Quantification of Nucleic Acid Concentration in the Nanoparticle

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Quantification of Nucleic Acid Concentration in the Nanoparticle/ polymer Conjugates using Circular Dichroism Spectroscopy Zhili Peng, Jiaojiao Li, Shanghao Li, Joel Pardo, Yiqun Zhou, Abdulrahman Obaid Al-Youbi, Abdulaziz Saleh Omar Bashammakh, Mohammad Soror El-Shahawi, and Roger M. Leblanc Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04621 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on December 28, 2017

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Analytical Chemistry

Quantification

of

Nucleic

Acid

Concentration

in

the

Nanoparticle/polymer Conjugates using Circular Dichroism Spectroscopy Zhili Penga,b, *, Jiaojiao Lic, Shanghao Li b, d, Joel Pardob, Yiqun Zhou b, Abdulrahman O. Al-Youbie, Abdulaziz S. Bashammakhe, Mohammad S. El-Shahawie, and Roger M. Leblancb,*

a

College of Pharmacy and Chemistry, Dali University, Dali, Yunnan, 671000, P. R. China

b

Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida 33146,

USA c

Department of Cellular Biology and Pharmacology, Herbert Wertheim College of Medicine, Florida

International University, 11200 S.W. 8th Street, Miami, USA d

MP Biomedicals, 3 Hutton Center Dr., # 100, Santa Ana, CA, 92707, USA

e

Department of Chemistry, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah

21589, Kingdom of Saudi Arabia

Keywords: Nanoparticles; Nucleic Acid; DNA; RNA; UV-Vis Spectroscopy; Circular Dichroism Spectroscopy; *Corresponding author (Z. P.) Tel.: +86–0872–2257401; E–mail: [email protected]. (R.M.L). Tel.: +1–305–284–2194; Fax: + 1–305–284–6367. E–mail: [email protected].

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Abstract The interface of nucleic acids and nanomaterials is among the most promising fields in recent years. Considerable efforts have been devoted for the development of novel systems based on the two components for various promising applications such as sensing, bioimaging, drug delivery and theranostics. However, the determination of nucleic acid concentration in these systems remains as a challenge due to the interference of nanoparticles. To this end, we developed a simple, yet reliable method to quantify the nucleic acid concentration in their nanoparticle conjugates based on circular dichroism (CD) spectroscopy. In this paper, three nucleic acids, namely deoxyribonucleic acid sodium salt from calf thymus (NaDNA), deoxyribonucleic acid from herring sperm (hsDNA) and ribonucleic acid from torula yeast (tyRNA), were noncovalently conjugated to three nanoparticles. The concentrations of the three nucleic acids in their nanoparticle conjugates were successfully determined based on CD spectra calibration curves.


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The interdisciplinary study of nucleic acids and nanomaterials is among the most promising fields in nanobiotechnology, in which the prominent properties of nanomaterials and the critical biological functions of nucleic acids are brought together. Systems based on both nanomaterials and nucleic acids hold exciting potentials in nanobiotechnology for applications such as biosensing, biocatalysis, bioimaging, diagnostics, drug delivery and theranostics development.1,2 Especially, the progress of the Human Genome Project (HGP) and other advancements in the elucidation of structure, organization, and functions of genes at molecular levels have opened new avenues for disease diagnostics, monitoring and treatment. Based on accurate target identification and genetic profiling, nucleic acidderived medication has the most revolutionary potential to pinpoint and tackle disease which is not feasible with traditional small molecule counterpart.3 The nucleic acid-based therapeutics is currently being driven by the intensive expectation of their revolutionary potentials in both research and applications. However, one critical challenge people often face is the poor bioavailability of nucleic acids, it is very hard for nucleic acids to pass the lipid biomembranes and make themselves available to the site of interest due to the thousands of years of evolution.4,5 To this end, significant efforts have been devoted to developing nanomaterials-based drug delivery systems (DDS) that could provide alternative transportation mechanism and thus increase the ability of nucleic acids to pass the bio-barriers.6,7 In these systems, nucleic acids are either physically adsorbed via non-covalent interactions or covalently bond to a DDS (i.e., gold nanoparticles, carbon dots, or biodegradable polymers) that is designed to selectively deliver them to the site of interest. In both strategies, one important aspect to consider is the accurate quantification of nucleic acid concentrations in these conjugates. It is critical to deliver known amount of nucleic acid drugs into the body since insufficient dose may not give desired efficacy while over dose could result in reverse effects (i.e., drug toxicity, immune reactions). 8 Traditionally, UV-Vis absorbance or dye-binding based fluorescence assays are used to determine the concentration of free nucleic acids (i.e., without any nanoparticles) in an assay based on a known extinction coefficient or a standard calibration curve (Figure 1A). For example, the UV-Vis absorbance at 260 nm is often used to determine the concentrations of nucleic acids based on a specific coefficient number.9 However, in most of the above-mentioned nanoparticle-nucleic acid systems, the UV-Vis spectroscopy based method generally cannot be applied due to the interference from the strong UV-Vis absorbance of nanoparticles (NPs), which often overlaps with the absorbance of nucleic acids or the dye. Also, nanoparticles (18 MΩ·cm at 20.0 ± 0.5 °C.



