Size-selective separation of DNA fragments by using lysine-functionalized silica particles

In this work, a facile and efficient approach has been demonstrated for size-selective separation of DNA fragments by using lysine-functionalized silica particles. At a given pH, the environmental ionic strength can be utilized to alter the electrostatic interactions of lysine-functionalized silica particles with DNA fragments and in turn the DNA fragments on the silica particle surfaces, which exhibits a clear dependence on the DNA fragment sizes. By carefully adjusting the environmental pH and salt concentration, therefore, the use of the lysine-functionalized silica particles allows effective separation of binary and ternary DNA mixtures, for example, two different DNA fragments with sizes of 101 and 1073 bp, 101 and 745 bp, 101 and 408 bp, respectively, and three different DNA fragments with sizes of 101, 408 and 1073 bp.

Adsorption and desorption of DNA fragments on the Lys-SiO 2 particles. Figure 2 shows the adsorption and desorption behaviors of DNA-101 and DNA-745 fragments on as-prepared Lys-SiO 2 particles as a function of pH. At pH 4.0, nearly 100% of both the DNA-101 and DNA-745 fragments can absorb onto the Lys-SiO 2 particles, while the absorption efficiency dramatically decreases to less than 10% at pH 5.0 and then remains little changed with the pH further increasing to pH 7.0. The desorption of these two differently sized DNA fragments from the Lys-SiO 2 particles is hardly observed in the pH range of 4.0-7.0. The desorption efficiency sharply increases from 10% to 98% when the pH increases from 7.0 to 10.0. The pH-sensitive DNA adsorption onto and desorption from the Lys-SiO 2 particles is in good line with the pH-sensitive zeta-potential of the particles. The phosphate groups of the DNA fragments remain negatively charged in a broad pH range 19,27,28 . Taken together, one can conclude that the adsorption of DNA fragments onto the Lys-SiO 2 particles is driven by their electrostatic attraction with the positively charged surfaces of the particles at pH < 7 due to the protonation of lysine amino groups. The desorption is driven by the electrostatic repulsion between the negatively charged DNA fragments and the negatively charged particle surfaces at pH > 7 due to the deprotonation of the lysine carboxyl groups. Based on the adsorption isotherm ( Supplementary Information, Figure S3), the DNA adsorption onto the Lys-SiO 2 particles follows the typical Langmuir adsorption model with the maximum adsorption capacity of about 9.0 mg/g. Note that in the current work, the DNA adsorption and desorption experiments were  carried out at pH 5.0 and 9.0, respectively, because of the relatively high efficiency, the relatively high stability of DNA fragments and the particles under these conditions, and the relatively weak electrostatic interaction between DNA fragments and the particle surfaces, which enable reversible tuning by the environmental ionic strength.
Selective separation of binary DNA mixtures by Lys-SiO 2 particles. It is expected that the electrostatic interactions of small charged species with oppositely charged matrices decrease with the increasing of ionic strength in solution [29][30][31] . Here, we studied the effect of the environmental ionic strength on the adsorption of DNA fragments with different sizes. DNA-101 and DNA-745 were chosen as a model and their adsorption behaviors were assessed in the aqueous solutions of NaCl at different concentrations. Figure 3 shows the difference in the adsorption efficiency between DNA-101 and DNA-745, which can be as large as 30% in the range of NaCl concentration from 0.4 to 0.6 M. In 0.4 M NaCl, for instance, the adsorption efficiency of DNA-101 on the Lys-SiO 2 particles is 36%, while that of DNA-745 is 69%. In 0.5 M NaCl, for instance, the adsorption efficiency of DNA-101 is 26%, while that of DNA-745 is 54%. This suggests the possibility to effectively separate DNA-101 and DNA-745 binary mixture in NaCl at the concentration range of 0.4-0.6 M. The ionic strength induced differentiation in adsorption efficiency between DNA-101 and DNA-745 could not be observed, when bare SiO 2 particles were used for the DNA adsorption (Supplementary Information, Figure S4). These results further highlight that the DNA adsorption to Lys-SiO 2 particles is driven by the electrostatic interactions between the DNA fragments and the lysine-functionalized particle surfaces.
