S1 Nuclease Activity on Poly- Adenine- Mediated Spherical Nucleic Acids

S1 Nuclease Activity on Poly- Adenine- Mediated Spherical Nucleic Acids | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article S1 Nuclease Activity on Poly- Adenine- Mediated Spherical Nucleic Acids Weiwei Shen, Letian Zhong, He Qian, Weirong Yao, Yahui Guo, Qiaoying Chang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3317362/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Recently, spherical nucleic acids ( SNA ) have attracted the attention of many professionals and scientists. However, due to the very limited and incomplete researches, the mechanism of the SNA and nuclease remains confusing and ambiguous. Methods and results In this work, the poly-adenine-modified spherical nucleic acid ( polyA-SNA ) was selected as the experimental subject for the first time to study the activity of S1 nuclease on the surface of gold nanoparticles. We have systematically investigated the regulation of S1 nuclease activity changing by many factors, including PEG, DNA combining form, particle size, DNA combining density and DNA strand length. Conclusion The mechanism of biomolecular - nanoparticle interface affecting S1 nuclease activity was preliminarily discussed. The results will also be helpful to understand and construct high stability SNA probes and lay a foundation for expanding their biological applications in complex environments. Spherical nucleic acid polyA S1 nuclease activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction In 1996, Mirkin team [ 1 ] and Alivisatos team [ 2 ] first reported the formation of DNA - AuNPs conjugates. Until now, the properties of nucleic acid functionalized gold nanoparticles have been continuously developed and applied. Nanostructures composed of DNA alone are short of clear functionality, while inorganic nanomaterials have excellent properties such as optics, electricity, magnetism, catalysis, etc. Spherical nucleic acid (SNA), one of DNA functionalized nanomaterials, as a functionalized multivalent DNA - gold nanoparticles bilayer complex, is mainly composed of core - gold nanoparticles and shell - high oriented oligonucleotides. SNA not only shows the physical and chemical characteristics of gold nanoparticles, but also satisfies the programmability, dynamic controllability and logic operation functions of the outer oligonucleotide chain. In general, SNA has the ability of molecular recognition and directional assembly, while AuNP provides signal transduction for DNA-based biosensors [ 3 , 4 ] . Zhou [ 5 ] realized one-step, salt - free aging and sulfhydride - free freezing labeling by using the DNA sequence labeled with polyadenylate (about 10 adenine bases at the end) and the affinity adsorption of polyA and AuNPs, which is also one of the most convenient and low - cost AuNPs biological probe labeling technologies reported so far. Compared with sulfhydryl mediated DNA, SNA mediated with polyA is not only cheaper, but also easier to regulate density. In recent decades, SNA has been widely used in the fields of biological analysis and biomedicine, such as the construction of colorimetric biosensors, surface plasmon resonance (SPR) sensors, surface enhanced Raman spectroscopy (SERS) sensors, cell imaging, drug delivery and disease treatment. Li [ 6 ] described a new plasma CRISPR-Cas12a detection method for visual colorimetric detection of grape virus infection. In the experiment, CRISPR - Cas12a was used to identify the target and activate the related ssDNase activity. After cutting the S chain of the single - chain substrate, the plasma DNA functionalized AuNPs were induced to color rendering. The S chain can be degraded only when the S chain of the virus gene marker was present, which would not lead to the cross - linking of AuNPs, making the solution remain red. Different from the above, Li [ 7 ] used the SNA fluorescence probe (Au nanoparticle-DNAzyme probe) to identify the specificity of Pb 2+ . He used CRISPR - Cas12a to amplify the signal and employed simple detection equipments - smart phones and portable ultraviolet lamps to enhance the fluorescence on the photonic crystal chip with bionic periodic arrangement, thus realizing the on - site detection of Pb 2+ . Liu [ 8 ] used the magnetic beads modified by the aptamer and SNA - CuNCs anchored by the aptamer to form a sandwich structure sensor with the target to detect low-abundance cancer markers, providing a promising platform for the detection of biomarkers with excellent stability and accuracy. In addition, SNA can also be applied to the detection of insulin [ 9 ] , vascular endothelial growth factor (VEGF) [ 10 ] , immune signaling molecule interlukin-6 (IL-6) [ 11 ] , the solid interlukin-2 receiver α (sIL-2R α) [ 12 ] , thrombin [ 13 ] , etc. As a widely used tool in the field of biological detection, the enzyme stability and activity of SNA in complex biological environment has become one of the important factors affecting the accuracy of detection results. Feng [ 14 ] prepared a super - structured DNA nanoprobe with high enzyme stability and signal background ratio and proposed that the DNA chain with higher density on the larger size of AuNPs may have higher local salt concentration around the surface of nanoparticles to resist enzyme degradation. The assembled nanoprobe can remain stable for even more than 24 hours in the presence of excessive nuclease. Kevin [ 15 ] systematically studied the catalytic activity of 10–23 type DNAzyme on the surface of gold nanoparticles and the effects of assembly density, linker and fixation sites on the enzyme activity. They found that DNAzyme was affected by steric hindrance and non-specific adsorption on the surface at the nano interface, resulting in the decrease of enzyme activity. Moreover, DNAzyme's enzyme activity after assembly by 5 'end modified sulfhydryl group and 3' end modified sulfhydryl group with nanogold is very different. They believed that this is because the active region of DNAzyme is close to the 5 'end of the DNA chain. Zhu [ 16 ] further explored the activity of 8–17E DNAzyme on nanogold by using the double-block DNA - AuNPs system with controllable assembly density and configuration. It was found that the enzyme catalytic activity of DNAzyme on the AuNPs surface can be effectively improved by eliminating the non - specific adsorption of the AuNPs surface by poly - A and adjusting the assembly density of DNAzyme on the nanointerface. For foreign enzymes, the surface properties of SNA can also influence the activities of different enzymes. Jeff C. [ 17 ] found that the interaction between enzyme and nanoparticles would cause a series of new phenomena. He has systematically studied the interaction between exonuclease (ExoIII) and endonuclease (Nt. BspQI) and gold nanoparticles coated with oligonucleotides (spherical nucleic acid, SNA). Through the kinetic experiment based on fluorescence, he has changed various parameters (including enzyme and substrate concentration), as well as the size of nanoparticles and the surface coverage of non - recycled and recycled forms, and found that the surface characteristics of SNA can regulate the nuclease activity. It is also shown that the module unit of SNA can be used to accelerate or inhibit nuclease kinetics. For purpose of further exploring the effect of the binding properties of DNA and gold nanoparticles on the activity of S1 nuclease, polyA-SNA was selected as the research object for the first time (Scheme 1 ). The fluorescence based kinetic tests were used as well. In this study, some binding properties including PEG, DNA combining form, particle size, DNA combining density and DNA strand length were investigated. The obtained results were believed to further replenish the interaction mechanism of biomolecule-nanoparticle interface. The results will also be helpful to understand and construct high stability SNA probes and lay a foundation for expanding their biological applications in complex environments. 