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Circular dichroism (CD) spectra were recorded using Jasco J-810 spectropolarimeter (Jasco Inc, USA). Demonstration of tyRNA concentration by UV-Vis spectrophotometer was performed using a Synergy H4 Hybrid Reader (BioTek, USA). Fluorescence of carbon dots was measured by a fluorescence spectrophotometer (Horiba Jobin Yvon Fluorolog–3) at 25 °C with a slit width of 5 nm for both excitation and emission. The UV/Vis absorption spectra of carbon dots were obtained using a Cary 100 UV-Vis spectrophotometer (Agilent Technologies, USA) with a 1 cm optical cell. Centrifugation was taken on an Allegra 64R from Beckman Coulter (Brea, CA, USA).

Ultraviolet-visible

spectroscopy measurements were obtained on an Agilent Cary 100 spectrophotometer (Santa Clara, CA, USA). Fluorescence measurements were carried out on a Horiba Jobin Yvon Fluorolog-3 (Kyoto, Japan) with slit widths set to 5 nm for both excitation and emission. Buffer solutions Na Acetate/Acetic acid buffer: To make 100 mL of 0.1 M (pH = 5.0) buffer solution, 871 mg of Na Acetate (6.4 mmole) and 206 µL of acetic acid (3.6 mmole) were mixed together and made into a 100 mL solution. This buffer solution was used to solubilize tyRNA. Tris buffer: To make 250 mL of 0.01 M Tris + 0.01 M NaCl (pH = 7.65) buffer solution, 394 mg of Tris.HCl.3H2O and 146 mg of NaCl were mixed together and made into a 250 mL solution, then the pH was adjusted to 7.65 by adding a NaOH solution. This buffer solution was used to solubilize hsDNA. Preparation of C-dots1 and C-dots2 C-dots1: C-dots1 used in this study was synthesized by following a previously reported procedure,25 then functionalized with ethylenediamine.26 Briefly, 1 g of carbon nanopowders in a mixture of sulfuric acid (36 mL) and nitric acid (12 mL) were heated at 110 °C for 15 h to yield C-dots. In order to isolate and purify C-dots, acids were neutralized by sodium hydroxide, the salt formed was then removed by crystallization in cold water, and this process was repeated three times. The solution was then further extracted by chloroform, and the aqueous layer obtained was subjected to dialysis with a semi membrane dialysis bag (MWCO 3500) against pure water for 4 days. C-dots in black powder form were obtained after the removal of water.25 To functionalize the obtained black powdered C-dots with ethylenediamine to yield C-dots1, black powdered C-dots (in aqueous PBS buffer, pH 7.4 solution) were first activated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) , before conjugated with excess amount of ethylenediamine to yield the desired amine-


