ISSN 1998-0124 CN 11-5974/O4 2019, 12(8): 1952–1958 https://doi.org/10.1007/s12274-019-2463-z

Research Article Research Construction of RNA nanotubes

Hui Li1,†,§, Shaoying Wang1,‡,§, Zhouxiang Ji1,§, Congcong Xu1, Lyudmila S. Shlyakhtenko2, and Peixuan Guo1 ()

1 Center for RNA Nanobiotechnology and Nanomedicine; Division of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy; Department of Physiology & Cell Biology, College of Medicine; Dorothy M. Davis Heart and Lung Research Institute and James Comprehensive Cancer Center, The Ohio State University, Columbus, OH, 43210, USA 2 UNMC Nanoimaging Core Facility, Department of Pharmaceutical Sciences, College of Pharmacy University of Nebraska Medical Center, Omaha, NE, 68182, USA † Present address: University of California, San Francisco, CA, 94158, USA ‡ Present address: P&Z Biological Technology, Newark, NJ, 07103, USA § Hui Li, Shaoying Wang, and Zhouxiang Ji contributed equally to this work.

© Tsinghua University Press and Springer-Verlag GmbH Germany, part of Springer Nature 2019 Received: 12 February 2019 / Revised: 6 June 2019 / Accepted: 17 June 2019

ABSTRACT Nanotubes are miniature materials with significant potential applications in nanotechnological, medical, biological and material sciences. The quest for manufacturing methods of nano-mechanical modules is in progress. For example, the application of carbon nanotubes has been extensively investigated due to the precise width control, but the precise length control remains challenging. Here we report two approaches for the one-pot self-assembly of RNA nanotubes. For the first approach, six RNA strands were used to assemble the nanotube by forming a 11 nm long hollow channel with the inner diameter of 1.7 nm and the outside diameter of 6.3 nm. For the second approach, six RNA strands were designed to hybridize with their neighboring strands by complementary base pairing and formed a nanotube with a six-helix hollow channel similar to the nanotube assembled by the first approach. The fabricated RNA nanotubes were characterized by gel electrophoresis and atomic force microscopy (AFM), confirming the formation of nanotube-shaped RNA nanostructures. Cholesterol molecules were introduced into RNA nanotubes to facilitate their incorporation into lipid bilayer. Incubation of RNA nanotube complex with the free-standing lipid bilayer membrane under applied voltage led to discrete current signatures. Addition of peptides into the sensing chamber revealed discrete steps of current blockage. Polyarginine peptides with different lengths can be detected by current signatures, suggesting that the RNA-cholesterol complex holds the promise of achieving single molecule sensing of peptides. KEYWORDS RNA nanotechnology, peptide sensing, RNA nanotube, nanobiotechnology

1 Introduction via bottom-up self-assembly. RNA is also more thermodynamically stable compared with DNA. Varieties of RNA architectures with The advancement of nanotechnology has inspired many applications defined shape, size and stoichiometry have been constructed for such as micro-electro mechanical systems (MEMs) [1, 2], transistors diverse applications, such as therapeutics delivery, cancer targeting, [3–5], chips [6–8], sensors [9, 10], circuits [11–13], nanorobotics [14], immunomodulation, and sensing [29, 30, 34–37]. logic gates [15, 16], resistive memory [17], medical apparatus [18, 19], Here we report the design, construction and characterization of and many other practices in nanotechnology, medical, biological molecularly defined RNA nanotubes. Nanotubes with hollow channels and material sciences [20–23]. The precise control over size and are fundamental structures in nanotechnology and nanoscience. length of nanostructures is highly important. For example, the Previously, different nanotubes such as carbon nanotubes [38, 39], precise control of the width of carbon nanotubes has been reported inorganic nanotubes [40], peptide nanotubes [41, 42] and nucleic while the length control remains challenging [24–26]. Similar to acid nanotubes [43, 44] have been widely studied. After insertion other organic and inorganic nanotubes, the length control heavily into a lipid bilayer membrane, nanotubes form artificial channels. relies on the growth time and physical/chemical method of isolation, Different analytes can pass through or interact with the channel, thus making the precise length control difficult [27, 28]. thereby producing electrochemical signals. By analyzing the recorded The last two decades have witnessed the development of the ionic current, it is possible to obtain distinct current signatures emerging field of RNA nanotechnology and its potentials for material for different analytes such as DNA and peptides. Owing to the sciences and biomedical applications [29–33]. As naturally occurring programmability of RNA biopolymer, we utilized RNA nano- polymers, RNA molecules can fold into unique three-dimensional technology to develop novel RNA nanotubes for applications in (3D) structures and play various biological roles in vitro and in vivo. peptide sensing. In our current study, stable RNA nanotubes were The diversity of RNA in structures and functions enables itself to designed in silico and fabricated via bottom-up self-assembly. The be used as building blocks for bottom-up self-assembly of various fabricated RNA nanotubes exhibited well-defined stoichiometry, size de novo architectures with unique physical/biochemical properties and structure characterized by polyacrylamide gel electrophoresis