2. Experimental 2.1. Materials and Instruments Chloroauric acid was purchased from Sigma - Aldrich (Buchs, Switzerland). Sodium citrate, concentrated hydrochloric acid, concentrated nitric acid and Polyethylene glycol 20000 (PEG20000) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Nuclease-free water and S1 nuclease were obtained from Thermo Fisher Scientific (Massachusetts, US). Tris (2 – carboxyethyl) phosphine (TCEP) solution was purchased from Macklin (Shanghai, China). All oligonucleotides were obtained from Sangon Biotech (Shanghai, China). The instruments involved in this experiment include transmission electron microscope, ultraviolet visible spectrophotometer, fluorescence photometer, water bath, vortex oscillator, centrifuge, etc. 2.2. Oligonucleotide Sequences All oligonucleotides were modified by FAM at the 5 'end. The same oligonucleotide sequence was used to compare the catalytic ability of S1 nuclease to the single chain (B1) bound on the surface of AuNPs and the single chain (F1) in the free state in solution. The 3 'end of B1 chain was modified with SH bond to bind to the surface of AuNPs, and the 3' end of F1 chain was modified with BHQ1 group to quench fluorescence. In order to compare the effect of the binding density of the single chain on the surface of AuNPs on the shear efficiency of S1 nuclease, we designed different lengths of polyA single chain (A5-20, A10-20, A15-20), in which the base sequence is identical except for polyA. Similarly, by fixing the number of polyA and designing different length single chains (A5-10, A5-20, A5-30) based on the above sequence, we can compare the effect of the length of single chain on the surface of AuNPs on the shear efficiency of S1 nuclease. DNA sequences are given in Supplemental Table S1 . 2.3. SNA Synthesis 2.3.1. AuNPs Synthesis Before preparation, the conical flask should be soaked in aqua regia (65% concentrated nitric acid, 35% concentrated hydrochloric acid) for 24 hours, and then washed with clean water and distilled water for at least 3 times before use, and then dried. AuNPs were prepared by sodium citrate reduction method, using sodium citrate as reducing agent and stabilizer, and HAuCl 4 as gold source to prepare citric acid-stabilized AuNPs. First, 0.01% (volume ratio) of chlorogold acid solution and 1% (mass ratio) of sodium citrate solution are prepared. Add 100 mL of gold chloride solution into the conical flask, stir with magnetic force until boiling, then quickly add 2.5 mL of sodium citrate solution, stir with magnetic force for 3 min, keep boiling for 15–20 min, cool to room temperature, and store at 4 ℃ for use. AuNPs with different particle sizes were prepared by changing the volume of sodium citrate solution. The UV absorption spectrum and particle size were measured by UV - Vis spectrophotometer and transmission electron microscope respectively. 2.3.2. Preparation of ssDNA Solution After fully centrifuging the purchased ssDNA powder tube, add ultra - pure water according to the instructions and prepare 10 µM ssDNA stock solution. Place the stock solution in a refrigerator at − 20 ℃ for long-term use. In order to reduce the generation of disulfide bonds and promote the binding of ssDNA to the surface of AuNPs to the greatest extent, the B1 chain modified with sulfhydryl group needs to be reduced by 0.1 M TCEP solution (2:1 by volume) for 30 minutes. All ssDNA stock solutions were properly diluted with ultrapure water and placed in a refrigerator at 4 ℃. 2.3.3. Preparation of SNA by Freezing Method Preparation with thiol DNA: 10 µL B1 (10 µM) and 10 µL PEG 2000 solution (0.3 g/mL) were added into AuNPs solution (100 µL), mixed well and frozen in a -20 ℃ refrigerator for 2 hours. The thawed solution was centrifuged at 13000 rpm at 4℃ for 15–20 minutes. Then the precipitation was redissolved to obtain the SNA solution. Preparation with polyadenine-containing DNA: AuNPs stock solution was centrifuged and concentrated to about 10 nM. AuNPs solution (20 µL) was concentrated and then added with 5 µL diluted polyadenine DNA solution (10 µM). The sample was added with ultrapure water to 100 µL. After full mixing, the sample was frozen at -20 ℃ for 2 h. The thawed solution was centrifuged at 13000 rpm at 4℃ for 15–20 minutes. Then the precipitation was redissolved to obtain the SNA solution. All prepared SNA solutions were stored at 4 ℃. 2.4. SNA Characterization 2.4.1. Particle Morphology and Size Characterization The ultraviolet absorption spectra of SNA solution were determined by UV-vis spectrophotometer to verify the successful binding of ssDNA to AuNPs surface. The particle morphology and size were observed by transmission electron microscope. The specific operation was as follows: the ordinary carbon film was placed on the multi-layer clean filter paper, and then SNA solution prepared by different methods was added to the carbon film. After the solution was absorbed by the lower filter paper, the drip process was repeated again. Then, the treated carbon film was dried under an infrared irradiation lamp until droplet free state for observation. 2.4.2. Surface DNA Quantification The fluorescence intensity of ssDNA solution at 489 nm/519 nm was measured by fluorescence photometer. The fluorescence standard curve was established with the known concentration of SH-DNA-FAM solution, and then the prepared SNA solution was centrifuged at high speed, and the surface DNA content was indirectly calculated by measuring the fluorescence intensity in the supernatant. 2.5. Enzyme Kinetic Assays A certain volume of SNAs solution and 30 µL S1 nuclease buffer solution were obtained from the microcentrifuge tube according to the gradient, and the ultra-pure water was added to 92 µL (the total volume of the reaction system was set to be 100 µL). After mixing well, the solution was placed in a water bath at 25 ℃ for 2 minutes, and finally 8 µL S1 nuclease solution (10 U/µL) was added. Thoroughly mixed with pipette gun and immediately tested with fluorescence photometer. Timing was started immediately after adding the enzyme solution, and the fluorescence intensity of the system at 489 nm/519 nm was measured every 2 minutes for 1 hour. 2.6. Data Analysis The original fluorescence data were analyzed in GraphPad Prism 9 software using Michaelis-Menten model to obtain Michaelis-Menten constant (Km) and maximum reaction rate (Vmax). 3. Results and discussion 3.1. Characterization of SNA Under transmission electron microscopy (TEM), it was observed that AuNPs without DNA modification were uniform in size (The mean diameter is about 13 nm and the concentration is 2.4 nM.) and evenly dispersed (Supplemental Figure S1 ). After assembling ssDNA, SNA remained well dispersed and stable in the buffer solution (Supplemental Figure S1 ). The absorption peak of AuNPs at 525 nm can be observed through the ultraviolet absorption spectrum. The prepared SNA solution was centrifuged at high speed, and it was found that the supernatant only had a DNA peak at 260 nm. Therefore, the pure SNA solution can be obtained by discarding the supernatant and dissolving the precipitate. The spectral red of SNAs shifted to 530 nm, and the characteristic peak of DNA appeared at 260 nm, indicating that DNA was successfully modified on the surface of AuNPs (Supplemental Figure S1 ). The standard fluorescence curve of B1 solution with known concentration was established, and the relationship between fluorescence intensity and DNA concentration was y = 14.568x + 1118 (R 2 = 0.9802). The prepared SNA solution was centrifuged at high speed. The fluorescence intensity of the supernatant at 489 nm / 519 nm was determined to be 33438. From this, it can be calculated that about 3021 pieces of DNA were attached to each AuNP. 3.2. Influence of SNAs on S1 Nuclease Activity With F1 strand in solution as control group, the cutting effect of S1 nuclease on DNA on SNAs and DNA in solution was compared under the same DNA concentration. Figure 1 A shows the fluorescence process curve of S1 nuclease with different enzyme concentrations shearing SNAs with the same concentration. Figure 1 B shows the fluorescence process curve of S1 nuclease with different enzyme concentrations shearing F1 single chain in solution. When there was no S1 nuclease, the fluorescence intensity of the system remained almost unchanged at about 1000 with the increase of time. With the addition of S1 nuclease, the FAM group modified at the end of DNA dropped and the fluorescence intensity increased. When the concentration of S1 nuclease increased, the fluorescence intensity increased as well. We observed that when S1 nuclease was activated at 80 U, the time to reach the plateau of the reaction was reduced to 60 minutes. On the contrary, S1 nuclease was able to complete F1 single strand in solution almost within 10 minutes, indicating that the functional modification of DNA on AuNPs surface significantly inhibited S1 nuclease activity. 