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functionalized carbon dots C-dots1. Size exclusion chromatography column (Sephacryl S-300) was then used for purification; solid C-dots1 was obtained after lyophilization.26 C-dots2: C-dots2 used in the experiments was prepared according to a previously reported procedure with slight modification.27 Briefly, a mixture of 2.5 g of glucose and 150 mg of tryptophan in 15.0 mL deionized water were heated in a domestic microwave at the maximum powder of 700W for 9 min. The resulted solution mixtures were allowed to cool down and dissolved in water. Then the aqueous solution was subject to centrifugation at 6,000 rpm for 10 min to remove the large particles. The resulted clear top layer solutions were then purified by dialysis (MWCO 1000) to get the desired carbon dots C-dots2. All of the characterizations are carefully compared against the reported values.27 Non-covalent conjugation of nucleic acids with NPs NaDNA: briefly, 5.0 mg of NaDNA were first solubilized in 5.0 mL of DI water; then 5.0 mL of 0.2 mg/mL C-dots1 solution in DI water were added. The mixture was then kept at ambient temperature under stir overnight, after that, the resulted mixture was subjected to centrifugation at 10,000 rpm at 4 °C for 60 min. The supernatant after centrifugation was discarded, and the precipitation was then resuspended into solution in appropriate volume for the CD measurements as discussed in the manuscript. hsDNA: briefly, 5.6 mg of haDNA were first solubilized in 5.0 mL of 0.01 M Tris + 0.01 M NaCl (pH = 7.65) buffer solution; then 5.0 mL of 0.2 mg/mL C-dots1 solution in the same buffer solution were added. The mixture was then kept at ambient temperature under stir overnight, after that, the resulted mixture was subjected to centrifugation at 10,000 rpm at 4 °C for 60 min. The supernatant after centrifugation was discarded, and the precipitation was then suspended into solution in appropriate volume for the CD measurements as discussed in the manuscript. tyRNA: briefly, 4.6 mg of tyRNA were first solubilized in 5.0 mL of 0.01 M Tris + 0.01 M NaCl (pH = 7.65) buffer solution; then 5.0 mL of 0.2 mg/mL C-dots1 solution in the same buffer solution were added. The mixture was then kept at ambient temperature under stir overnight, after that, the resulted mixture was subjected to centrifugation at 10,000 rpm at 4 °C for 60 min. The supernatant after centrifugation was discarded, and the precipitation was then suspended into solution in appropriate volume for the CD measurements as discussed in the manuscript. The other two particles followed similar experiment procedures.



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RESULTS AND DISCUSSIONS To investigate the possibility of using CD spectroscopy to determine the concentrations of nucleic acid in nucleic acid-NPs conjugates, it is important to make sure that the presence of NPs does not interfere with the CD signals of nucleic acids. In other words, to establish a calibration curve between the concentrations of nucleic acids and their CD signal intensities, the calibration curves obtained should be very similar to each other regardless of the absence or presence of NPs. To this end, three nucleic acids solutions with known concentrations were freshly mixed with different nanoparticles and polymer, and the CD signals resulting from the nucleic acid-NPs mixtures were analyzed against that of free nucleic acids. Three nucleic acids with different function and conformations were studied in this experiment, namely deoxyribonucleic acid sodium salt from calf thymus (NaDNA), deoxyribonucleic acid from herring sperm (hsDNA) and ribonucleic acid from torula yeast (tyRNA). In light of the wide applications in various biomedical fields such as bioimaging, diagnostic, drug delivery as well as theranostics, carbon dots28-30 and poly (ethylene glycol) bis (3-aminopropyl) terminated (PEG)31 were chosen as the standard NPs and polymer for this study. There are numerous methods available for the preparation of C-dots,32 among the various methods available, there are generally two major strategies to prepare C-dots, namely “top-down” and “bottom-up” approach. Thus, two types of carbon dots, one prepared from the top-down approach (C-dots1)25,26 and the other synthesized by the bottom-up approach (C-dots2)27 were selected in this study. To probe the connection between the concentration of a nucleic acid and its CD signal, aqueous solutions of each nucleic acid in various concentrations (0.025-1.00 mg/mL) were measured by CD spectroscopy. Then CD spectra of the nucleic acid of same concentrations (0.025-1.00 mg/mL) were studied in the presence of NPs and polymer. As can be seen from figure 2, the CD signals of NaDNA become stronger as the concentrations gradually increase from 0.025 to 1.0 mg/mL (Figure 2A). The spectra establish a positive correlation between the concentrations and CD signal intensities of NaDNA, indicating that the determination of nucleic acid concentration using CD spectroscopy is possible. To ensure the presence of NPs and polymer do not interfere with the CD spectroscopy, the CD spectra of NaDNA at same concentrations (0.025-1.00 mg/mL) were also measured in the presence of C-dots1 (0.05 mg/ml), C-dots2 (0.05 mg/ml) and PEG (0.05 mg/ml), respectively. As expected, a similar, positive correlation between the concentrations and the CD signal intensities of NaDNA were also observed in presence of the NPs (Figure 2B and C) and polymer (Figure 2D). A close look at the CD spectra of free NaDNA (Figure 2A), NaDNA with C-dots1 (Figure 2B), NaDNA with C-Dots2 (Figure 2C) and NaDNA with PEG (Figure 2D) revealed that these four spectra are almost identical to each other, indicating there was no obvious interference from the NPs or polymer