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(PAGE) and atomic force microscopy (AFM). The cell binding and 1.1.1 (Release Date: July 17, 2008). For the design of the first electrophysiology property of RNA nanotubes inserted in lipid bilayer generation RNA nanotube, six RNA strands were used to assemble were also investigated. With the addition of positively charged the tube by utilizing an approach of RNA origami similar to DNA peptides into the chamber, discrete steps of current blockage were origami’s [43–46], forming a 11 nm long hollow channel (Fig. S1 in observed. Therefore, we expected that the RNA nanotube-cholesterol the Electronic Supplementary Material (ESM)) with the inner complex inserted into the lipid membrane could be developed for diameter of 1.7 nm and the outside diameter of 6.3 nm (Figs. 1(a) the sensing of positively charged peptides and potentially used for and 1(b)). To assemble the RNA nanotube with cholesterol, two other applications. more chemically synthesized RNA strands with cholesterol at the end were used, and all synthesized RNA strands were mixed in 2 Results stoichiometric ratio and annealed in 1 × TMS buffer in a one-pot manner. Step-wise self-assembly of the RNA nanotubes with and 2.1 Two approaches for the design and construction of without cholesterol was examined by gel electrophoresis, confirming the formation of the higher-ordered RNA nanotubes (Figs. 1(c) and RNA nanotubes 1(d)). The formation of RNA nanotubes was also characterized by RNA nanotubes were designed by NanoEngineer software, version AFM (Fig. 1(e)). The size and shape of the observed RNA nanotubes

Figure 1 Design, construction and physiochemical characterization of origami designed RNA nanotube. (a) Two-dimensional (2D) sequence design of the RNA nanotube composed of 6 RNA strands labeled by six different colors. (b) Schematic side view of RNA nanopore structure. (c) 4% agarose gel electrophoresis showing the step-wise assembly of origami designed RNA nanotube. (d) 4% agarose gel electrophoresis showing the assembly of origami designed RNA nanotube with two cholesterol molecules. Ladder: low range DNA ladder. (e) AFM analysis of origami designed RNA nanotubes without cholesterol. AFM images of representative individual RNA nanotubes are shown in the left panel. AFM images of several RNA nanotubes in the same field are shown in the right panel.