3.3. Exploration of Influencing Factors of S1 Nuclease Activity 3.3.1. PEG Polyethylene glycol (PEG) is a water-soluble polyether with good biocompatibility, non-toxicity and low immunogenicity. It can be excreted through the kidney and will not accumulate in the body. PEG has a wide range of application prospects in the field of biomedicine. The surface stabilization of nanoparticles is mainly achieved by the addition of hydrophilic polymers or surfactants, among which PEG is the most studied. When PEG is modified to AuNPs, the stabilization mode of AuNPs will be changed from electrostatic stability to spatial stability, which is less affected by external factors. PEG modification on the surface of inorganic nanoparticles has been reported in many literatures to increase water solubility, improve biocompatibility, reduce non-specific adsorption of proteins, etc., which is of great significance in the application of drug carrier materials [ 18 , 19 , 20 , 21 ] . In the process of preparing SNAs, we found that the introduction of PEG has a great effect on the stability of solution. Compared with the SNAs modified with SH-DNA, the SNAs modified with polyA-DNA were more likely to aggregate, and the solution turned purple. However, after the addition of PEG in the middle of preparation, this aggregation was greatly reduced, and the solution turned red (Supplemenal Figure S2). In order to investigate the effect of PEG on the experiment, the reaction rates of S1 nuclease cleavage of SNA (synthesized using 20nm AuNPs and A10-20) in the presence and absence of PEG were first compared. We found that the maximum rate of the reaction with PEG was much higher than that without PEG (Fig. 2 C and D). On the other hand, the introduction of PEG also improved the dispersion and uniformity of the solution, and increased the affinity of S1 nuclease to the target (compared with Km). From the value of kcat/Km, the S1 nuclease activity of the system with PEG added was slightly higher than that of the system without PEG, but the ratio of the two was of the same order of magnitude, indicating that the surface ligand to some extent hindered the enzyme digestion slightly. Therefore, PEG was selected to improve the stability of the solution in the following experiment. The regulation of PEG on enzyme activity can be simplified into two aspects: one is steric hindrance effect, the other is spatial stability. For PEG 20000ated AuNPs, the surface was coated with steric hindrance, the S1 nuclease was difficult to cut DNA molecules, and the reaction rate was low. However, without PEG, the AuNPs in solution tended to aggregate, and the spatial stability of the solution prevailed. Although the addition of PEG reduced the cutting efficiency of a single enzyme, it could increase the accessibility of the enzyme and more enzymes would begin to work. 3.3.2. DNA combing form The DNA functionalization modification on the surface of gold nanoparticles can be carried out in two ways: one is to modify the sulfhydryl group on the DNA and carry out covalent modification with Au-S bond; the other is to make the DNA contain poly-adenine and connect the DNA to the surface of gold nanoparticles based on the principle of base adsorption. In order to compare the effect of binding mode on the activity of S1 nuclease, the B1 chain and A10-20 chain of the same concentration were modified on 13nm AuNPs. The synthesized SNA were named SNA-SH-20 and SNA-A10-20, respectively. According to the fluorescence process curve, the fluorescence growth trend of SH-DNA-SNA was more obvious than that of polyA-DNA-SNA. According to Fig. 3 C and D, the substrate conversion rate of S1 nuclease on SNA is similar (the same order of magnitude), but the affinity for sulfhydryl functionalized SNA is greater (the larger the Km, the smaller the affinity). This may be because adenine has a weaker adsorption affinity than mercaptan and DNA is more easily dissociated under long-term storage. In addition, if a DNA is rich in adenine or cytosine throughout its sequence, it is unlikely to attach via the expected polyA anchor alone. If the other segment also binds to gold, it may hinder the enzyme reaction [ 22 , 23 ] . 3.3.3. Particle Size According to step 2.3.1, AuNPs with diameter of 13 nm and 20 nm were prepared respectively (Supplemental Figure S3) and then SNA13-A10-20 and SNA20-A10-20 were synthesized by modifying A10-20 chains on the surface. Supplemental Figure S3. A and S3. B respectively show the Ultraviolet absorption spectrum of the above materials from 400 nm to 600 nm. As can be seen from Fig. 4 , the maximum absorption peak wavelength of SNA-13 redshifted from 518 nm to 526 nm, and the maximum absorption peak wavelength of SNA-20 redshifted from 525 nm to 530 nm. The increase of peak width indicated that the uniformity is less than that of SNA-13, which would affect the cutting of S1 nuclease on the surface. The steric effect may be more important in the regulation of nuclease activity by particle size. It is well known that when AuNPs are prepared by chloro-auric acid reduction, the diameter of AuNPs decreases with more sodium citrate added. We speculated that the residual salt concentration in SNA solution prepared by AuNPs with small particle size was higher. The excess cations could be used to balance the negative charge on the DNA surface. It reduced the curvature of DNA molecules and steric hindrance, increasing the nuclease activity. 3.3.4. DNA Combining Density In order to facilitate the regulation of the modification density of DNA on AuNPs, DNA containing polyA was selected and its density on AuNPs surface was reduced by increasing the polyA length at the end of single-stranded DNA. The letters of A5, A10 and A15 represent DNA with 5, 10 and 15 polyA bases, respectively. Under the same preparation method, the fluorescence intensity curve at 489 nm/519 nm was measured at the same enzyme concentration level. As can be seen from Fig. 5 D, when polyA length was short, DNA strands modified by AuNPs surface were densely distributed. The active center of S1 nuclease was difficult to get close to AuNPs surface with the lowest affinity, and the kcat/Km ratio was 3.6*10 − 7 nM-1·s-1. When the polyA chain became longer (Fig. 5 E), the binding density of DNA decreased and the kcat/Km ratio decreased. This may be because the longer polyA chain may not be able to completely adsorb on the same AuNPs surface, and a short segment of polyA may still be exposed for binding another AuNPs, resulting in the aggregation of AuNPs. However, as the length of poly-A chain continued to increase (Fig. 5 F), the kcat/Km ratio increased. The reason may be that although a longer polyA chain could connect to adjacent AuNPs, the length of adenine sequence exposed to AuNPs would also become longer with the increase of polyA chain length, that is to say, the spacing of AuNPs gathered increased, thus improving the dispersion of SNA and increasing the activity of S1 nuclease against it. In short, the longer polyA chain may induce aggregation of adjacent AuNPs, leading to a decrease in enzyme activity. However, as the polyA chain continues to lengthen, so does the length of the adenine sequence exposes to AuNPs. The increased distance between clustered AuNPs resulted in increased SNA dispersion and possibly increased S1 nuclease activity. 3.3.5. DNA Strand Length In order to explore the effect of surface DNA strand length on the conversion rate of S1 enzyme substrate, we designed A5-10, A5-20 and A5-30 chains composed of 10, 20 and 30 bases with the size of AuNPs (13 nm) and the number of adenine bases (5 bases) unchanged. It can be seen from the Fig. 6 A to Fig. 6 C that the more the number of bases, the more the fluorescence intensity of the system increases at the same time. Correspondingly, from the maximum reaction rate Vmax, the longer the DNA chain length, the greater the maximum reaction rate. However, according to the experimental data, S1 nuclease had a larger Km to A5-30, indicating that the longer the length of DNA chain, the lower the affinity for it. Therefore, by comprehensively comparing the kcat/Km ratio, we can think that the shorter the DNA strand bound on the surface of AuNPs, the stronger the activity of S1 nuclease to cut single strand under the condition of only changing the length of DNA strand. The length of DNA strands is closely related to enzyme activity. It was found that the longer the DNA strand bound to the surface of SNA, the weaker the S1 enzyme activity. There are two possible reasons for this. On the one hand, non-polyA DNA sequences may be disorganized on the AuNPs surface. The longer the DNA strand, the greater the steric hindrance. On the other hand, there is a negative charge on the surface of the DNA molecular chain. As the length of the molecular chain increases, the salt particles in the system cannot stabilize the charge and make the DNA molecule stretch further. 