A High-yield Method to Fabricate and Functionalize DNA

Subscriber access provided by STEPHEN F AUSTIN STATE UNIV

Article

A High-yield Method to Fabricate and Functionalize DNA Nanoparticles from the Products of Rolling Circle Amplification Xuexia Yuan, Fan Xiao, Haoran Zhao, Yishun Huang, Chen Shao, Leilei Tian, and Yossi Weizmann ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00238 • Publication Date (Web): 01 Aug 2018 Downloaded from http://pubs.acs.org on August 1, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Bio Materials

A High-yield Method to Fabricate and Functionalize DNA Nanoparticles from the Products of Rolling Circle Amplification Xuexia Yuan, † a Fan Xiao, † a Haoran Zhao, a Yishun Huang, a Chen Shao, a Yossi Weizmann*b and Leilei Tian *a

a

Department of Materials Science and Engineering, Southern University of Science and Technology,

1088 Xueyuan Blvd., Nanshan District, Shenzhen, Guangdong 518055, P. R. China. b Department of Chemistry, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA. †

There authors contributed equally to this work.

KEYWORDS. DNA nanostructures, condensation, rolling circle amplification, ratiometric fluorescence sensor, lysosomal pH tracker

ACS Paragon Plus Environment

ACS Applied Bio Materials 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 30

ABSTRACT

DNA condensation is a facile method to construct DNA nanostructure with high biostability and low cost, which is mainly used in DNA separation and gene transfection. The recent emerging condensed DNA nanostructures from the rolling circle amplification (RCA), i.e., the complexes between RCA products and magnesium pyrophosphate (RCA-MgPPi), have quickly become attractive biomedical materials with broad application potentials, because they combine the advantages of the designable and high-throughput isothermal amplification technique and the high stability of DNA condensation structures. However, we find that only approximately 10% of RCA products can be condensed after an RCA reaction, which limits the practical application of the RCA-MgPPi nanostructures. Therefore, in this paper, we investigate how to control the condensation efficiency of RCA-synthesized DNAs in depth. The very long RCA products, which show high charge densities, can be efficiently condensed by an excessive amount of Mg2+ to form RCA-MgPPi nanostructures at a yield approaching 100%. What's more, the new condensation approach is general and not limited to the RCA products, which can be applied to other polymeric DNAs. These RCA-MgPPi nanoparticles exhibit a high bio-stability and low toxicity, in addition, which can be efficiently functionalized with foreign components to create hierarchical properties. Finally, as a proof of concept, based on RCA-MgPPi nanostructures, a ratiometric fluorescence sensor system has been constructed and demonstrated to be an efficient lysosomal pH tracker.


Page 3 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Nucleic acids are macromolecules with simple but definite molecular structures; the combination of the four nucleotides can precisely encode nearly unlimited information, which is the most attractive feature of nucleic acids. Therefore, compared with other nanostructures, self-assembled DNA nanostructures can organize foreign bio-active components on the nanoscale in a precise and programmable way, realizing interesting biomedical functionality. Pioneered by Seeman’s work, welldesigned one-dimensional,1 two-dimensional,2 and three-dimensional3,4 artificial DNA nanostructures have been created by virtue of the elegant Watson-Crick base-pairing rule, which show broad applications in nanotechnology, biology, and material science.5-12 Toward the biomedical applications, DNA nanostructures show incomparable advantages with regards to their rich functionality, excellent biocompatibility, and low toxicity; however, they still face some challenges, such as their unsatisfactory biostability for intracellular and in vivo applications, complicated fabrication process, and a high cost for a large number of oligonucleotides.13 As a natural phenomenon, genomic materials in DNA condensation are tightly packed inside limited spaces, such as viral capsids and cell nuclei. DNA condensation in vitro can be induced with the help of multi-valent cations, positively charged surfactants, and neutral poor solvents,14-19 which is a facile method to construct DNA nanostructures with high biostability and low cost. As a result, it has been applied in the field of gene delivery and extraction.20,21 Recently, a novel DNA condensation system has been produced from rolling circle amplification (RCA).22 By RCA technique, a high quantity of meaningful sequences equipped with either designable functions or self-assembly capabilities are amplified.23 Moreover, the RCA process will simultaneously produce a high concentration of magnesium pyrophosphate (MgPPi), which will lead the flexible long RCA products condense and form organic-inorganic hybrid micro/nanostructures (RCANPs in abbreviation). Apart from the good biostability of the very condense structure and the good bio-compatibility due to the intrinsic low toxicity of MgPPi,24 the other important feature of the resulting DNA micro/nanostructures is that the ACS Paragon Plus Environment


Page 4 of 30

RCA products with well-defined sequences can act as structural scaffolds to realize programmable functionalization.25 Therefore, this kind of DNA nanostructures quickly attracted a great deal of research attentions in the biomedical field.26-29 Hammond and co-workers constructed RCANPs consisting of cleavable RNA strands, which were converted to small interfering RNAs (siRNAs) by the intracellular RNA machinery, therefore providing inherent protection for siRNA delivery and resulting in improved therapeutics.30-32 Tan and co-workers constructed many multifunctional RCANPs that were integrated with aptamers, bioimaging agents, and drug loading sites for multiplexed cellular imaging and targeted drug delivery.33,34 Although RCANPs have been widely used in the biomedical field, the formation mechanism is still under investigation. The RCANPs formed in situ after an RCA reaction (iRCANPs) are generally too big (∼1-2 µm) for biomedical applications (Figure S1a), and hence, most efforts have been paid to optimizing the RCA reactions (such as the template design, reaction time, and reactant concentrations) to decrease the particle sizes.13,34-37 In addition, we observed that only approximately 10% of RCA products can be converted to nanoparticles after an RCA reaction. Although the leftover products showed no difference from the condensed part, it was hard to improve the condensation yield through optimizing the reaction conditions. Also, the condensed structure could not be completely dissociated at a high temperature (95 °C), and therefore, it was difficult to modify iRCANPs with the functional components through DNA hybridization. Herein, we demonstrated a new method to fabricate RCANPs, which can realize a high condensation yield and more accessible functionalization (Scheme 1). For this new method, MgPPi produced by the RCA reaction was first removed. Consequently, the base-pairing capability of RCA products with well-defined sequences was restored, therefore, the purified RCA products can act as structural scaffolds to realize multiple functionalization. Next, we found that, prior to MgPPi precipitation, the addition of an excessive amount of Mg2+ to the purified RCA products was very important for the successful condensation. Reaching a high P/N ratio (the ratio between the positive charges of Mg2+ and the negative charges of the DNA phosphate backbone) will lead DNAs to collapse from the random coil to globule conformation. Thereafter, the excessive Mg2+ near the vicinity of the DNA chains would precipitate with the ACS Paragon Plus Environment