We incubated Lys-SiO 2 particles with the mixtures of DNA-101 and DNA-745 with the mass ratio of 1:1 in the aqueous solution of NaCl. Agarose gel electrophoresis imaging was used to assess the composition of the DNA mixtures adsorbed on the particles and the DNA left in solution. Figure 4A shows that there are few DNA fragments, either DNA-101 or DNA-745, left in the solution in the absence of NaCl after the adsorption by Lys-SiO 2 particles (Line 1a), while the amount of the DNA desorbed from the particles is comparable to that in the original binary DNA mixtures, as indicated by the same brightness of Line 1b and Line S. This result confirms the high adsorption efficiency of DNA onto the Lys-SiO 2 particles. When the adsorption onto Lys-SiO 2 particles is carried out in the presence of NaCl, the amount of DNA-101 left in the solution increases with the NaCl concentration increasing from 0.2 M to 0.5 M, as indicated by the increase in the brightness from Line 2a to Line 3a, Line 4a and Line 5a. After DNA desorption from the Lys-SiO 2 particles, the bright line corresponding to DNA-101 is clearly visible at the NaCl concentration of 0.2 and 0.3 M (Line 2b and Line 3b), and became invisible when the NaCl concentration increases to 0.4 and 0.5 M (Line 4b and Line 5b). After the DNA adsorption onto the Lys-SiO 2 particles, few DNA-745 is left in the solution at the NaCl concentration < 0.5 M (Line 2a, Line 3a, Line 4a and Line 5a). When the NaCl concentration increases above 0.6 M, a noticeable amount of both DNA-101 and DNA-745 are retained in the solution after the adsorption onto the Lys-SiO 2 particles (Line 6a, Line 7a and Line 8a). Thus, the optimal NaCl concentration for size-selective separation of DNA-101 and DNA-745 ought to be 0.4 M, at which almost all of DNA-745 fragments are adsorbed onto Lys-SiO 2 particles (Line 4b), while almost all of DNA-101 fragments are retained in the solution (Line 4a).
To further investigate the size-selective separation efficiency, the software of Bio-Rad Quantity One is utilized to quantify the contents of DNA-101 and DNA-745 in solution according to the brightness of the lines showed in Fig. 4A. Figure 4B reveals that after incubation of binary DNA mixtures with Lys-SiO 2 particles in 0.4 M NaCl, the mass fraction of DNA-101 remaining in the solution is ca. 93%, while that of DNA-745 is ca. 7% after the adsorption to the Lys-SiO 2 particles. After the desorption of DNA from the Lys-SiO 2 particles in 0.4 M NaCl, the mass fraction of DNA-745 released into the solution is ca. 87%, while that of DNA-101 is ca. 13% (Fig. 4C). This data analysis further confirms the effectiveness and efficiency of using Lys-SiO 2 particles for size-selective separation of DNA-101 from DNA-745 in 0.4 M NaCl.
For comparision, amine-functionalized silica particles (NH 2 -SiO 2 ) were also evaluated in size-selective separation of DNA-101 and DNA-745. Gel electrophoresis results ( Supplementary Information, Figure S5) showed that the effective adsorption of both DNA-101 and DNA-745 on NH 2 -SiO 2 under the typical conditions (pH 5.0, 0.4 M NaCl), and the adsorbed DNA could not be eluted at pH 9.0 due to lack of electrostatic repulsion between DNA and NH 2 -SiO 2 21 .