4. CONCLUSIONS In summary, we successfully prepared polyA-DNA modified SNA by freezing method. Through fluorescence based kinetic experiments, we found for the first time that this SNA has obvious inhibitory effect on the activity of S1 nuclease. And this inhibitory effect can be adjusted by changing the surface properties of SNA. However, this effect cannot be fully explained by a uniform and simple rule according to our results. Only by comprehensively considering the influence of various factors on enzyme activities can SNA probes with high anti-enzyme stability be prepared more reasonably. The SNA probe with high anti-enzyme stability can be prepared more reasonably only when the influence of various factors on enzyme activity is considered comprehensively. This paper provides reference for its biological application in complex systems. This experiment also encourages the discovery and application of organic-inorganic complex probes that promote endogenous nuclease activity. Declarations Acknowledgments This work was sponsored by the National Key Research and Development Program of China (No. 2022YFF1101103), the Investigation Project for the Basic Resources of National Science and Technology (2022FY101202), the Opening Foundation of Jiangsu Key Laboratory of Experimental & Translational Non-coding RNA Research (NO. 202284), and Jiangsu Training Program of Innovation and Entrepreneurship for Undergraduates (CXZ2023025). Author contributions Weiwei Shen contributed to the investigation and experiment. Weiwei Shen wrote the original draft and all authors commented on previous versions. Yahui Guo read and approved the final manuscript. Funding The National Key Research and Development Program of China (No. 2022YFF1101103), the Investigation Project for the Basic Resources of National Science and Technology (2022FY101202), the Opening Foundation of Jiangsu Key Laboratory of Experimental & Translational Non-coding RNA Research (NO. 202284), Jiangsu Training Program of Innovation and Entrepreneurship for Undergraduates (CXZ2023025). Data availability Supplementary data to this article can be found online. Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Ethical approval No ethical approval is required. 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Langmuir 18:3669-3675. https://doi.org/10.1021/la011393y Moffatt S, Cristiano RJ (2006) Uptake characteristics of NGR-coupled stealth PEI/pDNA nanoparticles loaded with PLGA-PEG-PLGA tri-block copolymer for targeted delivery to human monocyte-derived dendritic cells. Int J Pharm 321:143-154. https://doi.org/10.1016/j.ijpharm.2006.05.007 Perera YR, Xu JX, Amarasekara DL, Hughes AC, Abbood I, Fitzkee NC (2021) Understanding the Adsorption of Peptides and Proteins onto PEGylated Gold Nanoparticles. Molecules 26:5788. https://www.mdpi.com/1420-3049/26/19/5788 Zhou W, Wang F, Ding J, Liu J (2014) Tandem Phosphorothioate Modifications for DNA Adsorption Strength and Polarity Control on Gold Nanoparticles. ACS Appl Bio Mater 6:14795-14800. https://doi.org/10.1021/am504791b Shen J, Jiang X, Xu L, Ge Z, Li Q, Song B, Wang L, Song S (2019) Poly-Adenine-Engineered Gold Nanogaps for SERS Nanostructures. ACS Appl Nano Mater 2:3501-3509. https://doi.org/10.1021/acsanm.9b00473 Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files SupplementalMaterials.docx scheme1.png Scheme 1. Schematic diagram of the influencing factors of inhibiting S1 enzyme activity Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3317362","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":230479560,"identity":"bbf304db-bf66-4979-9334-68d7d35fe742","order_by":0,"name":"Weiwei Shen","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weiwei","middleName":"","lastName":"Shen","suffix":""},{"id":230479561,"identity":"31c4000c-558d-44ec-b895-8a7ede4ef13e","order_by":1,"name":"Letian Zhong","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Letian","middleName":"","lastName":"Zhong","suffix":""},{"id":230479562,"identity":"542fffc1-283b-4ac4-81db-d1991fa69f13","order_by":2,"name":"He Qian","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Qian","suffix":""},{"id":230479563,"identity":"f7de7e31-c051-499f-ab9a-ad0a3e85bafc","order_by":3,"name":"Weirong Yao","email":"","orcid":"","institution":"Jiangnan University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Weirong","middleName":"","lastName":"Yao","suffix":""},{"id":230479564,"identity":"8e3ef18f-c59f-441f-a04e-410be84ff2a7","order_by":4,"name":"Yahui Guo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABDElEQVRIiWNgGAWjYLACxgYGHn6Ggw1wAQmitEg2kKqFweAAkgBeLfIRycckfu44LGN88HDj54Jfd+TNGZgP3uZhsMvDpcXwRlqaZO+ZwzxmBw42S8/se2a4s4Et2ZqHIbkYp5YZOWYSvG1gLQ3SvD2HGTcc4DGT5mE4kNiAR4vkX6AW44aDzb+BWuw3HOD/hleLvESOmTTIFgOGg23SPD8OJwJtYcOrxYDnWbK1bFs6j8SBg23WvA2HkzccZjO2nGOQjNuW9uSDN9+2Wdvzzzj++DbPn8O2G443P7zxpsIOty0HGFggsSBxABhBbUAGM1gch3qQLQ0MzB/ALH6QqX9wqxwFo2AUjIKRCwClil5hpt1FjQAAAABJRU5ErkJggg==","orcid":"","institution":"Jiangnan University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Yahui","middleName":"","lastName":"Guo","suffix":""},{"id":230479565,"identity":"12d92130-c01c-4cc2-81f2-23b4c3097251","order_by":5,"name":"Qiaoying Chang","email":"","orcid":"","institution":"Chinese Academy of Inspection and Quarantine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qiaoying","middleName":"","lastName":"Chang","suffix":""}],"badges":[],"createdAt":"2023-09-01 13:59:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3317362/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3317362/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":42718587,"identity":"53a5a15b-f713-49f2-be9c-b502ab2028f0","added_by":"auto","created_at":"2023-09-06 14:20:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":64912,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Fluorescence process curve of the bound system (B) Fluorescence process curve of the free system\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3317362/v1/ba573ea3859c7d89ed997297.png"},{"id":42718588,"identity":"e4391db2-965b-4f83-8f32-1e5fad92eda0","added_by":"auto","created_at":"2023-09-06 14:20:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":98490,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Fluorescence progress curve of S1 nuclease cutting SNA-A10-20 with PEG (B) Fluorescence progress curve of S1 nuclease cutting SNA-A10-20 without PEG (C) Reaction rate curve of S1 nuclease cutting SNA-A10-20 with PEG (D) Reaction rate curve of S1 nuclease cutting SNA-A10-20 without PEG\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3317362/v1/d5cd82dfd1e6dfd0c72357a5.png"},{"id":42720621,"identity":"2bd889c3-6cd6-4cc1-b8b4-a85c31ddc355","added_by":"auto","created_at":"2023-09-06 14:28:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":82028,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Fluorescence progress curve of S1 nuclease cutting SNA-SH-20 (B) Fluorescence progress curve of S1 nuclease cutting SNA-A10-20 (C) Reaction rate curve of S1 nuclease cutting SNA-SH-20 (D) Reaction rate curve of S1 nuclease cutting SNA-A10-20\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3317362/v1/1dc1516301e5f72dce41f9df.png"},{"id":42718590,"identity":"0ed90c61-fe1f-4450-b8df-a460a62a3bd8","added_by":"auto","created_at":"2023-09-06 14:20:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":142591,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Fluorescence progress curve of S1 nuclease cutting SNA13-A10-20 (B) Fluorescence progress curve of S1 nuclease cutting SNA20-A10-20 (C) Reaction rate curve of S1 nuclease cutting SNA13-A10-20 (D) Reaction rate curve of S1 nuclease cutting SNA20-A10-20\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3317362/v1/3c5d050e9508b349bebccb45.png"},{"id":42718591,"identity":"a4a9acd5-2034-4dc7-a46d-7435b5144349","added_by":"auto","created_at":"2023-09-06 14:20:28","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":224628,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Fluorescence progress curve of S1 nuclease cutting SNA-A5-20 (B) Fluorescence progress curve of S1 nuclease cutting SNA-A10-20 (C) Fluorescence progress curve of S1 nuclease cutting SNA-A15-20 (D)Reaction rate curve of S1 nuclease cutting SNA-A5-20 (E) Reaction rate curve of S1 nuclease cutting SNA-A10-20 (F) Reaction rate curve of S1 nuclease cutting SNA-A15-20\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3317362/v1/a0cba0fb042441cbda8e7724.png"},{"id":42718593,"identity":"35d30e62-9dbb-4d3f-a28d-9dc5554fb97c","added_by":"auto","created_at":"2023-09-06 