Page 5 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


pyrophosphate ions (PPis), forming nano-scaled RCANPs with a well-defined morphology. Moreover, it is a universal method to prepare DNA nanoparticles from any polymeric DNAs, not only RCA products. Finally, these DNA nanoparticles (named u-RCANPs) with diameters of ∼300 nm were produced in a yield approaching 100%, which show very similar properties to i-RCANPs, such as a rough morphology, condensed inner-structure, good resistance to biological degradation, and low cytotoxicity. It is worth noting that, compared with the i-RCANPs, u-RCANPs can be more efficiently functionalized through base-pairing with foreign components, which will greatly promote the practical application of this technique. As a proof of concept, through template design, the RCA products comprised of different blocks were synthesized, which can hybridize with different complementary sequences labelled with different dyes (FAM and CY3). Well functionalized u-RCANPs exhibiting multi-color emissions were applied to live cell imaging. As the FAM dye is sensitive to pH and the CY3 dye is not,38,39 the resultant u-RCANPs were demonstrated to be a ratiometric fluorescence sensing system that were used to monitor the intracellular lysosomal pH.



Page 6 of 30

Scheme 1. Schematic illustration of the formation of i-RCANPs and u-RCANPs based on rolling circle amplification. EXPERIMENTAL SECTION Chemicals. MgCl2, Na4P2O7, Ethylene Diamine Tetraacetic Acid Disodium Salt (EDTA-Na2), Tris(hydroxymethyl)aminomethane (Tris), hydrochloric acid (HCl), phenol/chloroform /isoamyl alcohol (25:24:1), sodium acetate (NaAc), ethyl alcohol (99.9%), and 5× tris/boric acid/EDTA (TBE) buffer were purchased from Bioson Corporation (Beijing, China). GelRed (×10,000) nucleic acid stain was obtained from Biotium, Inc. (USA). Nigericin (sodium salt), thiazolyl blue tetrazolium bromide (MTT), and all the DNA sequences given in Table S1 were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Deoxyribonucleic acid sodium salt from salmon tests and deoxyribonucleic acid (low molecular weight) from salmon sperm were purchased from Sigma Life Science (Saint Louis, MO, USA). Phi 29 polymerase was purchased from Thermo Fisher (USA). T4 ligase, deoxyribonucleotide triphosphates (dNTPs), and 1 kb DNA ladder were purchased from Takara (China). LysoBriteTM Blue was purchased from AAT Bioquest, Inc. (Sunnyvale, California). Phosphate buffered saline (PBS) was prepared by solving the tablets obtained from Amresco (Solon, OH, USA) in ultrapure water according to the instruction manual (10 mM PBS, containing 137 mM Na+ and 2 mM K+). Agrose RA was purchased from AMRESCO (Solon, USA). Aqueous solutions were prepared with ultrapure water obtained from a Millipore synergy UV Ultrapure water purification system (Millipore Co., MA, USA). MCF-7 cells (human breast adenocarcinoma cell line) were friendly provided by Professor Ying Sun at the Department of Biology of Southern University of Science and Technology (SUSTech). Fetal bovine serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM), and penicillin/streptomycin were obtained from Gibco BRL Co., Ltd. (Grand Island, NY, USA). Other chemicals not mentioned here were used as received without further purification. Instruments. RCA reactions proceeded on Eppendorf ThermoMixer®C. Agarose gel electrophoresis was run on Bio-Rad miniprotern® Tetra system and imaged by Tannon 3500. Zeta potentials were ACS Paragon Plus Environment

Page 7 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


determined on NanoBrook ZetaPALS Potential Analyzer (Brookhaven). Microscopic images were taken on a Tescan Mira3 Field Emission Scanning Electron Microscopes (FE-SEM) and a Hitachi HT7700 Transmission Electron Microscope (TEM). A Cytation 3 microplate reader (BioTek) was used for determining the concentration of DNA, calibrating condensation yield and hybridization efficiency, and performing the MTT assay. The fluorescence images of cells and RCANPs were taken on Leica TCSSP8 laser scanning confocal microscope (LSCM) with various excitation wavelengths. Fluorescence spectra were recorded on FluoroMax-4 fluorescence spectroscopy was used to record (HORBIN Jobin Yvon). The ultrasonic treatment was conducted with a SB-5200 DTD Ultrasonic Cleaner (Ningbo Scientz). RCA reaction. There are three steps in a standard RCA reaction: (1) Annealing: DNA template (T1, T2, or T3, 0.3 µM) was mixed with its primer (P1, P2 or P3, 0.6 µM) in T4 ligase buffer [50 mM TrisHCl, 10 mM MgCl2, 10 mM Dithiothreitol (DTT), 1 mM ATP, pH=7.5]. After being heated at 90 °C for 10 min, the mixture was gradually cooled to room temperature. (2) Ligation: The template-primer hybrid was mixed with T4 DNA ligase (10.4 U µL-1), reacted at 16 °C for 16 h, and subsequently heated at 65 °C for 10 min to inactivate the ligase. (3) Amplification: the ligation product was mixed with dNTPs (2 mM each) and Phi 29 DNA polymerase (0.2 U µL-1) in RCA reaction buffer (50 mM TrisHCl, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT). The amplification reaction was processed at 30 °C for 24 h, and then heated at 85 °C for 10 min to inactivate the DNA polymerase. The RCA products were treated with EDTA (0.5 M) to dissolve MgPPi thoroughly to obtain non-viscous RCA products, which were analysed by 1% agarose gel electrophoresis. RCA products purification. First, RCA product (50 µL) was diluted by deionized water (25 µL), and EDTA (0.5 M, 10 µL) was slowly added to the above solution and mixed well. After that, DNA sample changed from turbid to transparent solution with a low viscosity. Deionized water (300 µL) was added into the sample for dilution and further purification. Equal volume of organic solvent (Phenol/Chloroform/Isoamyl alcohol=25:24:1) was fully mixed with DNA sample. Transfer the aqueous ACS Paragon Plus Environment


