Citation

Lu Chang, Zheng Jinkai, Liu Juewen. Copper coordination-driven self-assembly and encapsulation of PCR reagents[J]. Chin. Chem. Lett., 2024, 35(3): 108808  

Chang Lu^a,b, Jinkai Zheng^a,*, Juewen Liu^b,*     

Since its discovery in 1986, polymerase chain reactions (PCR) have been one of the most important techniques in molecular biology to exponentially amplify target DNA [1], which is commonly used in bioanalytical, biomedical and biotechnological fields for the detection of genetic diseases, identification of genetic fingerprints, diagnosis of infectious diseases, cloning of genes, and paternity testing [2-8]. In the recent Covid-19 pandemic, PCR is still the gold standard for screening early infections [9,10].

A PCR reaction contains not only a DNA polymerase and dNTPs but also a set of primers. These reagents are stored separately and mixed with buffer and a template DNA for reaction. PCR reagents need to be stored at −20 ℃ and for a typical detection reaction, a lot of mixing steps are needed [11]. To improve the efficiency and speed of the amplification in a parallel manner, some researchers encapsulated PCR reagents in liposomes, emulsion droplets or polyelectrolyte microcapsules, which can be used as "nanoreactors" to perform biomolecular reactions for gene delivery application [12-17]. Some labs also freeze-dried reagents stored in polymer PCR chips for long-term stability of the reagents [18,19]. Some other methods such as real-time PCR [20], PCR-immune colloidal gold strip technology [21], loop-mediated isothermal amplification [22], digital PCR [23], and reverse transcription quantitative PCR [24] were also developed for improvement of the conventional PCR method.

A typical PCR reaction is comprised of deoxynucleoside triphosphates (dNTP, ~0.2 mmol/L), primers (~1 µmol/L) and a DNA polymerase (~12.5 IU). The template or target DNA to be amplified is typically at very low concentrations (nmol/L or lower level) [25]. Therefore, the main components are dNTP and primers. It has been recently shown that various transition metal ions and lanthanides can assemble various nucleotides to form coordination materials [26-29]. In addition, DNA can also participate in such reactions, and the formation of Fe/DNA nanoparticles is an interesting example [30,31]. Metal coordination can in turn stabilize DNA at high temperatures [32]. Since both dNTP and primers are in high concentrations in PCR, we wondered whether it is possible to use these intrinsic components of PCR reagents to form a coordination material to contain all the reagents, which may be suitable for storage, handling and integration of PCR reactions.

To investigate whether metal ions can encapsulate PCR reagents by forming coordination nanomaterials while still allowing PCR amplification, a few common transition metal ions were respectively incubated with PCR reagents (dNTP, primers and DNA polymerase) at room temperature for 1 h and centrifuged to remove the nonencapsulated reagents in the supernatants (Fig. 1A). After that, EDTA was added to dissolve the nanoparticles for releasing the PCR reagents. An 80-mer template DNA and buffer were then added to the samples, and PCR reactions were carried out in a thermocycler as normal. Finally, agarose gel electrophoresis was performed for product analysis. As shown in Fig. 1B, a high yield of PCR products was achieved with Cu²⁺ but little with the other metals including Zn²⁺, Ni²⁺, Co²⁺, Ce³⁺, and Mn²⁺, indicating Cu²⁺ may assemble the PCR reagents to form coordination materials. The position of the PCR product bands was at the expected position based on the ladder. So, Cu²⁺ was chosen as an optimal metal for the subsequent studies. For the other metal ions, it is known that Ni²⁺, Co²⁺ and Ce³⁺ can strongly interact with DNA [33,34], whereas the interaction might be too weak for Mn²⁺ and Zn²⁺. Cu²⁺ has the optimal affinity.

Figure 1

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Fig. 1. (A) A scheme showing the assembly and encapsulation of PCR reagents using metal coordination. The non-incorporated molecules were removed. (B) A gel micrograph showing the performance of PCR reagents assembled using 4 mmol/L different metal ions. The template DNA concentration was 10 nmol/L.

To quantitatively understand the effect of PCR reagents assembly by Cu²⁺, we first optimized the concentration of Cu²⁺. Different concentrations of Cu²⁺were added for encapsulating PCR reagents and then PCR thermocycling was conducted. The yield of the PCR product peaked when 4 mmol/L Cu²⁺ was used, after which the yield decreased as Cu²⁺ was further increased (Figs. 2A and B). We reasoned that Cu²⁺participated in the formation of the coordination reactions. Thus, the more Cu²⁺, the more PCR reagents encapsulated. However, too much metal ions may also inhibit the PCR efficiency [35]. The inhibition effect of high concentrations of Cu²⁺ was verified by performing the normal PCR reaction in the presence of different concentrations of Cu²⁺, where 1 mmol/L Cu²⁺ significantly inhibited the reaction (Fig. S1 in Supporting information). Note that while 4 mmol/L Cu²⁺ was added to form coordination nanomaterials with dNTP and PCR primers, not all the added Cu²⁺ ions were incorporated. The encapsulated efficiency of Cu²⁺ was calculated to be about 20% (Fig. S2 in Supporting information), and thus about 0.8 mmol/L Cu²⁺ was incorporated, which did not inhibit the reaction.