14:20:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":217087,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Fluorescence progress curve of S1 nuclease cutting SNA-A5-10 (B) Fluorescence progress curve of S1 nuclease cutting SNA-A5-20 (C) Fluorescence progress curve of S1 nuclease cutting SNA-A5-30 (D) Reaction rate curve of S1 nuclease cutting SNA-A5-10 (E) Reaction rate curve of S1 nuclease cutting SNA-A5-20 (F) Reaction rate curve of S1 nuclease cutting SNA-A5-30\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3317362/v1/6d790bdcd463b5c7955177c2.png"},{"id":45449434,"identity":"030eda64-727f-4495-baca-bd84b00ccd7f","added_by":"auto","created_at":"2023-10-30 14:22:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1249483,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3317362/v1/dc65f7fe-3bfe-48c9-a81f-d2b176288bef.pdf"},{"id":42718595,"identity":"9aade7e0-3130-4e53-9574-5a64978fd699","added_by":"auto","created_at":"2023-09-06 14:20:29","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1512913,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-3317362/v1/ae293504e439e25df55a431a.docx"},{"id":42718592,"identity":"0d810beb-17fd-4f3b-a070-943835780c1b","added_by":"auto","created_at":"2023-09-06 14:20:29","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":253127,"visible":true,"origin":"","legend":"\u003cp\u003eScheme 1. Schematic diagram of the influencing factors of inhibiting S1 enzyme activity\u003c/p\u003e","description":"","filename":"scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-3317362/v1/f54c6739f40a26714fa42206.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"S1 Nuclease Activity on Poly- Adenine- Mediated Spherical Nucleic Acids","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn 1996, Mirkin team\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e and Alivisatos team\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e first reported the formation of DNA - AuNPs conjugates. Until now, the properties of nucleic acid functionalized gold nanoparticles have been continuously developed and applied. Nanostructures composed of DNA alone are short of clear functionality, while inorganic nanomaterials have excellent properties such as optics, electricity, magnetism, catalysis, etc. Spherical nucleic acid (SNA), one of DNA functionalized nanomaterials, as a functionalized multivalent DNA - gold nanoparticles bilayer complex, is mainly composed of core - gold nanoparticles and shell - high oriented oligonucleotides. SNA not only shows the physical and chemical characteristics of gold nanoparticles, but also satisfies the programmability, dynamic controllability and logic operation functions of the outer oligonucleotide chain. In general, SNA has the ability of molecular recognition and directional assembly, while AuNP provides signal transduction for DNA-based biosensors\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Zhou\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e realized one-step, salt - free aging and sulfhydride - free freezing labeling by using the DNA sequence labeled with polyadenylate (about 10 adenine bases at the end) and the affinity adsorption of polyA and AuNPs, which is also one of the most convenient and low - cost AuNPs biological probe labeling technologies reported so far. Compared with sulfhydryl mediated DNA, SNA mediated with polyA is not only cheaper, but also easier to regulate density.\u003c/p\u003e \u003cp\u003eIn recent decades, SNA has been widely used in the fields of biological analysis and biomedicine, such as the construction of colorimetric biosensors, surface plasmon resonance (SPR) sensors, surface enhanced Raman spectroscopy (SERS) sensors, cell imaging, drug delivery and disease treatment. Li\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e described a new plasma CRISPR-Cas12a detection method for visual colorimetric detection of grape virus infection. In the experiment, CRISPR - Cas12a was used to identify the target and activate the related ssDNase activity. After cutting the S chain of the single - chain substrate, the plasma DNA functionalized AuNPs were induced to color rendering. The S chain can be degraded only when the S chain of the virus gene marker was present, which would not lead to the cross - linking of AuNPs, making the solution remain red. Different from the above, Li\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e used the SNA fluorescence probe (Au nanoparticle-DNAzyme probe) to identify the specificity of Pb\u003csup\u003e2+\u003c/sup\u003e. He used CRISPR - Cas12a to amplify the signal and employed simple detection equipments - smart phones and portable ultraviolet lamps to enhance the fluorescence on the photonic crystal chip with bionic periodic arrangement, thus realizing the on - site detection of Pb\u003csup\u003e2+\u003c/sup\u003e. Liu\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e used the magnetic beads modified by the aptamer and SNA - CuNCs anchored by the aptamer to form a sandwich structure sensor with the target to detect low-abundance cancer markers, providing a promising platform for the detection of biomarkers with excellent stability and accuracy. In addition, SNA can also be applied to the detection of insulin\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e, vascular endothelial growth factor (VEGF)\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e, immune signaling molecule interlukin-6 (IL-6) \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e, the solid interlukin-2 receiver α (sIL-2R α) \u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e, thrombin \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e, etc.\u003c/p\u003e \u003cp\u003eAs a widely used tool in the field of biological detection, the enzyme stability and activity of SNA in complex biological environment has become one of the important factors affecting the accuracy of detection results. Feng\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e prepared a super - structured DNA nanoprobe with high enzyme stability and signal background ratio and proposed that the DNA chain with higher density on the larger size of AuNPs may have higher local salt concentration around the surface of nanoparticles to resist enzyme degradation. The assembled nanoprobe can remain stable for even more than 24 hours in the presence of excessive nuclease. Kevin\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e systematically studied the catalytic activity of 10\u0026ndash;23 type DNAzyme on the surface of gold nanoparticles and the effects of assembly density, linker and fixation sites on the enzyme activity. They found that DNAzyme was affected by steric hindrance and non-specific adsorption on the surface at the nano interface, resulting in the decrease of enzyme activity. Moreover, DNAzyme's enzyme activity after assembly by 5 'end modified sulfhydryl group and 3' end modified sulfhydryl group with nanogold is very different. They believed that this is because the active region of DNAzyme is close to the 5 'end of the DNA chain. Zhu\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e further explored the activity of 8\u0026ndash;17E DNAzyme on nanogold by using the double-block DNA - AuNPs system with controllable assembly density and configuration. It was found that the enzyme catalytic activity of DNAzyme on the AuNPs surface can be effectively improved by eliminating the non - specific adsorption of the AuNPs surface by poly - A and adjusting the assembly density of DNAzyme on the nanointerface. For foreign enzymes, the surface properties of SNA can also influence the activities of different enzymes. Jeff C.\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e found that the interaction between enzyme and nanoparticles would cause a series of new phenomena. He has systematically studied the interaction between exonuclease (ExoIII) and endonuclease (Nt. BspQI) and gold nanoparticles coated with oligonucleotides (spherical nucleic acid, SNA). Through the kinetic experiment based on fluorescence, he has changed various parameters (including enzyme and substrate concentration), as well as the size of nanoparticles and the surface coverage of non - recycled and recycled forms, and found that the surface characteristics of SNA can regulate the nuclease activity. It is also shown that the module unit of SNA can be used to accelerate or inhibit nuclease kinetics.