www.theNanoResearch.com∣www.Springer.com/journal/12274 | Nano Research 1954 Nano Res. 2019, 12(8): 1952–1958 under AFM are in agreement with the predicted structure as designed, results have shown cholesterol anchored RNA nanotube groups confirming the successful self-assembly of the RNA nanotubes. displayed strong Cy5 fluorescent signal on cell membranes indicating For the second generation RNA nanotube design containing cholesterol anchored RNA nanotubes mostly interacted with the three cholesterol molecules [47, 48], six RNA strands were designed cell membrane (Fig. (3)). As a result of the active uptake by the cell, to hybridize by base pairing and formed the six-helix channel. some internalized RNA nanotubes were also observed. Channel size similar to the first generation RNA nanotube design The electrophysiological property of RNA nanotubes within the was predicted since both designs were composed of six RNA helixes lipid membrane was further investigated to test whether the RNA hybridized together (Fig. 2(a)). Compared with the first generation nanotube can be inserted into the lipid membrane. The small design, this approach has several advantages such as ease of unilamellar vesicles/RNA nanotube with cholesterol complex were fabrication, higher yield of pores at higher concentration, and lower added to the cis-chamber and incubated with the lipid bilayer synthesis cost. RNA nanotubes without cholesterol successfully membrane under applied voltage of −50 mV. A current jump of assembled into high molecular weight RNA nanotubes step-wisely 150–200 pA was observed (Fig. 4(a)). It was noted that the frequency (Fig. 2(b)). However, after incorporating three strands with cholesterol of producing current jumps for the first generation RNA nanotube molecules, some resulting structures did not run into the gel and with two cholesterols was lower than the one of the second generation stuck in the loading well. This is possibly due to partial aggregated RNA nanotube with three cholesterols. It might indicate that nanotubes resulting from the hydrophobic interaction among increasing the number of cholesterol could enhance the interactions cholesterol molecules (Fig. 2(c)). between the RNA nanotube and the lipid membrane. The current 2.2 Insertion of the RNA-cholesterol complex into the jump was normalized and summarized by conductance (Fig. 4(b)). It was found that RNA nanotubes with three anchored cholesterols lipid bilayer resulting in discrete current jump caused current jumps even in the absence of liposome preparation. To study whether RNA nanotubes can interact with the cell membrane, In both approaches distribution of conductance is relatively wide, RNA nanotubes with and without cholesterol together with several possibly due to the aggregation of RNA nanotubes induced by other controls, were incubated with LnCap cell. Cy5 fluorophore cholesterol. This is also revealed in the assembly gel in which some was conjugated to these structures for tracking the interaction. The cholesterol-anchored RNA nanotubes got stuck in the well (Fig. 2(c)).

Figure 3 Binding of RNA nanotubes to the cellular membrane. The confocal microscope was used to examine LnCap cells after incubation with RNA nanotubes harboring cholesterol. RNA nanotubes without cholesterol, single strand RNA and cell only were used as the negative control as indicated in the image. The DAPI and Alexa 488 were used to stain the cell nuclei and actin. Cy5 fluorophore was used to track the binding of RNA nanotubes to the cellular membrane.

Figure 2 Design and assembly of the second generation RNA nanotubes. (a) 2D design of the second generation RNA nanotubes. Six RNA strands are bound together by base paring and each RNA strand forms one face of the six-helix channel. (b) 4% agarose gel electrophoresis showing the step-wise assembly of the second generation RNA nanotubes. (c) 4% agarose gel electrophoresis showing Figure 4 Characterization of RNA nanotube insertion into lipid bilayer. (a) the assembly of the second generation RNA nanotubes with three cholesterol Current trace of multiple RNA nanotube/liposome insertion. (b) Conductance molecules. Ladder: ultra low range DNA ladder. distribution of RNA nanotube/liposome. Buffer: 1 M KCl, 5 mM HEPES, pH 8.