Page 8 of 30

phase to a new tube, followed by adding 1/10 volume of NaAc (pH 5.2, 3 M). Another 2.5 volume of ice ethanol was added to precipitate DNAs. After being storing at -24 °C for 2 h, DNA precipitation was isolated from the mixed solution by centrifugation. The precipitation was re-dissolved in a certain buffer and quantified according to the absorption at 260 nm using a BioTek microplate reader. RCA/Mg2+ system conformations. The laser light scattering (LLS) experiment was performed on a laser scattering spectrometer (BI-200SM) equipped with a digital correlator (BI-9000AT) and a solid laser (35 mW and 637 nm). The radius of gyration (Rg) was obtained with the BI-SLSW software, which was studied over an angular range of 35∼155° and based on Berry Plot. The hydrodynamic radius (Rh) was obtained with the BI-DLSW software. Preparation of the u-RCANPs. Here we obtain u-RCANPs by dropwise adding the RCA products (concentration: 0.01 mg mL-1 ∼ 1 mg mL-1) into a solution containing a certain concentration of Mg2+ to reach a variety of P/N ratios (0 ∼ 1600), and the mixture was incubated at 30 °C for 12 h. Thereafter, NaPPi (0.5 ∼ 9 mM) was dropwise added the previous mixture, which was incubated at 85 °C for 20 min, cooled to 30 °C, and then kept undisturbed at 30 °C for 48 h until reaching the equilibrium. To fabricate the multi-color u-RCANPs, the purified RCA products were incubated with FAM and CY3 functionalized complementary sequences at 90 °C for 10 min prior to the other fabrication process. Cell culture. MCF-7 cells (human breast adenocarcinoma cell line) were cultured in DMEM medium supplemented with 10% (v/v) FBS, 1% penicillin/streptomycin at 37 °C in an INCO2/153 CO2 incubator (Memmert, Germany; 5% CO2). Cytotoxicity test. MCF-7 cells were seeded into a 96-well plate at a density of 8000 cells/well in DMEM (100 µL) containing 10% FBS and cultured at 37 °C under CO2 (5%, v/v) for 24 h. The cells were incubated with variable concentrations of u-RCANPs in DMEM containing 10% FBS for 12 h. 10 µL of the MTT solution (5 mg mL-1 in PBS) was added to each well and incubated for 4 h. Then, the medium was gently removed, and the formazan products were solubilized with 150 µL of dimethyl ACS Paragon Plus Environment

Page 9 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


sulfoxide (DMSO) for 10 min in a swag bed. Absorbance of each plate well was measured by a Bio-Tek Cytation 3 at 490 nm. Cell viability was expressed as a ratio of the absorbance 490 nm of u-RCANPs treated cells to that of the untreated controls. All measurements were performed in quintuplicate. Intracellular Detection. MCF-7 cells were seeded into a 96-well plate at a density of 5000 cells/well in DMEM (100 µL) containing 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin and incubated for 24 h. Then the medium was replaced by 100 µL of fresh medium containing the prepared multi-color uRCANPs (20 µg mL-1). After incubation for 3 h, cells were washed with warm PBS, and the cell images were taken under LSCM. The excitation wavelength used to detect the multi-color u-RCANPs was 488 nm, and the collected emissions of FAM and CY3 were 510 to 540 nm, 568 to 650 nm, respectively. For nigericin induction experiment, MCF-7 cells were seeded into a 96-well plate at a density of 5000 cells/well in DMEM (100 µL) containing 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin and cultured at 37 °C under CO2 (5%, v/v) for 24 h. The medium was replaced by 100 µL of fresh medium containing the multi-color u-RCANPs (20 µg mL-1). After incubation for 3 h, the culture medium was removed and the cells were washed three times with PBS. Subsequently, medium of pH 7.3 with nigericin (1 µg mL-1) were added to the well. With 1 h incubation, the fluorescence images were taken on the LSCM. Calculation of condensation yield and functionalization efficiency. The condensation yields of the RCA were calculated via the following equation (a). According the standard preparation protocol, the uRCANPs was synthesized, and the resultant solution was centrifuged at 8000 rpm to collect the supernatant, whose concentration was quantified according to the absorption at 260 nm using a BioTek microplate reader. Finally, the condensation yield was calculated according to the equation (a):

Condensation yield =

஼೚ ି஼ೞ ஼೚

× 100%

(a)

where C0 and CS are the original concentration of DNA and non-condensed DNA in the supernatant respectively.



Page 10 of 30

The functionalization efficiency of the u-RCANPs and i-RCANPs were calculated via the equation (b). For u-RCANPs, FAM-CS1 functionalized u-RCANPs were fabricated first. The purified RCA products were incubated with FAM-CS1 (the molar ratios of FAM-CS1 to the binding sites on RCA products were 0.1, 0.2, 0.5, 0.8, 1, and 1.5) at 90 °C for 10 min prior to the other fabrication process. After reaching the equilibrium, the mixtures were centrifuged and the fluorescence intensities of the supernatants were detected by the microplate reader. For i-RCANPs, the in situ formed i-RCANPs were incubated with FAM-CS1 (the molar ratios of FAM-CS1 to the binding sites on RCA products were 0.1, 0.2, 0.5, 0.8, 1, and 1.5) at 95 °C for 10 min and cooled down to room temperature. After reaching the equilibrium, the mixtures were centrifuged and the fluorescence intensities of the supernatants were detected by the microplate reader. Finally, the functionalization efficiency was calculated according to the equation (b):

Functionalization efficiency =

ூೀ ିூೄ ூೀ

× 100%

(b)

where Io is the original fluorescence intensity of FAM-CS1 solution; and Is is the fluorescence intensity of FAM-CS1 in the supernatant which has not been functionalized into u-RCANPs or i-RCANPs. pH titration. The emission spectra were measured in PBS buffer (10 mM). The PBS buffer with pH values at 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.2, 7.4, 7.6, 7.8 and 8.0 were prepared according to the standard procedures. For titration experiments, a test solution of u-RCANPs (0.1 mg mL-1) was prepared by placing it into PBS buffer (10 mM) in a quartz optical cell with 1.0 cm optical path length. NaOH (500 mM) or HCl (500 mM) were used to adjust the pH of the test solutions. All the resulting solutions were thoroughly mixing at 25 °C for 10 min before fluorescence spectral measurements. RESULTS AND DISCUSSION We first prepared i-RCANPs according to the literature method, i.e., running the rolling circle amplification in the presence of 2 mM dNTPs for 24 h long and heating at 85 °C for 10 min to quench the RCA reaction. Flower-like nanoparticles (i-RCANPs) with diameters of ∼2 µm (Figure S1a) were ACS Paragon Plus Environment

