Liao, Xiaoming Zhou,* Yu Fu, and Da Xing* MOE Key Laboratory of Laser Life Science and Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China

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S Supporting Information *

ABSTRACT: It is well known that graphene oxide (GO), a planar nanomaterial, is endowed with the capacity to immobilize short ssRNA via π−π stacking, thus enhancing its stability. However, whether large RNA molecules, such as total RNA, extracted from biological tissues can be protected using GO has not been investigated. It is usually believed that the protection of total RNA by GO is not effective because the lengths of total RNA, which range from a few to thousands of bases, are inclined to undergo desorption due to their complicated structure. Herein, the nanobiological effects of total RNA/GO are first investigated and demonstrate that the total RNA can be harbored on the surface of GO, thus resulting in a shield effect. This shield effect allows total RNA to highly resist RNase degradation and maintain RNA stability at room temperature up to 4 days, enabling the discovery of GO as the potential next-generation RNase nanoinhibitor. Furthermore, GO can be conjugated to nanomagnetic beads, defined as magnetic graphene oxide, enabling the rapid purification and protection of RNA from animal cells and tissues, whole blood, bacteria, and plant tissue. KEYWORDS: bifunctional material, graphene oxide, RNA protection, RNA extraction

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RNA purification.10,11 An irreplaceable constituent of the TRIzol method, guanidinium isothiocyanate12−14 (GIT, an aggressive chemical denaturant) simultaneously performs the functions of cell lysis and RNase denaturation. However, GIT must be eliminated to obtain the RNA extracts without chemical denaturants. The elimination of GIT leads to the exposure of RNA extracts to the ubiquitous RNase contamination. Subsequent purification or storage of RNA in vitro remains subject to enzymatic degradation. It reveals that the continuous protection during the whole process of RNA extraction and storage is important for RNA extraction. Obviously, TRIzol kit cannot provide continuous protection and subsequent steps increase the risk of RNase contamination, which generally reduces RNA yield and negatively impacts RNA fidelity. The acquisition of high-quality RNA remains a considerable challenge due to the insufficiency of RNA extraction techniques. Thus, innovations in RNA extraction methods are urgently needed to overcome the shortcomings of current methodology for high-quality RNA purification. Meanwhile, the recent efforts in the development of useful tools15−18 inspired us to construct an unconventional RNA protection and extraction method.

INTRODUCTION RNA performs various biological functions in addition to its role as a genetic material.1,2 Generally, RNA is extremely unstable in vitro. When exposed to air, RNA degrades irreversibly within few hours. This instability of RNA is mainly attributed to ubiquitous RNase contamination.3,4 The stable structure of RNase makes RNase inactivation through physical means extremely difficult.5,6 RNase remains active even after autoclave sterilization at 121 °C for 20 min.7 Thus, enzymatic degradation is a major contributor to RNA instability that perplexes the RNA manipulations. To protect RNA from enzymatic degradation, RNase denaturation and inhibition are widely employed. RNase denaturation is an aggressive method that introduces denaturants, such as diethylpyrocarbonate,8,9 to irreversibly inactivate RNase. This approach is limited because extreme chemical denaturants cannot be applied to biological systems. Meanwhile, the accompanying toxicity and carcinogenesis is a potential risk for the relative manipulators. In contrast, using RNase inhibitor is a more moderate method for RNA protection. However, RNase inhibitor only inhibits the activity of RNase and lacks sufficient strength to entirely prevent enzymatic degradation. Hence, the development of an effective, moderate RNA protection assay would be significant for routine RNA manipulation. Currently, RNA extraction with TRIzol, a technological means of routine RNA manipulations, is the gold standard for © XXXX American Chemical Society