The ionic-strength induced size-selectivity of DNA separation should arise from the fact that each deoxynucleotide of a given fragment has equal charge, so that total charge quantity of the DNA fragment is determined by its size and so is its electrostatic interaction with the surface of a Lys-SiO 2 particle [31][32][33] It is known that the distance between the charges decrease with increased salt concentration, resulting in less effective charge separation and thus screened electrostatic interactions 34,35 . Such screening effect should be more significant for short DNA fragments than for long ones under the same salt concentration. As a result, one can expect that at an optimal ionic strength, namely 0.4 M here, the electrostatic interaction between DNA-101 and Lys-SiO 2 particles is significantly reduced close to zero, while that between DNA-745 and Lys-SiO 2 particles remain substantially strong thanks to the large difference in the size and thus the charge quantity associated thereof. This size-selectivity is suppressed at high ionic strength, since the electrostatic attraction between the DNA-745 and Lys-SiO 2 particles can be reduced so significantly that the considerable amount of the DNA-745 remains in solution after the adsorption to the silica particles (Line 6a, Line 7a and Line 8a in Fig. 4A).
In order to generalize the present strategy, we utilized Lys-SiO 2 particles to separate other binary DNA mix-  while hardly from DNA-189 (Line 15). This indicates that the size selectivity requires large DNA fragments at least one time larger than small ones for electrostatic interaction-based DNA separation.

Selective separation of ternary DNA mixtures. Here we endeavored the use of the as-prepared
Lys-SiO 2 particles for separation of the ternary mixtures of DNA-1073, DNA-408 and DNA-101. At the first step, as shown in Fig. 6, DNA-101 is readily separated from DNA-408 and DNA-1073 in 0.4 M NaCl at pH 5.0 (Line 3) according to the aforementioned protocol. At the second step, the selective separation of DNA-408 from DNA-1073 can be implemented in 0.6 M NaCl at pH 5.0. As discussed above, the large size of the DNA-408 should account for the increased ionic strength value required for effective separation of DNA-408 from DNA-1073.

Discussion
Built on the aforementioned results, the mechanism for size-selective separation of DNA fragments by using the Lys-SiO 2 particles is depicted in Fig. 7. In the absence of NaCl, both short and long DNA fragments, which are negatively charged, can be effectively absorbed on the positively charged surfaces of the Lys-SiO 2 particles via the strong electrostatic attraction (top panel in Fig. 7). In the presence of NaCl, the electrostatic attraction between the negatively charged DNA and the positively charged particle surfaces is screened and becomes weaker with the ionic strength increasing, 34,35 thus decreasing the DNA adsorption efficiency onto the Lys-SiO 2 particles. Short DNA fragments is expected to be neutralized more easily than long ones with the NaCl concentration increase, so one expect an optimal NaCl concentration, at which the adsorption of the short DNA fragments onto the particle  surfaces may be completely blocked while the adsorption of the long DNA fragments may still take place in high efficiency (middle panel in Fig. 7). When the NaCl concentration is fairly higher than the optimal value, the electrostatic interaction of the long DNA fragments with the the surfaces of the Lys-SiO 2 particles may be suppressed, so both the short and long DNA fragments may remain in the NaCl solution (bottom panel in Fig. 7).
We demonstrate that Lys-silica particles can be utilized for size-selective separation of both binary and ternary DNA mixtures by properly adjusting the electrostatic interactions between DNA fragments and the lysine-functionalized surface of the silica particles with the environmental pH and especially ionic strength. The success of the present size-selective DNA separation strategy relies on the fact that the charge quantity of the DNA fragments and in turns their electrostatic interactions with the oppositely charged surface of the silica particles are strongly dependent on the DNA fragment sizes. Thanks to the operation ease, the present strategy should have promising potential in practical application in size-selective DNA extraction for downstream research.