Page 11 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


produced, which is in good accordance with the previous reports.26,27,34,36,40 Thus far, there is still the lack of an efficient and consistent method to control and tune the diameters of i-RCANPs, which are generally too large for biomedical applications. Besides, the yield of i-RCANPs is only approximately 10%, with the major part of RCA products left in the supernatant (Figure S1b). When analyzed by agarose gel electrophoresis, the RCA products in the precipitate and supernatant showed no difference in sequence length (Figure 1a). However, even though the reaction temperature (30 and 37 °C), reaction time (8, 12, 24 h), and RCA templates (T1, T2 and T3) and primers (P1, P2 and P3) have been carefully optimized, the condensation yields did not show any improvements (Figure S2 and Table S2). Further, the total RCA products were purified by an ethanol precipitation method, and the purified DNA was redispersed in the RCA buffer (containing 10 mM MgCl2) and incubated according to the same RCA reaction protocol. During the incubation process, sodium pyrophosphate (NaPPi) was slowly added to the DNA solution until a final concentration of 10 mM was reached. Although precipitate was generated, it was finally demonstrated to be pure MgPPi without condensed DNAs. This experiment indicated that, without the enzymatic amplification process, DNAs cannot condense in the RCA buffer; and the forming mechanism of i-RCANPs is still unclear. All these observations drive us to further investigate the formation mechanism of RCANPs. Occasionally, we revealed that the purified RCA products would go through a conformational transition in the presence of excessive Mg2 by laser light scattering (LLS), which is a classic method to study the conformations of bio-macromolecules.41,42 According to the LLS data (Figure 1b), as the P/N ratio increased from 0 to 1600, the radius of gyration (Rg) gradually decreased from 174.3 nm to 91.5 nm, indicating that the RCA products shrank in the presence of excessive Mg2+. At the same time, the Rg/Rh (Rh is the hydrodynamic radius) ratios changed from 1.58 for a P/N ratio of 0 (RCA products in pure water) to 0.71 for a P/N a ratio of 1600. Based on the established principle, a Rg/Rh ratio larger than 1.5 indicates a random-coil conformation, while a Rg/Rh ratio smaller than 0.7 corresponds to a solid-sphere conformation.43 The results coincide well with the facts that pure RCA products will adopt coil conformations due to the strong repulsion between their negatively charged phosphate backbone,44 ACS Paragon Plus Environment


Page 12 of 30

and these long ssDNAs was found to significantly shrink in the presence of excessive Mg2+. It is well known that DNA can be condensed by small multi-valent counter-ions, such as Co3+, Al3+, and Ga3+, which is the result of the charge neutralization of DNAs.45 However, as an intrinsic metal ion, Mg2+ will be more biocompatible compared with other di- or trivalent metal ions. Compared with the high valence salts (≥ 3), Mg2+ is less efficient for neutralization, and therefore, an excessive amount of Mg2+ is required to induce the full compaction of DNAs. To further characterize the compaction process, the RCA products with the presence of different amounts of Mg2+ were analyzed by a zeta potential test (Figure 1c). The RCA products in pure water showed a zeta potential of -30.4 mV, which turned to almost neutral when the P/N ratio reached 400. In the meantime, the compaction also reached its equilibrium, i.e., no significant reduction in Rg was observed when the P/N ratios further increased. The results confirmed that the compaction of RCA products in the presence of Mg2+ was mainly caused by charge neutralization. We also noted that the values of Rh did not decrease along with the increase in P/N ratios, but they increased a little bit when the P/N ratios surpassed 200. Since the intensities of scattering light quickly increased as more Mg2+ ions were mixed with the RCA products (Figure S3), we assumed that the compaction was probably a multi-chain process.46 As the DNA compaction was accompanied by multi-chain aggregations, as a result, the Rh value showed no significant change. Furthermore, with the addition of NaPPi, the accumulated Mg2+ near the vicinity of the DNA chain would precipitate, which further stabilized the compacted DNAs. The exciting result is that the RCA DNAs were completely condensed to reach a production yield of 100% (Figure 2a), and the resultant nanoparticles fabricated by this new method are called u-RCANPs. As shown in Figure 2c and 2d, the SEM and TEM images indicated that u-RCANPs showed very similar morphologies to i-RCANPs, and both displayed condensed interiors and rough surfaces. Unlike “flower-like” i-RCANPs, u-RCANPs were more like “claw-like” structures. More importantly, the diameter of u-RCANPs was reduced to ∼300 nm, making the nanoparticles more suitable for biomedical applications. The elemental compositions of these two types of RCANPs were analyzed by energy dispersive X-ray spectroscopy (EDS), as shown in Figure S4. The results revealed that u-RCANPs showed a very similar composition ACS Paragon Plus Environment

Page 13 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


to that of i-RCANPs, including carbon, nitrogen, oxygen, magnesium, and phosphorus, which were the composites between DNA and MgPPi.

Figure 1. (a) Agarose gel electrophoresis analysis of an RCA product: Lane 1: DNA ladder, lane 2: iRCANPs, and lane 3: the RCA products leftover in supernatant. (b) Laser light scattering data of RCA products (0.1 mg mL-1) in the presence of different amounts of Mg2+; P/N ratio is the ratio between the positive charges of Mg2+ and the negative charges of the DNA phosphate backbone; Rg is the radius of gyration and Rh is the hydrodynamic radius. (c) Zeta potentials of various Mg2+/DNA complexes under different P/N ratios. Error bars indicate standard error of triplicate tests. (d) An illustration of DNA condensation process in the presence of Mg2+. Furthermore, the new fabrication method was investigated in depth. Figure 2a describes the condensation yields of different types of DNAs (0.1 mg mL-1), including RCA products (long ssDNA), ACS Paragon Plus Environment


Page 14 of 30

long dsDNA (DNAs from fish sperm >2000 bp abbreviated as FSDNA2000), and short DNAs (“crude oligonucleotides” 1 mM) of PPi was used. With regard to the morphology (Figure S7), undispersed precipitate was collected when 0.5 mM PPi was used and ill-defined aggregates started to form when the concentration of PPi was increased to 1 mM. Well-defined u-RCANPs were formed when 3 mM PPi ACS Paragon Plus Environment

Page 15 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


was used, however, further increasing the concentration of PPi (> 6 mM) was found to cause crosslinking between the nanoparticles. (3) The properties of the DNAs are also important for nanoparticle formation. We observed that the short FSDNA50 cannot be condensed by this method. As Mg2+ is a relatively weaker condensation agent compared with other high-valence cations, long DNAs showing high charge density could facilitate the condensation process. On the other hand, we found that FSDNA2000 could be condensed by this method (Figure S1c), which indicated that this method was a universal method applicable for any polymeric DNAs. It is of note that the condensation of RCA products (long ssDNA) was much easier than that of FSDNA2000 (long dsDNA), i.e., the FSDNA2000 requires a P/N ratio larger than 100 to realize a 100% condensation, while the corresponding P/N ratio for RCA products is 60. This is reasonable because ssDNA exhibits a much smaller persistence length compared with dsDNA.47