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Addition of peptides into the cis-chamber revealed discrete steps and self-assembled in a single-step production. The fabricated RNA of current blockage. Interestingly, the addition of poly-arginine nanotubes were further characterized by gel electrophoresis peptide could reduce the current jump (Fig. 5) generated by the and AFM, confirming the spontaneous one-pot assembly of RNA liposome-RNA-cholesterol complex. The poly-arginine peptides with nanostructures. Cholesterol molecules were introduced into the different lengths (R2, R4, R6, R8, R10) showed reduced current jump, RNA nanotube to facilitate their incorporation into the lipid bilayer. indicating the liposome-RNA-cholesterol complex could potentially Incubation of RNA nanotubes with the isolated lipid bilayer be applied for peptide sensing. Moreover, the addition of negatively membrane resulted in a reproducible discrete current signature. This charged peptide poly-glutamic acid (EEE EE) or neutral charged unique current change might be caused by ions passing through the peptide poly-glycine (GGG GG) caused little current blockages hollow structure or the margin between the RNA nanotube and the compared with the positively charged peptide TAT (CYG RKK RRQ lipid membrane. Cholesterol modification could facilitate the process RRR; Fig. S2 in the ESM). of RNA insertion into the lipid membrane. With three anchored cholesterol molecules, RNA nanotubes could be directly inserted into lipid membrane, which was also observed in DNA nanopores [49]. In both approaches, distribution of conductance is relatively wide, possibly due to the aggregation of RNA nanotube induced by cholesterol. This is also revealed in the assembly gel in which some cholesterol–anchored RNA nanotubes stuck in the well (Fig. 2(c)). The discrete steps of current blockage was observed after the addition of peptides with different lengths into the cis-chamber. This current blockage may be caused by the interaction of a positively charged peptide with a negatively charged RNA tube or by the passing through of the peptide. Other studies have reported that translocation of analytes or binding of analytes with membrane proteins could affect ionic flow of the channel, causing the reduction of current jump [50–54]. Current blockage was not observed in the presence of negatively charged peptides. We suspected that negatively charged analytes could not enter the negatively charged RNA nanotube. Therefore, we proposed the electrostatic interactions between RNA and poly-arginine peptides may play a role in the sensing of positively charged peptides by RNA nanotubes. Though the charge-charge interaction may decrease the significance of this sensing platform, this limitation could possibly be overcome by using chemically- Figure 5 Sensing of poly-arginine peptide. Typical current traces with the modified materials such as peptide nucleic acid (PNA) which may addition of polyarginine R2, R4, R6, R8, R10. R represents arginine. Voltage: reduce the negative charge of RNA. No matter whether the peptide −50 mV. Buffer: 0.4 M KCl, 5 mM HEPES, pH 8. has passed through the channel or simply blocked the pore, the generation of uniform and repeated current blockage revealed the 3 Discussion potential in applying the system for peptide detection, sensing, differentiation, and disease diagnosis. In this study, we have successfully designed, constructed and assembled RNA nanotubes. The assembled RNA nanotube was further characterized by gel assay and AFM imaging, confirming the 4 Conclusion spontaneous self-assembly of the RNA nanotube. The cell binding RNA nanotubes were successfully assembled by two different strategies and electrophysiological property of RNA within lipid bilayer were in this study. Change of migration rate in gel electrophoresis and also characterized. formation of tube-like structures revealed by AFM imaging con- The de novo designed RNA nanostructure can provide numerous firmed the spontaneous one-pot assembly of RNA nanostructures. advantages, such as tunable size, predictable functionality, ease of Incorporation of cholesterol into the RNA nanotube facilitated their construction, and site-directed modification. Owing to its unique insertion into the lipid bilayer, resulted in discrete current jumps physical/biochemical properties, RNA nanotubes can be further under applied voltage. The addition of poly-arginine peptides caused explored as biosensors [9, 10], MEMs [1, 2] and many other devices current blockage, suggesting that the RNA-cholesterol complex has with potential applications in analytical chemistry, biomedical the potential for peptide sensing at the single molecule level. engineering, drug delivery and tissue engineering. Compared with DNA, RNA has stronger thermodynamic stability and more sophisticated versatility concerning both structure and function. 5 Experimental Compared with protein, RNA has greater control over the self-assembly 5.1 Materials of its 3D structure due to the relative simplicity of RNA base pairing among four different bases. Thus, RNA nanotubes have their own The phospholipid 1,2-diphytanoyl-sn-glycerol-3-phosphocholine uniqueness and advantages compared with other nanotubes. Moreover, (DPhPC) was obtained from Avanti Polar Lipids, Inc. Organic the developments of RNA nanotubes may also pave the way to solvents (n-decane and chloroform) were purchased from Fisher better understand the fundamental mechanisms for many other Scientific, Inc. and TEDIA, Inc., respectively. TAT (CYG RKK RRQ nanotubes assembled by different biomolecules such as DNA and RRR), poly-glutamic acid (EEE EE), poly-glycine (GGG GG) and peptide nanotubes. The assembly strategies described here may also poly-arginine peptides with 2, 4, 6, 8, 10 amino acids were custom- be further expanded and applied to construct more sophisticated ordered from GenScript, Inc. n-octyl-oligo-oxyethylene was purchased 3D RNA nanostructures with different size and shapes. from Enzo Life Sciences, Inc. All other reagents were purchased Herein, RNA nanotubes were designed by modular strategy from Sigma or Fisher, if not specified.