Received: July 24, 2018 Accepted: August 21, 2018 Published: August 21, 2018 A

DOI: 10.1021/acsami.8b12522 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 1. Verification of GO as an effective total RNA protection agent. (A) Electrophoresis experiments assessing the effect of GO-assisted RNA protection. Commercial GO was preprocessed to remove RNase and impurities. Lane “a” shows the newly extracted RNA; lane “b” shows newly extracted RNA + 40 units RNase; lane “c” shows the newly extracted RNA + 40 units RNase + 10 μg GO; lane “d” shows the newly extracted RNA exposed to air for 24 h; lane “e” shows the newly extracted RNA + 10 μg GO exposed to air for 24 h. (B) Evaluation of the RNA degradation ratio by comparing the grayscale intensity of the corresponding bands. b, c, d, and e represent the lanes b, c, d, and e, respectively. in a volume of 50 μL containing the following: 10 μL of extracted RNA; 200 mM dATP, dCTP, dGTP, and dTTP; and 0.4 mM of each primer. The RT-PCR amplification conditions consisted of 1 cycle at 60 °C for 35 min for RT of the RNA. During this step, the AMV Reverse Transcriptase (New England Biolabs) was used. This step was followed by a temperature cycling routine that consisted of denaturation at 94 °C for 30 s, annealing at 54 °C for 30 s, and extension at 72 °C for 1 min. After 35 cycles, a final extension at 72 °C was performed for 5 min and the tubes cooled to 4 °C. This process yielded a 119-bp fragment from the mRNA of the iap gene. The primer sequences are listed in Table S1. Synthesis of Magnetism-Functionalized GO. Magnetismfunctionalized GO was synthesized via the formation of an amide bond between the carboxyl of GO (1 μm) and amino of MB (200 nm). The detailed synthesis route is shown in Figure 4A. Ten milligrams GO was first dissolved in 30 mL ddH2O and treated with ultrasonic processing for 1.5 h to disperse the fragments of solid GO adequately. Then, 4 mg N-hydroxysuccinimide and 5 mg N-(3dimethylaminopropyl)-N′-ethylcarbodiimide were simultaneously added to the GO solution. The mixture was stirred at 37 °C for 1 h and treated with ultrasonic processing for 0.5 h. Ten milligrams amino-coated MB was added to the above solution of activated GO and incubated at 37 °C for 12 h. GO without MB was definitively eliminated by magnetic separation and washing three times. Total RNA Extraction Using MGO. The protective RNA extraction assay consists of sample pretreatment, “cell disruption”, RNA capture, and washing steps. The sample pretreatment step should be adjusted based on the sample source. In general, 107 cells need to be immersed in 1 mL sample protector for 1 h. For cultured cells or bacteria, centrifugation is required before adding the sample protector. Gram-positive bacteria must be disrupted by 20 mg/mL lysozyme (200 μL for 107 cells). For Gram-negative bacteria that possess thicker cell walls, such as Staphylococcus, lysostaphin is effective. Grinding with liquid nitrogen is effective for cell disruption of plant and animal tissue. The disruption step uses cell lysis buffer and proteinase K to lyse cells and degrade proteins. The cell debris is eliminated by centrifugation. Adding RNase or DNase to the supernatant of dissociation can specifically yield pure genomic DNA or RNA extracts. A total of 100 μg MGO was added to the supernatant (107 cells) to capture nucleic acids. After 30 min, the MGO−nucleic acid compound was washed with 1× phosphatebuffered saline buffer three times. Then, the obtained MGO−nucleic acid compound was stored in a freezer at −20 °C.

In recent years, the extensive applications of graphene oxide (GO), such as for imaging,19,20 drug delivery,21,22 biosensing,23−25 and even antimicrobial purposes,26,27 have attracted significant interests. It is well known that GO, a planar nanomaterial,28,29 is endowed with the capacity to immobilize nucleic acids (including RNA) via π−π stacking.30,31 It has been demonstrated that the stability of short RNAs (usually ∼20 bp in length) can be enhanced through GO/RNA interaction.32 However, whether large RNA molecules, such as total RNA (23S, 16S, and 5S RNA in procaryotic organism, and 28S, 18S, and 5S in eucaryotic organism), can be protected using GO has not been investigated. It is usually believed that the protection of total RNA by GO is impossible due to the lengths of the total RNA, which range from a few to thousands of bases, and their complicated structure, which is inclined to undergo desorption. Interestingly, we discovered that GO can effectively protect total RNAs in a highly efficient manner, even when the RNA is exposed to air for 4 days; such protection is more effective than that by protein-based RNA inhibitors. Additionally, the protection mechanism is experimentally observed. Furthermore, GO can be conjugated to nanomagnetic beads, defined as magnetic graphene oxide (MGO). We speculated that the grid structure of GO provides a capture section for RNA molecules and the RNA protection capacity of GO affords RNA continuous protection during the extraction and storage processes. To simplify the separation process of RNA extraction, amino-coated magnetic beads (MBs) were linked with GO to construct magnetism-functionalized GO (MGO) via the formation of an amide linkage between the carboxyl of GO and the amino of the MBs. It enables the construction of a rapid and high-performance nucleic acid extraction assay.