\u003c/p\u003e \u003cp\u003eFor purpose of further exploring the effect of the binding properties of DNA and gold nanoparticles on the activity of S1 nuclease, polyA-SNA was selected as the research object for the first time (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The fluorescence based kinetic tests were used as well. In this study, some binding properties including PEG, DNA combining form, particle size, DNA combining density and DNA strand length were investigated. The obtained results were believed to further replenish the interaction mechanism of biomolecule-nanoparticle interface. The results will also be helpful to understand and construct high stability SNA probes and lay a foundation for expanding their biological applications in complex environments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials and Instruments\u003c/h2\u003e \u003cp\u003eChloroauric acid was purchased from Sigma - Aldrich (Buchs, Switzerland). Sodium citrate, concentrated hydrochloric acid, concentrated nitric acid and Polyethylene glycol 20000 (PEG20000) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Nuclease-free water and S1 nuclease were obtained from Thermo Fisher Scientific (Massachusetts, US). Tris (2 \u0026ndash; carboxyethyl) phosphine (TCEP) solution was purchased from Macklin (Shanghai, China). All oligonucleotides were obtained from Sangon Biotech (Shanghai, China).\u003c/p\u003e \u003cp\u003eThe instruments involved in this experiment include transmission electron microscope, ultraviolet visible spectrophotometer, fluorescence photometer, water bath, vortex oscillator, centrifuge, etc.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Oligonucleotide Sequences\u003c/h2\u003e \u003cp\u003eAll oligonucleotides were modified by FAM at the 5 'end. The same oligonucleotide sequence was used to compare the catalytic ability of S1 nuclease to the single chain (B1) bound on the surface of AuNPs and the single chain (F1) in the free state in solution. The 3 'end of B1 chain was modified with SH bond to bind to the surface of AuNPs, and the 3' end of F1 chain was modified with BHQ1 group to quench fluorescence. In order to compare the effect of the binding density of the single chain on the surface of AuNPs on the shear efficiency of S1 nuclease, we designed different lengths of polyA single chain (A5-20, A10-20, A15-20), in which the base sequence is identical except for polyA. Similarly, by fixing the number of polyA and designing different length single chains (A5-10, A5-20, A5-30) based on the above sequence, we can compare the effect of the length of single chain on the surface of AuNPs on the shear efficiency of S1 nuclease. DNA sequences are given in Supplemental Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. SNA Synthesis\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. AuNPs Synthesis\u003c/h2\u003e \u003cp\u003eBefore preparation, the conical flask should be soaked in aqua regia (65% concentrated nitric acid, 35% concentrated hydrochloric acid) for 24 hours, and then washed with clean water and distilled water for at least 3 times before use, and then dried. AuNPs were prepared by sodium citrate reduction method, using sodium citrate as reducing agent and stabilizer, and HAuCl\u003csub\u003e4\u003c/sub\u003e as gold source to prepare citric acid-stabilized AuNPs. First, 0.01% (volume ratio) of chlorogold acid solution and 1% (mass ratio) of sodium citrate solution are prepared. Add 100 mL of gold chloride solution into the conical flask, stir with magnetic force until boiling, then quickly add 2.5 mL of sodium citrate solution, stir with magnetic force for 3 min, keep boiling for 15\u0026ndash;20 min, cool to room temperature, and store at 4 ℃ for use. AuNPs with different particle sizes were prepared by changing the volume of sodium citrate solution. The UV absorption spectrum and particle size were measured by UV - Vis spectrophotometer and transmission electron microscope respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Preparation of ssDNA Solution\u003c/h2\u003e \u003cp\u003eAfter fully centrifuging the purchased ssDNA powder tube, add ultra - pure water according to the instructions and prepare 10 \u0026micro;M ssDNA stock solution. Place the stock solution in a refrigerator at \u0026minus;\u0026thinsp;20 ℃ for long-term use. In order to reduce the generation of disulfide bonds and promote the binding of ssDNA to the surface of AuNPs to the greatest extent, the B1 chain modified with sulfhydryl group needs to be reduced by 0.1 M TCEP solution (2:1 by volume) for 30 minutes. All ssDNA stock solutions were properly diluted with ultrapure water and placed in a refrigerator at 4 ℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Preparation of SNA by Freezing Method\u003c/h2\u003e \u003cp\u003ePreparation with thiol DNA: 10 \u0026micro;L B1 (10 \u0026micro;M) and 10 \u0026micro;L PEG 2000 solution (0.3 g/mL) were added into AuNPs solution (100 \u0026micro;L), mixed well and frozen in a -20 ℃ refrigerator for 2 hours. The thawed solution was centrifuged at 13000 rpm at 4℃ for 15\u0026ndash;20 minutes. Then the precipitation was redissolved to obtain the SNA solution. Preparation with polyadenine-containing DNA: AuNPs stock solution was centrifuged and concentrated to about 10 nM. AuNPs solution (20 \u0026micro;L) was concentrated and then added with 5 \u0026micro;L diluted polyadenine DNA solution (10 \u0026micro;M). The sample was added with ultrapure water to 100 \u0026micro;L. After full mixing, the sample was frozen at -20 ℃ for 2 h. The thawed solution was centrifuged at 13000 rpm at 4℃ for 15\u0026ndash;20 minutes. Then the precipitation was redissolved to obtain the SNA solution. All prepared SNA solutions were stored at 4 ℃.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.4. SNA Characterization\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Particle Morphology and Size Characterization\u003c/h2\u003e \u003cp\u003eThe ultraviolet absorption spectra of SNA solution were determined by UV-vis spectrophotometer to verify the successful binding of ssDNA to AuNPs surface. The particle morphology and size were observed by transmission electron microscope. The specific operation was as follows: the ordinary carbon film was placed on the multi-layer clean filter paper, and then SNA solution prepared by different methods was added to the carbon film. After the solution was absorbed by the lower filter paper, the drip process was repeated again. Then, the treated carbon film was dried under an infrared irradiation lamp until droplet free state for observation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Surface DNA Quantification\u003c/h2\u003e \u003cp\u003eThe fluorescence intensity of ssDNA solution at 489 nm/519 nm was measured by fluorescence photometer. The fluorescence standard curve was established with the known concentration of SH-DNA-FAM solution, and then the prepared SNA solution was centrifuged at high speed, and the surface DNA content was indirectly calculated by measuring the fluorescence intensity in the supernatant.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Enzyme Kinetic Assays\u003c/h2\u003e \u003cp\u003eA certain volume of SNAs solution and 30 \u0026micro;L S1 nuclease buffer solution were obtained from the microcentrifuge tube according to the gradient, and the ultra-pure water was added to 92 \u0026micro;L (the total volume of the reaction system was set to be 100 \u0026micro;L). After mixing well, the solution was placed in a water bath at 25 ℃ for 2 minutes, and finally 8 \u0026micro;L S1 nuclease solution (10 U/\u0026micro;L) was added. Thoroughly mixed with pipette gun and immediately tested with fluorescence photometer. Timing was started immediately after adding the enzyme solution, and the fluorescence intensity of the system at 489 nm/519 nm was measured every 2 minutes for 1 hour.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Data Analysis\u003c/h2\u003e \u003cp\u003eThe original fluorescence data were analyzed in GraphPad Prism 9 software using Michaelis-Menten model to obtain Michaelis-Menten constant (Km) and maximum reaction rate (Vmax).