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5.2 Design, synthesis, and self-assembly of RNA nanotubes Y693F mutant T7 polymerase and 2’-F modified cytosine (C) and uracil (U) nucleotides were used to synthesize these 2’F-modified The NanoEngineer program was used to facilitate the design of the RNA strands. RNA nanotubes. Unmodified RNA strands were synthesized by in vitro T7 transcription and purified by 8 M urea, 8% denaturing c. The second generation RNA nanotubes with three cholesterol PAGE. DNA templates for transcribing the RNA strands were made molecules: by polymerase chain reactions (PCR). DNA oligonucleotides for PCR were directly ordered from IDT, Inc. Cholesterol modified RNA Strand 1: 5’- GGAUACGGGU AGG AAAA ACAGCUCAUA strands were ordered from IDT or synthesized by Azco Oligo-800 CCCAGCACAU UG AAAA GCCCUUCUC -3’ – cholesterol; DNA and RNA synthesizer. Strand 2: 5’- GGUCGACAUU AGC AAAA CCUACCCGUA The RNA nanotubes were self-assembled in a one-pot manner by UCCGAGAAGG GC AAAA GCACAUCGU -3’; mixing the synthesized RNA strands in equimolar concentrations Strand 3: 5’- GGCGGACGAU GAC AAAA GCUAAUGUCG in 1× TMS buffer and heated to 95 °C and slowly cooled down to ACCACGAUGU GC AAAA ACUCGACUA -3’- cholesterol; room temperature. The step-wise self-assembly of the RNA nanotubes Strand 4: 5’- GGCUGCCUCA UGC AAAA GUCAUCGUCC was examined by agarose gels. The gels were stained with ethidium GCCUAGUCGA GU AAAA UCCAACUCC -3’; bromide (EB) and imaged by Typhoon FLA 7000 (GE Healthcare). Strand 5: 5’- GGGUGAACGU UGA AAAA GCAUGAGGCA The detailed sequences of the designed RNA nanotubes are GCCGGAGUUG GA AAAA CUGAUGCAC -3’ – cholesterol; shown below: Strand 6: 5’- GGGUAUGAGC UGU AAAA UCAACGUUCACCC GUGCAUCAG AAAA CAAUGUGCU -3’. a. The first generation RNA nanotubes: 5.3 AFM Strand 1: 5’- GGGUCUG CUGUGUC CGUAUCACCA UGGG GUCUUCC ACAUGCCAAC GUUGA CAUGCGAG UUACAAGCCG The RNA nanotubes were imaged by AFM, following previously UGUAGC GCCCAUCC UGUCGAGU -3’ ; described methods [55]. Briefly, RNA nanotubes were placed on Strand 2: 5’- GGACAGCUGG AUGGGCAUAG GCUCUUCCCG the APS-modified mica surface and excess samples were washed AAAA GUUACAG UGCAUCC ACUCGACA CUCAACUG with DEPC water and dried before imaging. AFM imaging was CCCAUGG UGAUACG AAAA GACACAGCAG GUUC -3’; performed by using the MultiMode AFM NanoScope IV system Strand 3: 5’- GGAUGCA CCACGAU UCCGCCUGCU ACUG (Veeco) operated in tapping mode. AUCGUAG GUAAUCG AAAA CGAUUAC AGCUGGU GCUACACG GCUUGUAA GGCGAUU CAGACCC -3’; 5.4 In vitro binding of RNA nanotubes to cancer cells Strand 4: 5’- GGGUUGAGGA AGACGGAGUA CGUCGAGCAG Prostate cancer LnCap cells were grown on glass cover slides in AUCGUGGUAU ACUC AAAA GAGUAUA CUGUAAC CGGGAAG 24-well plates in RPMI-1640 medium with 10% FBS at 37 °C in AGCCUAU ACCAGCU UGCGUAU -3’; humidified air containing 5% CO2 overnight. 