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EXPERIMENTAL SECTION

Reagents. GO (1 mg/mL, RNase free), RNase inhibitor, Taq DNA polymerase, and a total RNA extraction kit were used as received. The detailed parameters and manufacturers are listed in the Experimental Section of the Supporting Information. RNA Protection Assay. Generally, newly extracted RNA from 107 cells was mixed with 10 μg of RNase-free GO. The resultant mixture was vigorously shaken to ensure sufficient adsorption. The mixture was then incubated for 10 min before use. Reverse-Transcription Polymerase Chain Reaction (RTPCR). The RT-PCR of Listeria monocytogenes mRNA was performed

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RESULTS AND DISCUSSION Confirmation of RNA Protection by GO. We used the newly extracted total RNAs from L. monocytogenes to evaluate B


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Figure 2. Quantitative analysis of GO-assisted RNA protection and identification of RNA usability. (A) Time frame of GO-assisted RNA protection. The 23S RNA and 16S RNA were recovered from the corresponding electrophoretic bands. (B) RNase tolerance of GO-protected RNA. One unit of RNase is defined as the amount of enzyme required to fully digest 1 pmol of synthetic 33-mer single-stranded RNA in a total reaction volume of 10 μL in 15 min. (C) Quantitative comparisons of RNA-degradation rate between the GO and inhibitor groups. (D) Assessment of RNA usability using PCR. The iap gene can also be amplified effectively by reverse-transcription quantitative PCR.

RNA-Protection Capacity of GO. We further investigated the long-term RNA stability in the GO system. The time frame of RNA protection was investigated (Figure 2A). After electrophoresis, RNA bands were recovered and analyzed by UV−vis absorption spectroscopy. The degradation levels of 23S RNA and 16S RNA indicated that GO (10 μg) provided a long-acting protection for 4 days in air. The tolerance to RNase assay (Figure 2B) revealed that 10 μg of GO inhibited the degradation against 40 units of RNase. These experiments simultaneously demonstrated that GO holds its strong potency of RNA protection. Meanwhile, we explored the effect of GO particle size on the RNA-protection capacity using commercial GO (Figures S1 and S2). The results showed that three sizes of GO (1 μm, 500 nm, and 200 nm) were all able to protect RNA. A GO particle size of 1 μm was optimal for RNA protection. According to previous studies, the adsorption capacity of GO depends on the grain diameter,33 which consequently affects the protection capacity. Furthermore, we designed the concentration gradient of GO for a certain amount of RNA. The results in Figure S3 indicate that the degradation rate decreased with the concentration of GO. And, a plateau occurs at 10 μg.

whether GO could be an effective protection agent for large RNA molecules. The ability of GO to protect RNA was primarily evaluated by artificially introducing RNase to newly extracted RNA. The RNA stability in the absence and presence of GO was compared. The results showed that the RNA mixed with GO could significantly resist RNase degradation (electrophoretic lanes b and c in Figure 1A). The degree of RNA degradation was compared by calculating the grayscale intensity ratio of the bands representing the newly extracted and treated total RNAs. More than 75% of the RNA was degraded when RNase was applied, and more than 85% RNA still maintained its integrity in the GO-assisted system (b and c in Figure 1B). RNA is also quite unstable when exposed to air without specific protection. Experiments using total RNAs exposed to air in the presence or absence of GO were executed to evaluate the protection effects provided by GO under normal conditions. It is shown that after exposure to air for 24 h, the total RNAs (mixed with GO, lane e of Figure 1A) exhibited remarkable stability compared with the control group (lane d of Figure 1A). Grayscale analysis showed that only approximately 10% of the RNA was degraded when GO was applied. C