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Characterization of SNA\u003c/h2\u003e \u003cp\u003eUnder transmission electron microscopy (TEM), it was observed that AuNPs without DNA modification were uniform in size (The mean diameter is about 13 nm and the concentration is 2.4 nM.) and evenly dispersed (Supplemental Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). After assembling ssDNA, SNA remained well dispersed and stable in the buffer solution (Supplemental Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The absorption peak of AuNPs at 525 nm can be observed through the ultraviolet absorption spectrum. The prepared SNA solution was centrifuged at high speed, and it was found that the supernatant only had a DNA peak at 260 nm. Therefore, the pure SNA solution can be obtained by discarding the supernatant and dissolving the precipitate. The spectral red of SNAs shifted to 530 nm, and the characteristic peak of DNA appeared at 260 nm, indicating that DNA was successfully modified on the surface of AuNPs (Supplemental Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe standard fluorescence curve of B1 solution with known concentration was established, and the relationship between fluorescence intensity and DNA concentration was y\u0026thinsp;=\u0026thinsp;14.568x\u0026thinsp;+\u0026thinsp;1118 (R\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.9802). The prepared SNA solution was centrifuged at high speed. The fluorescence intensity of the supernatant at 489 nm / 519 nm was determined to be 33438. From this, it can be calculated that about 3021 pieces of DNA were attached to each AuNP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Influence of SNAs on S1 Nuclease Activity\u003c/h2\u003e \u003cp\u003eWith F1 strand in solution as control group, the cutting effect of S1 nuclease on DNA on SNAs and DNA in solution was compared under the same DNA concentration. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA shows the fluorescence process curve of S1 nuclease with different enzyme concentrations shearing SNAs with the same concentration. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB shows the fluorescence process curve of S1 nuclease with different enzyme concentrations shearing F1 single chain in solution. When there was no S1 nuclease, the fluorescence intensity of the system remained almost unchanged at about 1000 with the increase of time. With the addition of S1 nuclease, the FAM group modified at the end of DNA dropped and the fluorescence intensity increased. When the concentration of S1 nuclease increased, the fluorescence intensity increased as well. We observed that when S1 nuclease was activated at 80 U, the time to reach the plateau of the reaction was reduced to 60 minutes. On the contrary, S1 nuclease was able to complete F1 single strand in solution almost within 10 minutes, indicating that the functional modification of DNA on AuNPs surface significantly inhibited S1 nuclease activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Exploration of Influencing Factors of S1 Nuclease Activity\u003c/h2\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1. PEG\u003c/h2\u003e \u003cp\u003ePolyethylene glycol (PEG) is a water-soluble polyether with good biocompatibility, non-toxicity and low immunogenicity. It can be excreted through the kidney and will not accumulate in the body. PEG has a wide range of application prospects in the field of biomedicine. The surface stabilization of nanoparticles is mainly achieved by the addition of hydrophilic polymers or surfactants, among which PEG is the most studied. When PEG is modified to AuNPs, the stabilization mode of AuNPs will be changed from electrostatic stability to spatial stability, which is less affected by external factors. PEG modification on the surface of inorganic nanoparticles has been reported in many literatures to increase water solubility, improve biocompatibility, reduce non-specific adsorption of proteins, etc., which is of great significance in the application of drug carrier materials\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the process of preparing SNAs, we found that the introduction of PEG has a great effect on the stability of solution. Compared with the SNAs modified with SH-DNA, the SNAs modified with polyA-DNA were more likely to aggregate, and the solution turned purple. However, after the addition of PEG in the middle of preparation, this aggregation was greatly reduced, and the solution turned red (Supplemenal Figure S2). In order to investigate the effect of PEG on the experiment, the reaction rates of S1 nuclease cleavage of SNA (synthesized using 20nm AuNPs and A10-20) in the presence and absence of PEG were first compared. We found that the maximum rate of the reaction with PEG was much higher than that without PEG (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and D). On the other hand, the introduction of PEG also improved the dispersion and uniformity of the solution, and increased the affinity of S1 nuclease to the target (compared with Km). From the value of kcat/Km, the S1 nuclease activity of the system with PEG added was slightly higher than that of the system without PEG, but the ratio of the two was of the same order of magnitude, indicating that the surface ligand to some extent hindered the enzyme digestion slightly. Therefore, PEG was selected to improve the stability of the solution in the following experiment.\u003c/p\u003e \u003cp\u003eThe regulation of PEG on enzyme activity can be simplified into two aspects: one is steric hindrance effect, the other is spatial stability. For PEG 20000ated AuNPs, the surface was coated with steric hindrance, the S1 nuclease was difficult to cut DNA molecules, and the reaction rate was low. However, without PEG, the AuNPs in solution tended to aggregate, and the spatial stability of the solution prevailed. Although the addition of PEG reduced the cutting efficiency of a single enzyme, it could increase the accessibility of the enzyme and more enzymes would begin to work.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2. DNA combing form\u003c/h2\u003e \u003cp\u003eThe DNA functionalization modification on the surface of gold nanoparticles can be carried out in two ways: one is to modify the sulfhydryl group on the DNA and carry out covalent modification with Au-S bond; the other is to make the DNA contain poly-adenine and connect the DNA to the surface of gold nanoparticles based on the principle of base adsorption. In order to compare the effect of binding mode on the activity of S1 nuclease, the B1 chain and A10-20 chain of the same concentration were modified on 13nm AuNPs. The synthesized SNA were named SNA-SH-20 and SNA-A10-20, respectively. According to the fluorescence process curve, the fluorescence growth trend of SH-DNA-SNA was more obvious than that of polyA-DNA-SNA. According to Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and D, the substrate conversion rate of S1 nuclease on SNA is similar (the same order of magnitude), but the affinity for sulfhydryl functionalized SNA is greater (the larger the Km, the smaller the affinity).\u003c/p\u003e \u003cp\u003eThis may be because adenine has a weaker adsorption affinity than mercaptan and DNA is more easily dissociated under long-term storage. In addition, if a DNA is rich in adenine or cytosine throughout its sequence, it is unlikely to attach via the expected polyA anchor alone. If the other segment also binds to gold, it may hinder the enzyme reaction\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3. Particle Size\u003c/h2\u003e \u003cp\u003eAccording to step 2.3.1, AuNPs with diameter of 13 nm and 20 nm were prepared respectively (Supplemental Figure S3) and then SNA13-A10-20 and SNA20-A10-20 were synthesized by modifying A10-20 chains on the surface. Supplemental Figure S3. A and S3. B respectively show the Ultraviolet absorption spectrum of the above materials from 400 nm to 600 nm. As can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, the maximum absorption peak wavelength of SNA-13 redshifted from 518 nm to 526 nm, and the maximum absorption peak wavelength of SNA-20 redshifted from 525 nm to 530 nm. The increase of peak width indicated that the uniformity is less than that of SNA-13, which would affect the cutting of S1 nuclease on the surface.