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(PEG) when analyzing NaDNA using CD spectroscopy. (B)

(A) NaDNA concentration at: 1.0 mg/mL 0.8 0.65 0.5 0.4 0.25 0.2 0.125 0.1 0.0625 0.05 0.03125 0.025

mdeg

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0.05 mg/mL C-dots2 + NaDNA concentration at: 1.0 mg/mL 0.8 0.65 0.5 0.4 0.25 0.2 0.125 0.1 0.0625 0.05 0.03125 0.025

-20

200

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280

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Wavelength (nm)

(D) 60

40

mdeg

(C) 60

0.05 mg/mL C-dots1 + NaDNA concentration at: 1.0 mg/mL 0.8 0.65 0.5 0.4 0.25 0.2 0.125 0.1 0.0625 0.05 0.03125 0.025

60

mdeg

60

mdeg

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20

0

0.05 mg/mL PEG + NaDNA concentration at: 1.0 mg/mL 0.8 0.65 0.5 0.4 0.25 0.2 0.125 0.1 0.0625 0.05 0.03125 0.025

-20

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Figure 2. Circular dichroism spectra of NaDNA at various concentrations (0.025 – 1.0 mg/mL) in water (A), in the presence of 0.05 mg/mL of C-dots1 (B), in the presence of 0.05 mg/mL of C-dots2 (C) and in the presence of 0.05 mg/mL of PEG (D). We were glad to find that similar results were observed in the study using hsDNA (Figure 3). Briefly speaking, the CD intensities of hsDNA also demonstrated significant positive correlation to the concentrations (Figure 3A); and as expected, the introduction of carbon dots (C-dots1 and C-dots2) or polymer (PEG) did not change the CD spectra of hsDNA in all concentrations tested (Figures 3B, C and D). Similarly, positive correlations between the CD intensities and concentrations of hsDNA were observed in the presence of NPs and PEG (Figure 3). The similar trend was also observed when tyRNA was used in the study (Figure SI 1). It’s worth to mention, although only 0.05 mg/mL of NPs or PEG were used in figure 2, in principle, much higher concentrations of NPs could be applied in the experiment without interfering the CD signal of NaDNA as long as enough light could reach the CD



detector after passing through the NPs-rich sample. Most importantly, samples, in principle, could be diluted to appropriate concentrations before CD measurement to make sure enough light could reach the CD detector, thus an accurate determination could be generally delivered. In our experiments, the concentration of NPs or polymer (PEG) could go as high as 0.2 mg/mL without any obvious interference in the CD spectra of the nucleic acids (see Figure SI 4 for the spectra in presence of 0.2 mg/mL of C-dots1). (B)

(A) hsDNA concentration at: 1.0 mg/mL 0.8 0.65 0.5 0.4 0.25 0.2 0.125 0.1 0.0625 0.05 0.03125 0.025

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mdeg

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(D) 0.05 mg/mL C-dots2 + hsDNA concentration at: 1.0 mg/mL 0.8 0.65 0.5 0.4 0.25 .2 .125 .1 .0625 0.05 0.03125 0.025

10

280

Wavelength (nm)

(C)

20

0.05 mg/mL C-dots1 + hsDNA concentration at: 1.0 mg/mL 0.8 0.65 0.5 0.4 0.25 0.2 0.125 0.1 0.0625 0.5 0.03125 0.025