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Figure 3. Probable mechanism for GO-assisted RNA protection. (A) Immobilization of RNase by GO. (B) Adsorption of total RNAs by GO. (C) Co-immobilization of RNase and RNA by GO. (D) Experiments verified that immobilization of RNase by GO contributes to RNA protection to a certain extent. “T1” represents the untreated initial RNA, T2 represents RNA mixed with free RNase, and T3 represents RNA incubated with GOimmobilized RNase. (E) Adsorption of total RNAs by GO provided a preemptive protection shield. “G1” refers to the GO-adsorbed total RNAs without RNase and “G2” refers to the GO-adsorbed total RNAs with RNase.

endowed with the ability to adsorb protein.34−37 Specifically, the active site of RNase is located in the interior of its threedimensional structure.38−41 The immobilization of RNA on the rigid GO plane may act as a shield to prevent RNA from accessing the RNase active site. To validate whether such an effect is responsible for the observed inhibition, the enzymatic activity of GO-immobilized RNase was evaluated (Figure 3A) and the possible situation after immobilization of RNase on GO was summarized (Figure S4). The RNase was adequately saturated for immobilization on GO and centrifuged to remove the suspended RNase. Total RNA was added as the substrate. After 2 h of incubation, we examined the integrity of the total RNAs. The results in Figure 3D indicate that the immobilized RNase lost most of its enzymatic activity (“T3” group in Figure 3D) compared with the dissociative RNase (“T2” group in Figure 3D). Thus, the speculation that RNase immobilization

The protection provided by commercial RNase inhibitor and that by GO was also directly compared. As shown in Figure 2C, the degradation ratio of RNA + RNA inhibitor and RNA + GO against 40 units of RNase shows a striking difference. All three kinds of RNAs gained satisfactory stability (55% of RNA lost its stability in a RNA inhibitor protection strategy, again confirming the superiority of GO as a RNA stabilizer. In addition, reverse-transcription PCR (RT-PCR) was implemented using GO-protected RNA templates to investigate whether GO adsorption limited the subsequent use of RNA. The iap gene was amplified and detected using electrophoresis. As shown in Figure 2D, the RT-PCR products yielded obvious electrophoretic bands. Principles of GO-Assisted RNA Protection. These findings indicate that GO markedly suppressed the enzymatic activity of RNase. Previous studies have indicated that GO is D


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Figure 4. Synthetic route of MGO and the scheme of RNA extraction assay. (A) Amino-coated magnetic beads (MBs) were linked with GO via the formation of amide bond between carboxyl of GO and amino of magnetic beads. (B) The character of MB, GO, and MGO. (C) The scheme of RNA extraction assay.

Figure 5. Steps used in the extraction of RNA by MGO. (A) The protective RNA extraction assay consists of sample pretreatment, cell disruption, RNA capturing, and washing steps. The cell disruption step uses the cell lysis buffer and proteinase K to lyse cells and degrade proteins. (B) AFM characterization of intact L. monocytogenes. (C) AFM characterization of lysed L. monocytogenes. (D) Electrophoresis results of RNA extracted with MGO. T1 and T2 are two parallel experiments.

the centrifugation process), and the possible situation after RNA was immobilized by GO was also summarized (Figure S5). The RNase (30 units) was subsequently added to this mixture and incubated for 2 h (at 37 °C). The stability of the adsorbed RNA was then evaluated. In Figure 3E, the G1 group (without RNase) and G2 group (with RNase) produced identical results, which indicated that GO effectively protected the adsorbed RNA. Because GO is conventionally used in excess, the RNAprotection effects of GO must reflect the joint contributions of

reduced the freedom of RNase and sealed its active site was supported by these data. At the same time, it is well known that GO holds strong ability to adsorb nucleic acids via the π−π stacking effect.42,43 It is not difficult to infer that immobilization of RNA on a rigid GO plane may prevent it from gaining access to the active site of RNase, which likely interferes with its enzymatic degradation (Figure 3B). To support the above assumptions, we mixed GO with excess RNA to generate a saturated adsorption interface (unbound RNA was almost eliminated by E