Methods
Materials. Tetraethoxysilane (TEOS), anhydrous ethanol, ammonium hydroxide (25 wt.% NH 3 in water), lysine, sodium chloride, acetic acid (HAc), sodium acetate (NaAc), sodium carbonate, and sodium hydrogen carbonate were purchased from Beijing Chemical Company. Glycidoxypropyltrimethoxysilane (GLYMO) and salmon sperm DNA were purchased from Aldrich. DNA marker, buffer and 2*Taq PCR (Polymerase Chain Reaction) Green Mix were purchased from Beijing Dingguo Changsheng Biotechnology Co. Ltd. DNA primers and nucleic acid dye were purchased from Sangon Biotech Co. Ltd. Genomic DNA was extracted from human blood as a template for PCR. DNA purification kit was purchased from Geneon Biotech Company. The blood samples were collected from the Second Hospital of Jilin University (Changchun, Jilin, China). A form of consent was obtained from the donor. The experiments were approved by the Ethical Committee of the Hospital and carried out in accordance with the approved guidelines and regulations. TEOS was distilled prior to use and other commercial reagents were used without further purification. Water with a resistivity of 18 MΩ ·cm was prepared via a Pall Purelab Plus system and used in all experiments.  Figure S1) were prepared by the Stöber method 36 . To synthesize lysine-functionalized silica particles, the pH of the aqueous solution of lysine-derived silane was adjusted to 3.0. The SiO 2 particles (400 mg) were dispersed into the resulting solution, followed by heating at 90 °C for 3 h under stirring (150 rpm). After water washing for six times with the aid of centrifugation (8000 rpm), Lys-SiO 2 particles were obtained. DNA adsorption Isotherm. The different amounts of DNA fragments (10~300 ng/μ L) were added into 1.5 mL tubes containing 0.5 mg of Lys-SiO 2 particles, followed by addition of NaAc/HAc buffer solution (0.02 M, pH 5.0). The total volume of all the samples was set to be 120 μ L. The samples were incubated for 2 h at room temperature (25 °C) under shaking (150 rpm). After centrifugation (8000 rpm, 10 min), the supernatants were transferred into new tubes. The amounts of the non-adsorbed DNA remained in the solution were quantified by measuring the UV absorbance at 260 nm. Adsorption and desorption of DNA at different pH. In adsorption experiments, 0.50 mg of Lys-SiO 2 particles were added into the solutions containing 4000 ng DNA fragments (Supplementary Information, Figure  S6 and Figure S7), and then different buffer solutions with pH ranging from 4.0 to 10.0 were added. The final volume of the mixtures was set to be 120 μ L. After 2 h incubation under shaking (150 rpm) and then centrifugation (8000 rpm, 10 min), amounts of DNA adsorbed onto Lys-SiO 2 particles at different pH were determined by measuring the UV absorbance at 260 nm before and after the adsorption. In desorption experiments, 0.50 mg of Lys-SiO 2 particles with the DNA adsorbed on the surfaces were redispersed in 120 μ L of buffer solutions with pH ranging from 4.0 to 10.0, followed by 2 h incubation under shaking (150 rpm). After centrifugation (8000 rpm, 10 min), the amounts of the DNA fragments released from the Lys-SiO 2 particles were determined by measuring the absorbance at 260 nm before and after the desorption.

Synthesis of lysine
Selective DNA separation by using Lys-SiO 2 particles. As-prepared Lys-SiO 2 particles (0.50 mg) were firstly used for selective separation of the binary mixtures of DNA-101 (2000 ng) and DNA-745 (2000 ng). The total volume of the resulting mixtures was set to 100 μ L and the NaCl concentration in the samples was varied varied from 0 to 1.0 M by adding NaCl. Afterwards, NaAc/HAc buffer solution (0.02 M, pH 5.0) was added into the suspensions, followed by 2 h incubation under shaking (150 rpm). In a control experiment, the Lys-SiO 2 particles (0.25 mg) were incubated with DNA-101 (2000 ng) and DNA-745 (2000 ng), respectively. The total volume of the resulting mixtures was set to 50 μ L. After centrifugation (15 min, 8000 rpm), the collected particles were re-dispersed in 50 μ L NH 3 Figure S6 and Figure S7). Gel electrophoresis images were taken by a Bio-Rad Gel electrophoresis imager. Quantitative analyses of DNA were carried out by using the Bio-Rad Quantity One software based on brightness of the gel electrophoresis images.