\u003c/p\u003e \u003cp\u003eThe steric effect may be more important in the regulation of nuclease activity by particle size. It is well known that when AuNPs are prepared by chloro-auric acid reduction, the diameter of AuNPs decreases with more sodium citrate added. We speculated that the residual salt concentration in SNA solution prepared by AuNPs with small particle size was higher. The excess cations could be used to balance the negative charge on the DNA surface. It reduced the curvature of DNA molecules and steric hindrance, increasing the nuclease activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.3.4. DNA Combining Density\u003c/h2\u003e \u003cp\u003eIn order to facilitate the regulation of the modification density of DNA on AuNPs, DNA containing polyA was selected and its density on AuNPs surface was reduced by increasing the polyA length at the end of single-stranded DNA. The letters of A5, A10 and A15 represent DNA with 5, 10 and 15 polyA bases, respectively. Under the same preparation method, the fluorescence intensity curve at 489 nm/519 nm was measured at the same enzyme concentration level. As can be seen from Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, when polyA length was short, DNA strands modified by AuNPs surface were densely distributed. The active center of S1 nuclease was difficult to get close to AuNPs surface with the lowest affinity, and the kcat/Km ratio was 3.6*10\u0026thinsp;\u0026minus;\u0026thinsp;7 nM-1\u0026middot;s-1. When the polyA chain became longer (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), the binding density of DNA decreased and the kcat/Km ratio decreased. This may be because the longer polyA chain may not be able to completely adsorb on the same AuNPs surface, and a short segment of polyA may still be exposed for binding another AuNPs, resulting in the aggregation of AuNPs. However, as the length of poly-A chain continued to increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), the kcat/Km ratio increased. The reason may be that although a longer polyA chain could connect to adjacent AuNPs, the length of adenine sequence exposed to AuNPs would also become longer with the increase of polyA chain length, that is to say, the spacing of AuNPs gathered increased, thus improving the dispersion of SNA and increasing the activity of S1 nuclease against it.\u003c/p\u003e \u003cp\u003eIn short, the longer polyA chain may induce aggregation of adjacent AuNPs, leading to a decrease in enzyme activity. However, as the polyA chain continues to lengthen, so does the length of the adenine sequence exposes to AuNPs. The increased distance between clustered AuNPs resulted in increased SNA dispersion and possibly increased S1 nuclease activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.3.5. DNA Strand Length\u003c/h2\u003e \u003cp\u003eIn order to explore the effect of surface DNA strand length on the conversion rate of S1 enzyme substrate, we designed A5-10, A5-20 and A5-30 chains composed of 10, 20 and 30 bases with the size of AuNPs (13 nm) and the number of adenine bases (5 bases) unchanged. It can be seen from the Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA to Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC that the more the number of bases, the more the fluorescence intensity of the system increases at the same time. Correspondingly, from the maximum reaction rate Vmax, the longer the DNA chain length, the greater the maximum reaction rate. However, according to the experimental data, S1 nuclease had a larger Km to A5-30, indicating that the longer the length of DNA chain, the lower the affinity for it. Therefore, by comprehensively comparing the kcat/Km ratio, we can think that the shorter the DNA strand bound on the surface of AuNPs, the stronger the activity of S1 nuclease to cut single strand under the condition of only changing the length of DNA strand.\u003c/p\u003e \u003cp\u003eThe length of DNA strands is closely related to enzyme activity. It was found that the longer the DNA strand bound to the surface of SNA, the weaker the S1 enzyme activity. There are two possible reasons for this. On the one hand, non-polyA DNA sequences may be disorganized on the AuNPs surface. The longer the DNA strand, the greater the steric hindrance. On the other hand, there is a negative charge on the surface of the DNA molecular chain. As the length of the molecular chain increases, the salt particles in the system cannot stabilize the charge and make the DNA molecule stretch further.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. CONCLUSIONS","content":"\u003cp\u003eIn summary, we successfully prepared polyA-DNA modified SNA by freezing method. Through fluorescence based kinetic experiments, we found for the first time that this SNA has obvious inhibitory effect on the activity of S1 nuclease. And this inhibitory effect can be adjusted by changing the surface properties of SNA. However, this effect cannot be fully explained by a uniform and simple rule according to our results. Only by comprehensively considering the influence of various factors on enzyme activities can SNA probes with high anti-enzyme stability be prepared more reasonably. The SNA probe with high anti-enzyme stability can be prepared more reasonably only when the influence of various factors on enzyme activity is considered comprehensively. This paper provides reference for its biological application in complex systems. This experiment also encourages the discovery and application of organic-inorganic complex probes that promote endogenous nuclease activity.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThis work was sponsored by the National Key Research and Development Program of China (No. 2022YFF1101103), the Investigation Project for the Basic Resources of National Science and Technology (2022FY101202), the Opening Foundation of Jiangsu Key Laboratory of Experimental \u0026amp; Translational Non-coding RNA Research (NO. 202284), and Jiangsu Training Program of Innovation and Entrepreneurship for Undergraduates (CXZ2023025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e Weiwei Shen contributed to the investigation and experiment. Weiwei Shen wrote the original draft and all authors commented on previous versions. Yahui Guo read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThe National Key Research and Development Program of China (No. 2022YFF1101103), the Investigation Project for the Basic Resources of National Science and Technology (2022FY101202), the Opening Foundation of Jiangsu Key Laboratory of Experimental \u0026amp; Translational Non-coding RNA Research (NO. 202284), Jiangsu Training Program of Innovation and Entrepreneurship for Undergraduates (CXZ2023025).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e Supplementary data to this article can be found online.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e No ethical approval is required.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eMirkin CA, Letsinger RL, Mucic RC, Storhoff JJ (1996) A DNA-based method for rationally assembling nanoparticles into macroscopic materials. 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ACS Appl Nano Mater 2:3501-3509. https://doi.org/10.1021/acsanm.9b00473\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Spherical nucleic acid, polyA, S1 nuclease, activity","lastPublishedDoi":"10.21203/rs.3.rs-3317362/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3317362/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eRecently, spherical nucleic acids ( SNA ) have attracted the attention of many professionals and scientists. However, due to the very limited and incomplete researches, the mechanism of the SNA and nuclease remains confusing and ambiguous.\u003c/p\u003e\u003ch2\u003eMethods and results\u003c/h2\u003e \u003cp\u003eIn this work, the poly-adenine-modified spherical nucleic acid ( polyA-SNA ) was selected as the experimental subject for the first time to study the activity of S1 nuclease on the surface of gold nanoparticles. We have systematically investigated the regulation of S1 nuclease activity changing by many factors, including PEG, DNA combining form, particle size, DNA combining density and DNA strand length.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe mechanism of biomolecular - nanoparticle interface affecting S1 nuclease activity was preliminarily discussed. The results will also be helpful to understand and construct high stability SNA probes and lay a foundation for expanding their biological applications in complex environments.\u003c/p\u003e","manuscriptTitle":"S1 Nuclease Activity on Poly- Adenine- Mediated Spherical Nucleic Acids","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-06 14:20:24","doi":"10.21203/rs.3.rs-3317362/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7650ceae-dcc3-4882-be63-95fe82f7cfcf","owner":[],"postedDate":"September 6th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-10-30T14:14:33+00:00","versionOfRecord":[],"versionCreatedAt":"2023-09-06 14:20:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3317362","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3317362","identity":"rs-3317362","version":["v1"]},"buildId":"7rjqhiLT3MXkJMwkYKINL","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}