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Figure 6. MGO shows high RNA-extraction efficiency and stability. (A) Electrophoresis experiments assessing RNA protection by MGO. The initial concentration of total RNA was 100 ng/μL. After the RNA was exposed to air, the RNAs in different groups were examined using 1% agarose electrophoresis and stained with 1× SYBR Gold. (1) MGO-extracted RNA and DNA exposed to air for 24 h. (2) Newly MGO-extracted RNA and DNA. (3) TRIzol-extracted RNA and DNA exposed to air for 24 h. (4) Newly TRIzol-extracted RNA and DNA. (B) Comparison of the degradation rates of RNA extracted by MGO and TRIzol methods. Newly extracted total RNA by MGO and TRIzol methods were exposed to air for 24 h and analyzed by electrophoresis. The degradation rates of RNA were assessed by comparing the grayscale values with those of the control group. (C) Time series of MGO-assisted RNA protection. The 23S RNA and 16S RNA were purified from total RNA extracts of L. monocytogenes. (D) RNase tolerance of MGO-protected RNA. (E) Representative RNA extracts from animal cells and tissues, blood, and plants using MGO. (1) LO2 cells; (2) MCF7 cells; (3) liver of mouse; (4) heart of mouse; (5, 6) whole blood of mouse; (7) tender leaf of papaya; (8) Arabidopsis; and (9) asparagus.

immobilization of RNase and immobilization of RNA (as depicted in Figure 3C). The immobilization of RNA, which is the primary cause of RNA protection, generates a preemptive shield that prevents the enzymatic degradation process. The immobilization of RNase, which enhances the RNA protection of GO, executes the function of passive inhibition. The relative characterization data are shown in Figure S6. RNA-Extraction Assay Using Magnetism-Functionalized GO. The tight interaction between GO and total RNAs prompted us to explore the function of GO for capturing and extracting the RNA. We speculated that the grid structure of GO provides a capture section for RNA molecules and the RNA-protection capacity of GO affords a RNA continuous protection during extraction and storage processes. To simplify the separation process of RNA extraction, aminocoated magnetic beads (MBs) were linked with GO to construct magnetism-functionalized GO (MGO) via the formation of an amide linkage between the carboxyl of GO and the amino of the MBs (Figure 4A). The GO solution was yellow, whereas MGO appeared black and darker than the MBs; MGO also aggregated when a magnetic field was applied (Figure 4B). Atomic force microscopy (AFM) data, particle size, and ζ-potential simultaneously revealed that MGO was successfully constructed (Figure S7). MGO shows excellent

dispersibility, thus providing a foundation for subsequent RNA extraction (Figure S8). The protective RNA extraction assay includes sample pretreatment, cell disruption, RNA capture, and washing steps (Figure 4C). A protective RNA-extraction procedure from L. monocytogenes is described in Figure 5A. The AFM results for intact and lysed L. monocytogenes are shown in Figure 5B,C. After the cell disruption, capturing, and washing steps, the extracts were evaluated by electrophoresis, whose results in Figure 5D indicate that the protective RNA extraction assay based on MGO can reliably and easily extract intact RNA from L. monocytogenes. Meanwhile, the obvious genomic DNA bands appearing in the wells at the top of the gel indicate that this assay can simultaneously extract genomic DNA, which is mainly attributed to the contribution of the adsorption induced by MGO. Thus, it seems that this assay can simultaneously extract RNA and genomic DNA. Adding RNase or DNase to the supernatant of the cell disruption step can preferentially isolate pure genomic DNA or total RNA. Evaluations of the Protective Capability of MGO. The RNA protection provided by MGO was evaluated to verify whether the structural change in GO (linkage with MBs) interfered with the RNA protection property. Analogously, we F