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mdeg

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

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0

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Figure 3. Circular dichroism spectra of hsDNA at various concentrations (0.025 – 1.0 mg/mL) in water (A), in the presence of 0.05 mg/mL of C-dots1 (B), in the presence of 0.05 mg/mL of C-dots2 (C) and in the presence of 0.05 mg/mL of PEG (D). Figure 2 clearly demonstrated the qualitative correlation between the intensities of NaDNA CD signal and its concentrations regardless of the absence or presence of NPs or polymer (PEG). It’s worth to note, no sign of conformational change of NaDNA was observed based on the CD spectroscopy analysis in all of the above experiments. The CD signal intensity of the nucleic acids at 275 nm (270


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nm for tyRNA) were then selected for the quantitative analysis of the CD intensities against the concentrations of nucleic acids. Based on the analysis, calibration curves correlating concentrations of nucleic acids with their CD signals were successfully established (Figure 4). As shown in the graph, the calibration curves for pure NaDNA (Figure 4A, black line), NaDNA with C-dots1 (Figure 4A, red line), NaDNA with C-dots2 (Figure 4A, blue line) and NaDNA with PEG (Figure 4A, magenta line) overlap well with each other. All of the four calibration curves are perfectly linear with a slope ranging from 22.67 to 22.78 (Figure 4A). Based on the calibration curves, one is confident to conclude that the presence of carbon dots or PEG does not interfere with the CD signal of NaDNA, and its analysis using CD spectroscopy. Very similar conclusions were also drawn in the experiments using hsDNA (Figure 4B) and tyRNA (Figure 4C). (A)

(B)

25

25

(C)

40

Calibration curves of NaDNA

Calibration curve of hsDNA

20

tyRNA calibration Curves

20

10 NaDNA only: y = -0.11 + 22.78x C-dots1 + NaDNA: y = -0.10 + 22.78x C-dots2 + NaDNA: y = -0.24 + 22.67x PEG + NaDNA: y = -0.20 + 22.68x

5

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1.0

CD at 270 nm

15

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30

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


15

10 hsDNA only: y = -0.10 + 22.39x C-dots1 + hsDNA: y = -0.10 + 21.98x C-dots2 + hsDNA: y = -0.12 + 21.64x PEG + hsDNA: y = -0.15 + 21.80x

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0 0.0

0.2

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tyRNA only: y = 0.03 + 35.94x C-dots1 + tyRNA: y = -0.07 + 35.71x C-dots2 + tyRNA: y = -0.12 + 36.44x PEG + tyRNA: y = -0.01 + 35.10x

10

0 0.0

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Figure 4. Calibration curves of nucleic acids using CD signal intensity versus concentration: (A), NaDNA at 275 nm, (B), hsDNA at 275 nm and (C), tyRNA at 270 nm. The concentrations of C-dots1, C-dots2 and PEG are all at 0.050 mg/mL. The path length of the quartz cell is 1 mm. With the successful establishment of the calibration curves, we then moved to the conjugation of the nucleic acids with the NPs and PEG. Since all the nucleic acids were negatively charged due to the presence of phosphate backbone; and both the carbon dots (zeta potential: C-dots1, +7.8 and C-dots 2, + 10.6) and PEG (presence of terminal amines) are slightly positively charged. We think it should be feasible for us to conjugate the nucleic acids with the NPs and PEG through noncovalent electrostatic interactions. After conjugation, the zeta potentials of the particles were shifted to negative values (Figure SI 2), which indicated the successful absorption of the negatively charged nucleic acids onto the particles. Since the NPs and polymer (PEG) tested were not optically active in terms of CD spectroscopy, and we have shown that their presence at lower concentrations did not interfere with the CD spectra of nucleic acids, the calibration curves of the free nucleic acids, in principle, could be used to determine the concentrations of the nucleic acids in presence of these particles. In our experiments, the conjugates were diluted accordingly so that their concentrations could fall into the range of the calibration curve. The CD spectra of the free nucleic acids, and the conjugates of these nucleic acids



with C-dots and PEG were then carefully measured (Figure 5). According to the spectra, NaDNA (Figure 5A) and hsDNA (Figure 5B) are typical B-form nucleotides while tyRNA (Figure 5C) is Aform.23 (C)