Page 16 of 30

Figure 2. Characterization of u-RCANPs. (a) The condensation yield of RCA products (red line), long double-strand DNAs (> 2000 bp) from fish sperm FSDNA2000 (black line), and short double-strand DNAs (< 50 bp) FSDNA50 (blue line), during the fabrication of nanoparticles at different P/N ratios. (b) The condensation yield of u-RCANPs at a fixed P/N ratio of 1600 but different concentrations of NaPPi and DNA. (c)-(d) SEM and TEM images of u-RCANPs. RCA is an isothermal amplification method with thousands of amplifications in quantity. It produces long single-stranded DNAs, which show periodic sequences with each of their repetitive fragments complementary to the template sequence. The RCA products with well-defined sequences can act as structural scaffolds to realize the programmable functionalization of the resultant DNA nanostructures. As a proof of concept, template T1 (Table S1) was designed to produce an RCA product with distinct ACS Paragon Plus Environment

Page 17 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


binding sites. Two complementary sequences, FAM-CS1 and CY3-CS2 (whose sequences are shown in Table S1), were synthesized and used for functionalization, which were labelled with FAM and CY3, respectively. Both FAM-CS1 and CY3-CS2 are 18-mer sequences which can hybridize on different sites of the RCA products. First, we studied the efficiency of using a complementary sequence (FAM-CS1) to functionalize the RCANPs. The purified RCA products were annealed with FAM-CS1 at different molar ratios. Thereafter, the resultant hybrids were processed by the steps for u-RCANPs fabrication. The dye-functionalized u-RCANPs were centrifuged and collected, and the functionalization efficiency was calculated by measuring the fluorescence of the remaining supernatant. As shown in Figure 3, the functionalization efficiency approached a maximum of ∼95% when the ratio was 1:1, which began to decline as more FAM-CS1 sequences were added. The result indicated that the very efficient functionalization was mainly formed by means of base-pairing interactions. On the contrary, the in situ formed i-RCANPs showed a very low functionalization efficiency of ∼6%. To reveal the reason, iRCANPs were heated to 95 °C and instantaneously cooled on ice, and thereafter, the sample was characterized by SEM. As shown in Figure S8, the SEM images of i-RCANPs before and after the thermal treatment showed no apparent change in morphology. This confirmed our assumption that iRCANPs could not unfold upon heating, and as a result, they could not be efficiently functionalized via structural re-organization. It is likely that only the sequences exposed on the surface of i-RCANPs could be hybridized with FAM-CS1. Therefore, the images taken by confocal laser scanning microscope (CLSM) showed that u-RCANPs were more fluorescent compared with i-RCANPs upon functionalization with FAM-CS1. Tan and co-workers have used dye-labelled deoxyuridinetriphosphate (dUTP) to run the RCA reaction and successfully functionalize the i-RCANPs.34-36 Our new method showed a high flexibility and efficiency in functionalization, which will make the RCANPs more useful in biomedical applications.



Page 18 of 30

Figure 3. (a) Schematic illustration of FAM functionalized u-RCANPs. (b) Functionalization efficiency of u-RCANPs (black line) and i-RCANPs (red line), the scale bars are 50 µm. Multi-fluorescent nano-systems generally display high sensitivity in imaging with improved spatial and temporal resolution in live specimens. Therefore, the u-RCANPs functionalized with two different dyes, FAM and CY3, were applied as a multi-color cell-imaging agent. It is well-known that FAM is a pH-sensitive dye,48 while CY3 shows pH-independent fluorescence,49,50 and thus the pH sensitivity of the resultant u-RCANPs was investigated. As shown in Figure 4a, the images taken in the green emission channel showed bright fluorescence in the neutral (pH 7.0) and basic buffers (pH 8.0), but negligible fluorescence in the acidic buffer (pH 5.8). Meanwhile, the images taken in the yellow emission channel showed unchanged fluorescence in all the three buffers. As shown in Figure 4b, the emissions of u-RCANPs were detected at 518 nm (for FAM) and 652 nm (for CY3), respectively, throughout a physiological pH range (5.8-8.0). The emission from FAM showed a linear decrease as the pH decreased from 8.0 to 5.8; and at a pH of 5.8, the fluorescence from FAM was almost completely diminished. However, the emission from CY3 was basically constant in the pH range from 8.0 to 5.8. It ACS Paragon Plus Environment

Page 19 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


has been demonstrated that the properties of the two dyes have been well inherited by the functionalized u-RCANPs. Moreover, the emission ratio (I518 nm/ I562 nm) could reversibly respond to pH switches from 8.0 to 5.8 (Figure 4c), which demonstrated the good reliability of u-RCANPs working as a ratiometric fluorescence probe. Upon all these in vitro results, the possibility of using u-RCANPs as a pH indicator for live-cell monitoring was further detected. First the cytotoxicity was evaluated using the MTT assay. MCF-7 cells were incubated with various concentrations of u-RCANPs (5, 10, 20, 50 and 100 µg mL-1) in DMEM containing 10% FBS for 12 h. As shown in Figure S9, the u-RCANPs exhibited no obvious cytotoxicity or any side effects in live cells. Nanostructures with a good capability against nuclease degradation are very crucial for the intracellular application. u-RCANPs were incubated with FBS (which has an intrinsic DNase activity) at 37 °C for 3 h, 24 h, and 6 days, respectively, and they were washed a couple of times prior to taking the SEM images. As shown in Figure S10, these u-RCANPs remained intact in the presence of FBS, even with prolonged incubation times of up to 6 days, indicating that the u-RCANPs have good stability in biological environments.