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investigated the RNA protection property of MGO by exposing RNA and DNA extracted with MGO to air for 24 h (group “1” in Figure 6A). Compared with newly MGOextracted RNA and DNA (group “2” in Figure 6A), the electrophoresis bands from group 1 did not exhibit marked degradation, which confirmed that MGO was endowed with the ability to protect the RNA. In contrast, the RNAs extracted with conventional TRIzol kits exhibited obvious degradation when exposed to air for 24 h (group “3” in Figure 6A) compared with the newly TRIzol-extracted RNA (group “4” in Figure 6A). Meanwhile, the degradation rate also confirmed the RNA protection capacity of MGO (Figure 6B). In addition to exploring the RNA protection capacity of MGO, the time frame and RNase tolerance of MGO-assisted RNA protection were simultaneously evaluated by analyzing the integrity of the 23S and 16S RNAs. The results recorded in Figure 6C,D indicate that the efficacies of RNA protection provided by MGO and GO were almost identical. Thus, the ability of MGO to protect RNA was fully verified. The RNA extraction efficiencies of the TRIzol- and MGO-based extraction assays are compared in Figure S9 and indicate that the extraction efficiency of the MGO-based assay was almost equal to that of TRIzol extraction. Notably, the MGO-based protective RNA extraction assay also provided effective protection for RNA, which the TRIzol kit did not. We also investigated the performance of the MGO-based RNA-extraction strategy for samples derived from animal cells and tissue, blood, and plant tissue. DNase was added to the supernatant of the cell disruption step to eliminate genomic DNA. Cultured animal cells were first processed with the protective RNA extraction method. As shown in Figure 6E, the electrophoresis bands of group 1 and group 2 (1 was normal LO2 cells; 2 was tumor MCF7 cells) indicated that this strategy can effectively extract RNA from animal cells. Subsequently, animal tissues including mouse liver (group 3) and heart (group 4) were processed. The electrophoresis results showed that the protective RNA-extraction strategy was capable of extracting RNA from animal tissues. In addition, protective RNA extraction strategy was also applicable to blood samples. In addition, mouse blood samples (group “5” and group “6” in Figure 6E) were tested. The results recorded in Figure 6E reveal that the the protective RNA-extraction strategy can reliably extract RNAs from plant samples (group “7”, papaya; group “8”, Arabidopsis; group “9”, asparagus). To exclude other factors, we investigated whether MB possess the capability of RNA protection and extraction. The results in Figure S10 indicate that RNA mixed with MB showed intense degradation after exposure to air for 24 h. It proved that the MB did not possess the capability to protect the RNA. The results in Figure S11 indicate that the MB possesses the capability of RNA extraction. But, the yield was much lower than the MGO.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b12522. Characterization and supporting data, principle ratiocination, extraction efficiency comparisons (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (X.Z.). *E-mail: [email protected]. Tel: (+86-20) 8521-0089. Fax: (+86-20) 8521-6052 (D.X.). ORCID

Yuhui Liao: 0000-0003-4702-9516 Xiaoming Zhou: 0000-0001-5597-4804 Da Xing: 0000-0002-5098-0487 Author Contributions

The manuscript was written through contributions of all the authors. All the authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant 21475048), the National Science Fund for Distinguished Young Scholars of Guangdong Province (Grant 2014A030306008), the Project of Guangzhou Science and Technology Plan (Grant 201508020003), the Program of the Pearl River Young Talents of Science and Technology in Guangzhou (Grant 2013J2200021), the Special Support Program of Guangdong Province (Grant 2014TQ01R599), and the Outstanding Young Teacher Training Program of Guangdong Province (Grant HS2015004).

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CONCLUSIONS We demonstrate here that GO could provide a remarkable RNA-protection effect, which may be attributed to the ability of GO to act as a preemptive shield. The experimental results indicate that GO provides substantially better RNA protection than commercial RNase inhibitors do. We also constructed MGO to collect high-quality RNA efficiently with long-acting and continuous protection. Hence, GO and MGO, which can act as potent RNA-stabilizing and -extracting agents, possess a high potential for applications in RNA fields. G


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Graphene Oxide as a Bifunctional Material toward Superior RNA

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