(B)

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0

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Figure 5. CD spectra of free nucleic acids (black), nucleic acid- C-dots1 conjugates (red), nucleic acid- C-dots2 conjugates (blue), nucleic acid-PEG conjugates (magenta): (A) NaDNA (0.50 mg/mL); (B) hsDNA (0.56 mg/mL); (C) tyRNA (0.46 mg/mL). Please note that the concentration of all the nanoparticles in the mixture is 0.10 mg/mL. Table 1. Ratio (absolute value) of the CD intensity at key wavelengths of NaDNA, hsDNA, and tyRNA in their pure form and nanoparticle/polymeric conjugates NaDNA275/245

hsDNA2275/248

tyRNA270/238

Pure nucleic acids

0.84

1.24

4.61

C-dots1

0.85

1.21

4.44

C-dots2

0.84

1.22

4.70

PEG

0.87

1.20

4.81

The key for the accurate concentration determination is to make sure there is no obvious conformational change after the conjugation, since a conformational change might significantly alter the CD spectral fingerprint of a nucleic acid. Thus the potential conformational alternation of nucleic acids in the conjugates was monitored by the ratio (absolute value) of the CD intensity at key wavelengths (Table 1). For example, the ratio (absolute value) of the CD intensity at 275 and 245 nm was used to evaluate the potential conformational changes of NaDNA after conjugation. The absolute values of Int275/240 in the free NaDNA, NaDNA-C-dots1 conjugate, NaDNA-C-dots2 conjugate, and NaDNA-PEG conjugate were determined to be 0.84, 0.85, 0.84 and 0.87, respectively (Table 1). These ratios are within 4% of difference, indicating the conformation of NaDNA remained almost unchanged


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after the conjugation with the NPs and polymer PEG. Very similar results were also obtained for hsDNA and tyRNA (Table 1). Further, these noncovalent conjugates are rather stable; we did not observe any significant conformational changes of the nucleic acids after one-week incubation at ambient temperature. Next, the CD intensities at 275 nm (270 nm for tyRNA) of the conjugates were applied into the calibration curves established previously (Figure 4) to determine the concentrations of the three nucleic acids in their conjugates. The NaDNA concentrations in the C-dots1, C-dots2, and PEG conjugates were determined as 0.483, 0.484, and 0.494 mg/mL, respectively (entry 1, Table 2). The actual concentration of NaDNA in these samples were calculated to be 0.50 mg/mL by dividing the mass of NaDNA used over the total final volume of the conjugated sample. As can be seen, concentrations for NaDNA in the NPs conjugates determined based on the calibration curve matched very well with the actual concentration: the differences between the two are within 4% for all the conjugates (entry 1, Table 2). For hsDNA, the actual concentration was calculated to be 0.56 mg/mL; the concentration in C-dots1, C-dots2 and PEG conjugates obtained based on the CD calibration curves at 275 nm was 0.569, 0.568, and 0.576 mg/mL, respectively. Thus, all the concentrations of hsDNA determined by CD calibration curves are within 3% difference to the actual concentration (entry 2, Table 2). As expected, concentrations for tyRNA from CD curve were also very close to the actual value, all within a 4% difference (entry 3, Table 2). In all of the samples tested, the difference (in percentage) between the actual concentration and the one determined by calibration curve was ranging from -3.70 to 3.48 %, demonstrating the feasibility of using CD spectroscopy for the concentration determination of nucleic acids in their NPs/polymeric conjugates. It’s worth to mention, although only carbon dots and polymer PEG were used as the model carriers in our study, in principle, this approach could be applied to inorganic NP-conjugates relying on either covalent coupling or simple coordination-driven conjugation. As previously discussed, most of the NPs were CD inactive, thus this CD spectroscopy based approach should be generally free from the interference of NPs; as long as the process of conjugation of NPs with nucleic acids does not alter the conformation of nucleic acids, one should be able to apply this approach in the analysis of NPs nucleic acid conjugates. Table 2. Concentrations of various nucleic acids in their nanoparticle/polymeric conjugates determined by CD spectra calibration curves.1 entry

sample

actual con.