Page 20 of 30

Figure 4. (a) Fluorescence images (with scale bars of 100 µm) of multi-color u-RCANPs cofunctionalized with FAM and CY3 in PBS buffers under different pH values. (b) Ratiometric fluorescence of u-RCANPs under different pH values. The black line: the fluorescence intensities at 518 nm (from FAM) of u-RCANPs were excited at 488 nm; and the red line: the fluorescence intensities at 562 nm (from CY3) of u-RCANPs were excited at 530 nm; All fluorescence intensities were normalized with respect to the fluorescence intensity at pH 7.0. (c) The reversible change of fluorescence intensity ratio (I518 nm/I562 nm) of u-RCANPs in response to pH switches between 8.0 and 5.8. For cell-imaging application, the u-RCANPs (20 µg mL-1) were first incubated with MCF-7 cells for 3 h, and then washed three times with PBS. According to the CLSM images of the resultant cells (Figure 5a), only yellow fluorescence was detected, while no green fluorescence was observed. We assumed that the u-RCANPs have been trapped in some acidic organelles. Lysosomes are dynamic, membranebound organelles, which contain a variety of digestive enzymes working in an acidic environment (pH 3.8-5.0).42 To prove this assumption, nigericin (1 µg mL-1) was added to the cell-culture media to elicit a rapid external and internal pH equilibrium at 7.4.51 Through such a standard approach to calibrate the pH value ex vivo, both green and yellow emissions were observed from the up-taken nanoparticles, as shown in Figure 5a. As more straightforward evidence, a commercial lysosome tracker, LysoBrite Blue, was incubated with MCF-7 cells together with the u-RCANPs to perform a colocalization experiment. 52,53

As shown in Figure 5b, cell images confirmed that the yellow signals from u-RCANPs and the blue

signals from LysoBrite originated from approximately the same cellular region. Combining all the evidence, we can conclude that the multi-color u-RCANPs mainly accumulated in the acidic lysosomes. Real-time monitoring of the lysosomal pH is of great importance, as abnormal lysosomal pH values can result in cellular dysfunction and some consequent diseases including cancer, shock, Alzheimer’s disease and rheumatoid arthritis. Based on their good tracking specificity and high resolution from the ratiometric fluorescence imaging, the functionalized u-RCANPs could act as a potential candidate for a lysosomal tracking agent (Figure 5c). ACS Paragon Plus Environment

Page 21 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. (a) Living cell fluorescence images (with scale bars of 50 µm) of MCF-7 cells probed by uRCANPs in non-treated natural state and in nigericin-treated state. (b) CLSM images of intracellular colocalization of u-RCANPs and LysoBrite in MCF-7 cells: the blue channel to detect LysoBrite was excited at 405 nm and collected at 420-470 nm; the yellow channel to detect CY3 was excited at 552 nm and collected at 568-650 nm (scale bars = 75 µm). (c) Schematic illustration of the working principle of multi-color u-RCANPs as a lysosome tracker.



Page 22 of 30

CONCLUSIONS Once the RCA-MgPPi composite nanostructures were occasionally observed as surprise products of an RCA reaction, they quickly attract a lot of attentions in the field of nanotechnology and biotechnology due to their application potentials in gene/drug-delivery. As the DNA sequences amplified by RCA are designable and can act as templates for further modifications, the resultant nanostructures show some similarities to DNA origami but are more stable in the bio-environment and cost efficient. In this paper, we fundamentally explored the synthesis strategy of RCA-MgPPi nanostructures and successfully achieved a 10-fold improvement in synthesis yield, a good control to particle size, as well as a very high functionalization efficiency. Moreover, the forming mechanism of RCA-MgPPi nanostructures in this new method has been well understood, providing directions for the future development of this technique. As the resultant nanoparticles can be efficiently modified through base-pairing interactions, and, as a proof of concept, multi-color DNA nanoparticles for pH sensing have been developed and applied to live cell imaging. In summary, based on all these improvements, we believe this high-yield synthesis method will promote the practical applications of this kind of interesting DNA nanoparticles in the future. ASSOCIATED CONTENT Supporting Information. The following files are available free of charge. A list of DNA sequences; the morphology of i-RCANPs and the leftover RCA products in the supernatant; the condensation yields of i-RCANPs under different optimized conditions; during LLS analysis, the intensity changes of scattering light along with P/N ratio changes; EDS analysis of the elementary compositions of i-RCANP and u-RNCANP; the influences of P/N ratios, the concentrations of DNA, and the concentrations of PPi on the morphologies of u-RCANPs; SEM characterization of the morphology changes during the annealing treatment to i-RCANPs; the cytotoxicity of u-RCANPs under


Page 23 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


different concentrations; the stability of u-RCANPs in serum. (PDF)

AUTHOR INFORMATION Corresponding Author [email protected] * [email protected] ACKNOWLEDGMENT The work was supported by grants from the National Natural Science Foundation of China (No. 51503096), Shenzhen Fundamental Research Programs (JCYJ20160226193029593), and Guangdong Innovative and Entrepreneurial Research Team Program (No. 2016ZT06G587). This work is also partially supported by the NSF CAREER Award (DMR-1555361) to Y.W. REFERENCES (1) Chen, G.; Liu, D.; He, C.; Gannett, T. R.; Lin, W.; Weizmann, Y. Enzymatic Synthesis of Periodic DNA Nanoribbons for Intracellular pH Sensing and Gene Silencing. J. Am. Chem. Soc. 2015, 137, 3844-3851. (2) Ke, Y.; Meyer, T.; Shih, W. M.; Bellot, G. Regulation at a Distance of Biomolecular Interactions Using a DNA Origami Nanoactuator. Nat. Commun. 2016, 7, 10935-10942. (3) Zhan, P.; Dutta, P. K.; Wang, P.; Song, G.; Dai, M.; Zhao, S.-X.; Wang, Z.-G.; Yin, P.; Zhang, W.; Ding, B. Reconfigurable Three-Dimensional Gold Nanorod Plasmonic Nanostructures Organized on DNA Origami Tripod. ACS Nano 2017, 11, 1172-1179. (4) Pei, H.; Zuo, X.; Zhu, D.; Huang, Q.; Fan, C. Functional DNA Nanostructures for Theranostic Applications. Acc. Chem. Res. 2013, 47, 550-559. (5) Seeman, N. C.; Kallenbach, N. R. Design of Immobile Nucleic Acid Junctions. Biophys. J. 1983, 44, 201-209.