2’F-modified RNA Strand 5: 5’- GGCAUGU GCAUCAG AAAA CGGUAUUGGA nanotubes harboring cholesterol and labeled by Cy5 were diluted in CGGCCUCGCA UGAUGGCUCC AUACGCACUA CGAU AAAA optium-MEM medium to 100 nM and incubated with the cells for CAGUAGC AGGCGGA UCAGCUCA UCAACGUU -3’; 2 h at 37 °C. RNA nanotubes without cholesterol and single strand Strand 6: 5’- GGAGCCAU UGAGCUGA CUGCUCGA RNA were used as the negative control. After the incubation, the cells CGUACUCC CAGUUGAG AGCUGUCC GAACCUG AAUCGCC were washed with PBS and fixed by 4% paraformaldehyde. Alexa GCCGUCC AAUACCG CUGAUGC UCAACCC -3’. 488 Phalloidin (Life Technologies) was used to stain the cellular actin. The Prolong Gold antifade reagent with DAPI (Life Technologies) were b. The first generation RNA nanotubes with two cholesterol then used to stain the cell nucleuses and mount the cells to the glass molecules: slides. The confocal images were recorded by the FluoView FV1000- Filter Confocal Microscope System (Olympus). Strand 1: 5’- GGGUCUG CUGUGUC CGUAUCACCA UGGG GUCUUCC ACAUGCCAAC GUUGA CAUGCGAG UUACAAGCCG 5.5 Electrophysiology property of RNA characterization UGUAGC GCCCAUCC UGUCGAGU -3’; Strand 2: 5’- GGCCCG AAAA GUUACAG UGCAUCC within lipid bilayer ACUCGACA CUCAACUG CCCAUGG UGAUACG AAAA Planar bilayer lipid membranes (BLMs) were generated in a BCH-1A GACACAGCAG GUUC -3’; horizontal BLM cell (Eastern Scientific). A Teflon partition with a Strand 3: 5’- GGAUGCA CCACGAU UCCGCCUGCU ACUG 200 μm aperture was placed in the apparatus to separate the BLM AUCGUAG GUAAUCG AAAA CGAUUAC AGCUGGU GCUACACG cell into cis- (top) and trans- (bottom) compartments. A planar GCUUGUAA GGCGAUU CAGACCC -3’; lipid bilayer was formed by painting the aperture with 0.5 μL of 3% Strand 4: 5’- GGGUUGAGGA AGACGGAGUA CGUCGAGCAG (w/v) DPhPC in n-decane. A conducting buffer (1 M KCL, 5 mM AUCGUGGUAU ACUC AAAA GAGUAUA CUGUAAC CGGGAAG HEPES, pH 8) was added to both the top and bottom compartments AGCCUAU ACCAGCU UGCGUAU -3’; of the BLM cell, and Ag/AgCl electrodes were placed in the buffer Strand 5: 5’- GGCAUGU GCAUCAG AAAA CGGUAUUGGA of each compartment. The electrode in the trans-compartment was CGGCCUCGCA UGAUGGCUCC AUACGCACUA CGAU AAAA connected to the headstage of an Axopatch 200B amplifier (Axon CAGU -3’; Instruments, Inc.), and the electrode in the top compartment was Strand 6: 5’- GGAGCCAU UGAGCUGA CUGCUCGA grounded. CGUACUCC CAGUUGAG AGCUGUCC GAACCUG AAUCGCC For direct incubation between RNA and lipid membrane, a GCCGUCC AAUACCG CUGAUGC UCAACCC -3’; mixture of cholesterol-anchored RNA nanotubes and 0.5% n-octyl- Strand 7: 5’- GGACAGCUGG AUGGGCAUAG GCUCUU – oligo-oxyethylene dissolved in conducting buffer was added to the cholesterol - 3’; cis side of the bilayer to a final concentration of 100 nM nanotubes. Strand 8: 5’- cholesterol - AGC AGGCGGA UCAGCUCA For liposome-guiled interaction between RNA and lipid membrane, UCAACGUU -3’. a two-step procedure was employed [56] for this design. DPhPC For cell binding experiments, strands 1, 2, 3, 4, 5 and 6 were lipids in chloroform were dehydrated to eliminate solvents first and 2’F-modified to increase their stability in the cell culture medium. then rehydrated with buffer containing 250 mM sucrose and purified

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