1

NaDNA

0.50

C-dots1

C-dots2

PEG

con.

diff. (%)

con.

diff. (%)

con.

diff. (%)

0.483

-3.40

0.484

-3.20

0.494

-1.20



Page 14 of 17

2

hsDNA

0.56

0.569

1.61

0.568

1.43

0.576

2.86

3

tyRNA

0.46

0.443

-3.70

0.476

3.48

0.454

-1.30

4

2

tyRNA

0.46

0.475

3.26

0.474

3.04

0.480

4.35

5

3

tyRNA

0.46

0.513

11.52

0.436

-5.22

0.450

-2.17

2,3

tyRNA

0.46

0.576

25.22

0.449

-2.39

0.448

-2.61

6 1

Concentration of NPs or PEG is 0.1 mg/mL unless otherwise indicated; the data shown were average values of five tests. The concentration of NPs or PEG is 0.2 mg/mL in the conjugates. 3 The concentration was determined using a commercial UV-Vis spectrophotometer. 2

In comparison, we also used a commercial UV-Vis spectrophotometer (BioTek, Synergy H4 Hybrid Reader) to determine the concentration of tyRNA in its NPs/polymeric conjugates. UV-Vis spectroscopy based analytical methods for nucleic acid concentration measurement have been widely used; it is based on the absorption of the measured sample at 260 nm. For each different type of nucleic acid (i.e., dsDNA, ssDNA or RNA), one unit absorption at 260 nm corresponds to different concentrations.9 Since this method is based on the UV-Vis absorption at 260 nm, the determination of nucleic acid concentration would be significantly affected if NPs have strong absorption in this region. In our experiment, the concentration of tyRNA in C-dots1, C-dots2 and PEG conjugates was determined to be 0.513, 0.436, and 0.450 mg/mL, respectively (entry 5, Table 2). Considering the actual concentration is 0.46 mg/mL, there is an 11.52% difference in the concentration of tyRNA-Cdots1 conjugate determined by UV-Vis spectrophotometer compared to the actual concentration. The significant difference is presumably due to the interference from C-dots1 since C-dots1 has very intense absorption around 260 nm (supporting information, Figure SI 3). Indeed, when we increased the concentration of NPs in the conjugates from 0.1 mg/mL to 0.2 mg/mL, there was no significant change in the concentrations determined by CD calibration curve (entry 4, Table 2, for the CD spectra of tyRNA in presence of 0.2 mg/mL C-dots1, see Figure SI 4) while the results from UV-Vis spectrophotometer were significantly affected (entry 6, Table 2). These findings support the potential application of CD spectroscopy in many fields such as materials science, biomedical science and clinical field where the quantitative analysis of nucleic acids in presence of NPs is often desired. CONCLUSION In summary, three different nucleic acids NaDNA, hsDNA and tyRNA were noncovalently conjugated to carbon dots and PEG in our study. It was shown that the presence of low to moderate concentrations of NPs or polymer (PEG) did not interfere the CD spectra of the nucleic acids as long as sufficient photons could reach the detector for accurate measurements of the CD signal. Based on this finding, we were able to quantify the nucleic acid concentration in their nanoparticle/polymeric conjugates using CD spectra calibration curves. We also have shown that traditional analytical method (i.e., UV-


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Vis spectrophotometer) does not work well for the nucleic acid concentration determination in the presence of NPs due to the overlapping of absorption in the analysis region (around 260 nm). Thus, our study provides a simple yet convenient alternative for the quantification of nucleic acids concentration in their nanoparticle conjugates, where UV-Vis absorbance or dye-binding assays based traditional analytical methods do not work efficiently due to the interference of NPs.

ACKNOWLEDGEMENTS R.M.L. gratefully acknowledges the support from King Abdulaziz University, Saudi Arabia, the University of Miami, USA, and the National Institute of Health grant R21 AR072226.

Supporting Information Description of the materials, experimental procedures and other data. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. The authors declare no competing financial interest. AUTHOR INFORMATION *Corresponding author (Z. P.) Tel.: +86–0872–2257401; E–mail: [email protected]. (R.M.L). Tel.: +1–305–284–2194; Fax: + 1–305–284–6367. E–mail: [email protected].

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Topic of Content


Quantification of Nucleic Acid Concentration in the Nanoparticle

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