Page 24 of 30

(6) Iwaki, M.; Wickham, S. F.; Ikezaki, K.; Yanagida, T.; Shih, W. M. A Programmable DNA Origami Nanospring that Reveals Force-Induced Adjacent Binding of Myosin VI Heads. Nat. Commun. 2016, 7, 13715-13724. (7) Lee, D. S.; Qian, H.; Tay, C. Y.; Leong, D. T. Cellular Processing and Destinies of Artificial DNA Nanostructures. Chem. Soc. Rev. 2016, 45, 4199-4225. (8) Shen, X.; Song, C.; Wang, J.; Shi, D.; Wang, Z.; Liu, N.; Ding, B. Rolling up Gold Nanoparticledressed DNA Origami into Three-Dimensional Plasmonic Chiral Nanostructures. J. Am. Chem. Soc. 2011, 134, 146-149. (9) Song, L.; Jiang, Q.; Liu, J.; Li, N.; Liu, Q.; Dai, L.; Gao, Y.; Liu, W.; Liu, D.; Ding, B. DNA Origami/Gold Nanorod Hybrid Nanostructures for the Circumvention of Drug Resistance. Nanoscale 2017, 9, 7750-7754. (10) Tan, L. H.; Xing, H.; Lu, Y. DNA as a Powerful Tool for Morphology Control, Spatial Positioning, and Dynamic Assembly of Nanoparticles. Acc. Chem. Res. 2014, 47, 1881-1890. (11) Polsky, R.; Gill, R.; Kaganovsky, L.; Willner, I. Nucleic Acid-functionalized Pt Nanoparticles: Catalytic Labels for the Amplified Electrochemical Detection of Biomolecules. Anal. Chem. 2006, 78, 2268-2271. (12) Huang, K.; Martí, A. A. Recent Trends in Molecular Beacon Design and Applications. Anal. Bioanal. Chem. 2012, 402, 3091-3102. (13) Linck, L.; Reiß, E.; Bier, F.; Resch-Genger, U. Direct Labeling Rolling Circle Amplification as a Straightforward Signal Amplification Technique for Biodetection Formats. Anal. Methods 2012, 4, 1215-1220.


Page 25 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(14) Vijayanathan, V.; Lyall, J.; Thomas, T.; Shirahata, A.; Thomas, T. Ionic, Structural, and Temperature Effects on DNA Nanoparticles Formed by Natural and Synthetic Polyamines. Biomacromolecules 2005, 6, 1097-1103. (15) Vijayanathan, V.; Thomas, T.; Shirahata, A.; Thomas, T. DNA Condensation by Polyamines: a Laser Light Scattering Study of Structural Effects. Biochemistry 2001, 40, 13644-13651. (16) Phengchat, R.; Takata, H.; Morii, K.; Inada, N.; Murakoshi, H.; Uchiyama, S.; Fukui, K. Calcium Ions Function as a Booster of Chromosome Condensation. Sci. Rep. 2016, 6, 3828138290. (17) Wang, X.; Shi, J.; Li, Z.; Zhang, S.; Wu, H.; Jiang, Z.; Yang, C.; Tian, C. Facile One-pot Preparation of Chitosan/Calcium Pyrophosphate Hybrid Microflowers. ACS Appl. Mater. Interfaces 2014, 6, 14522-14532. (18) Xu, L.; Wang, Y.; Wei, G.; Feng, L.; Dong, S.; Hao, J. Ordered DNA-Surfactant Hybrid Nanospheres Triggered by Magnetic Cationic Surfactants for Photon-and Magneto-Manipulated Drug Delivery and Release. Biomacromolecules 2015, 16, 4004-4012. (19) Grueso, E.; Cerrillos, C.; Hidalgo, J.; Lopez-Cornejo, P. Compaction and Decompaction of DNA Induced by the Cationic Surfactant CTAB. Langmuir 2012, 28, 10968-10979. (20) Angell, C.; Xie, S.; Zhang, L.; Chen, Y. DNA Nanotechnology for Precise Control over Drug Delivery and Gene Therapy. Small 2016, 12, 1117-1132. (21) Alipour, M.; Hosseinkhani, S.; Sheikhnejad, R.; Cheraghi, R. Nano-Biomimetic Carriers are Implicated in Mechanistic Evaluation of Intracellular Gene Delivery. Sci. Rep. 2017, 7, 4150741719.



Page 26 of 30

(22) Ali, M. M.; Li, F.; Zhang, Z.; Zhang, K.; Kang, D.-K.; Ankrum, J. A.; Le, X. C.; Zhao, W. Rolling Circle Amplification: A Versatile Tool for Chemical Biology, Materials Science and Medicine. Chem. Soc. Rev. 2014, 43, 3324-3341. (23) Cheglakov, Z.; Weizmann, Y.; Braunschweig, A. B.; Wilner, O. I.; Willner, I. Increasing the Complexity of Periodic Protein Nanostructures by the Rolling‐Circle‐Amplified Synthesis of Aptamers. Angew. Chem. Int. Ed. 2008, 47, 126-130. (24) Martin, T. G.; Dietz, H. Magnesium-Free Self-Assembly of Multi-Layer DNA Objects. Nat. Commun. 2012, 3, 1103-1108. (25) Li, Y.; Du, H.; Wang, W.; Zhang, P.; Xu, L.; Wen, Y.; Zhang, X. A Versatile Multiple Target Detection System Based on DNA Nano-Assembled Linear FRET Arrays. Sci. Rep. 2016, 6, 26879-26884. (26) Mohsen, M. G.; Kool, E. T. The Discovery of Rolling Circle Amplification and Rolling Circle Transcription. Acc. Chem. Res. 2016, 49, 2540-2550. (27) Ali, M. M.; Kanda, P.; Aguirre, S. D.; Li, Y. Modulation of DNA‐Modified Gold‐Nanoparticle Stability in Salt with Concatemeric Single‐Stranded DNAs for Colorimetric Bioassay Development. Chem. Eur. J. 2011, 17, 2052-2056. (28) Kim, E.; Zwi‐Dantsis, L.; Reznikov, N.; Hansel, C. S.; Agarwal, S.; Stevens, M. M. One‐Pot Synthesis of Multiple Protein‐Encapsulated DNA Flowers and Their Application in Intracellular Protein Delivery. Adv. Mater. 2017, 29, 1701086-1701095. (29) Roh, Y. H.; Deng, J. Z.; Dreaden, E. C.; Park, J. H.; Yun, D. S.; Shopsowitz, K. E.; Hammond, P. T. A Multi‐RNAi Microsponge Platform for Simultaneous Controlled Delivery of Multiple Small Interfering RNAs. Angew. Chem. Int. Ed. 2016, 55, 3347-3351.


Page 27 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(30) Lee, J. B.; Hong, J.; Bonner, D. K.; Poon, Z.; Hammond, P. T. Self-Assembled RNA Interference Microsponges for Efficient siRNA Delivery. Nat. Mater. 2012, 11, 316-322. (31) Shopsowitz, K. E.; Roh, Y. H.; Deng, Z. J.; Morton, S. W.; Hammond, P. T. RNAi‐ Microsponges Form through Self‐Assembly of the Organic and Inorganic Products of Transcription. Small 2014, 10, 1623-1633. (32) Roh, Y. H.; Lee, J. B.; Shopsowitz, K. E.; Dreaden, E. C.; Morton, S. W.; Poon, Z.; Hong, J.; Yamin, I.; Bonner, D. K.; Hammond, P. T. Layer-by-Layer Assembled Antisense DNA Microsponge Particles for Efficient Delivery of Cancer Therapeutics. ACS Nano 2014, 8, 97679780. (33) Lu, D.; He, L.; Zhang, G.; Lv, A.; Wang, R.; Zhang, X.; Tan, W. Aptamer-Assembled Nanomaterials for Fluorescent Sensing and Imaging. Nanophotonics 2017, 6, 109-121. (34) Zhu, G.; Hu, R.; Zhao, Z.; Chen, Z.; Zhang, X.; Tan, W. Noncanonical Self-Assembly of Multifunctional DNA Nanoflowers for Biomedical Applications. J. Am. Chem. Soc. 2013, 135, 16438-16445. (35) Hu, R.; Zhang, X.; Zhao, Z.; Zhu, G.; Chen, T.; Fu, T.; Tan, W. DNA Nanoflowers for Multiplexed Cellular Imaging and Traceable Targeted Drug Delivery. Angew. Chem. Int. Ed. 2014, 126, 5931-5936. (36) Lv, Y.; Hu, R.; Zhu, G.; Zhang, X.; Mei, L.; Liu, Q.; Qiu, L.; Wu, C.; Tan, W. Preparation and Biomedical Applications of Programmable and Multifunctional DNA Nanoflowers. Nat. Protoc. 2015, 10, 1508-1524. (37) Lee, S. Y.; Kim, K.-R.; Bang, D.; Bae, S. W.; Kim, H. J.; Ahn, D.-R. Biophysical and Chemical Handles to Control the Size of DNA Nanoparticles Produced by Rolling Circle Amplification. Biomater. Sci. 2016, 4, 1314-1317. ACS Paragon Plus Environment


Page 28 of 30

(38) Mujumdar, R. B.; Ernst, L. A.; Mujumdar, S. R.; Lewis, C. J.; Waggoner, A. S. Cyanine Dye Labeling Reagents: Sulfoindocyanine Succinimidyl Esters. Bioconjugate Chem. 1993, 4, 105-111. (39) Chauhan, V. M.; Orsi, G.; Brown, A.; Pritchard, D. I.; Aylott, J. W. Mapping the Pharyngeal and Intestinal pH of Caenorhabditis Elegans and Real-Time Luminal pH Oscillations Using Extended Dynamic Range pH-Sensitive Nanosensors. ACS Nano 2013, 7, 5577-5587. (40) Jin, Y.; Li, Z.; Liu, H.; Chen, S.; Wang, F.; Wang, L.; Li, N.; Ge, K.; Yang, X.; Liang, X.-J. Biodegradable, Multifunctional DNAzyme Nanoflowers for Enhanced Cancer Therapy. NPG Asia Mat. 2017, 9, e365. (41) Zhang, W.; Shi, L.; An, Y.; Gao, L.; Wu, K.; Ma, R.; He, B. Initial Copolymer Concentration Influence on Self-Assembly of PS 38-b-P (AA 190-co-MA 20) in Water. Phys. Chem. Chem. Phys. 2004, 6, 109-115. (42) Rolland-Sabaté, A.; Guilois, S.; Grimaud, F.; Lancelon-Pin, C.; Roussel, X.; Laguerre, S.; Viksø-Nielsen, A.; Putaux, J.-L.; D’Hulst, C.; Potocki-Véronèse, G. Characterization of Hyperbranched Glycopolymers Produced in vitro Using Enzymes. Anal. Bioanal. Chem. 2014, 406, 1607-1618. (43) Chu, B.; Liang, D.; Hadjiargyrou, M.; Hsiao, B. S. A New Pathway for Developing in vitro Nanostructured Non-Viral Gene Carriers. J. Phys.: Condens. Matter. 2006, 18, S2513. (44) Dunlap, D. D.; Maggi, A.; Soria, M. R.; Monaco, L. Nanoscopic Structure of DNA Condensed for Gene Delivery. Nucleic Acids Res. 1997, 25, 3095-3101. (45) Zhou, W.; Saran, R.; Liu, J. Metal Sensing by DNA. Chem. Rev. 2017, 117, 8272-8325. (46) Zhou, T.; Llizo, A.; Wang, C.; Xu, G.; Yang, Y. Nanostructure-Induced DNA Condensation. Nanoscale 2013, 5, 8288-8306.


Page 29 of 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(47) Park, S. H.; Finkelstein, G.; LaBean, T. H. Stepwise Self-Assembly of DNA Tile Lattices Using dsDNA Bridges. J. Am. Chem. Soc. 2008, 130, 40-41. (48) Ke, G.; Zhu, Z.; Wang, W.; Zou, Y.; Guan, Z.; Jia, S.; Zhang, H.; Wu, X.; Yang, C. J. A CellSurface-Anchored Ratiometric Fluorescent Probe for Extracellular pH Sensing. ACS Appl. Mater. Interfaces 2014, 6, 15329-15334. (49) Kroutil, O. e.; Romancová, I.; Šíp, M.; Chval, Z. k. Cy3 and Cy5 Dyes Terminally Attached to 5′ C End of DNA: Structure, Dynamics, and Energetics. J. Phys. Chem. B. 2014, 118, 13564-13572. (50) Cao, H.; Xiong, Y.; Wang, T.; Chen, B.; Squier, T. C.; Mayer, M. U. A Red Cy3-B

ased

Biarsenical Fluorescent Probe Targeted to a Complementary Binding Peptide. J. Am. Chem. Soc. 2007, 129, 8672-8673. (51) Perkins, L. A.; Yan, Q.; Schmidt, B. F.; Kolodieznyi, D.; Saurabh, S.; Larsen, M. B.; Watkins, S. C.; Kremer, L.; Bruchez, M. P. Genetically Targeted Ratiometric and Activated pH Indicator Complexes (TRApHIC) for Receptor Trafficking. Biochemistry 2018, 57, 861-871. (52) Tran, M. N.; Rarig, R.-A. F.; Chenoweth, D. M. Synthesis and Properties of Lysosome-Specific Photoactivatable Probes for Live-Cell Imaging. Chem. Sci. 2015, 6, 4508-4512. (53) Zhou, L.; Lv, F.; Liu, L.; Wang, S. Polarity Conversion of Conjugated Polymer for Lysosome Escaping. ACS Appl. Mater. Interfaces 2017, 9, 27427-27432.



Graphical Abstract


Page 30 of 30

A High-yield Method to Fabricate and Functionalize DNA

Recommend Documents