Figure 2

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Fig. 2. (A) A gel micrograph showing the PCR products using the Cu2+-assembled reagents with different Cu2+ concentrations. (B) Quantification of the results in (A) and 4 mmol/L Cu2+ is optimal. UV–vis and fluorescence spectra to calculate the encapsulation efficiency of (C) the primers, (D) FITC-BSA as a polymerase surrogate, and (E) dNTP. (F) The encapsulation efficiencies.

We also optimized the concentration of primer and dNTP for the normal PCR and PCR using Cu²⁺-coordination nanoparticles (Fig. S3 in Supporting information). The results showed the yield of PCR products increased as the primer or dNTP concentration increased. Without Cu²⁺, the optimal dNTP concentration was 0.2 mmol/L, and up to 4 µmol/L primer can be used. For the Cu²⁺-mediated nanoparticles, more products were formed with higher dNTP concentrations, while the optimal primer concentration was 2 µmol/L. Overall, the optimal condition for the Cu²⁺ mediated PCR and normal PCR was quite similar.

We then used a FAM-labeled 24-mer DNA oligonucleotide to evaluate the encapsulate efficiency of DNA primers. The FAM-labeled DNA (1 µmol/L) and primer DNAs (1 µmol/L) were mixed and incubated with Cu²⁺, dNTP and DNA polymerase to form the coordination nanomaterials. The samples were then centrifuged and the fluorescence of supernatant and dissolved precipitates were measured. The fluorescence of the dissolved precipitates was much higher than that of supernatant and the DNA encapsulate efficiency was calculated to be as high as 98%, indicating almost all the DNA primers were in the precipitant nanoparticles (Fig. 2C).

To evaluate polymerase encapsulation efficiency, we used FITC-labeled bovine serum albumin (BSA) to estimate the protein encapsulate efficiency for the formed coordination nanomaterial. Using the same method, about 98% BSA was in the precipitant, indicating a high encapsulate efficiency of protein (Fig. 2D). From this, we speculated the formed coordination nanomaterials can encapsulate nearly all the polymerase to conduct the PCR reaction. A high protein incorporation efficiency was also observed for other metal/nucleotide complexes [36]. It is reported that the phosphate and the base in DNA nucleotides can be involved in metal coordination [37], and Cu²⁺ has an optimal affinity to DNA [38], which can result in full coordination of DNA with Cu²⁺ ions.

Finally, the encapsulate efficiency of dNTP was measured by UV–vis absorption spectroscopy as dNTPs display a characteristic absorption peak at 260 nm. Since almost all the DNA primers were encapsulated in the precipitant, the absorption peak at 260 nm can be assigned to dNTP. Fig. 2E shows that only a small amount of dNTP was in the precipitant and the encapsulate efficiency was about 10%. The low encapsulate efficiency of dNTP may be attributed to the excess dNTP in solution, but the small amount dNTP was still enough for the PCR reaction. We summarized the encapsulation efficiency of each component in Fig. 2F. According to the data, before forming the nanomaterials, the ratio of enzyme, primer, and dNTP was 12.5 IU: 2 µmol/L: 200 µmol/L, whereas after forming the integrated PCR nanomaterials, the ratio was 12.5 IU: 2 µmol/L: 20 µmol/L.

The morphology and size of the coordination nanoparticles were further characterized by TEM (Fig. 3). The obtained nanoparticles had a rough surface and were about 100 nm in size. It seemed that many small nanoparticles clustered together to form the assembled nanoparticles. DLS showed the size of the nanoparticle to be about a few hundred nanometers, which was consistent with the TEM experiment. The measured size became smaller after drying and re-dispersion (Fig. S4A in Supporting information). The size of the coordination nanoparticles is also influenced by Cu²⁺, i.e., the more copper, the larger the particle size (Fig. S5 in Supporting information).

Figure 3

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Fig. 3. A TEM micrograph of the Cu2+ and PCR reagents formed nanoparticles.

After treated with 1 mmol/L EDTA, the nanoparticles were dissolved. We used 1 mmol/L EDTA to chelate Cu²⁺, but some Mg²⁺ still remained for the PCR reaction, since the formation constant of EDTA/Cu²⁺ is about 10 orders of magnitude larger than that of EDTA/Mg²⁺. A low concentration of EDTA actually increased the PCR efficiency, but with 2 mmol/L EDTA, the PCR efficiency was significantly inhibited (Fig. S6A in Supporting information). This result can be explained by EDTA chelation of Mg²⁺in the PCR reaction buffer. Mg²⁺ was required in the PCR reaction, but a high concentration of Mg²⁺ may inhibit the reaction by mechanisms like binding to dNTP or increasing the melting temperature of DNA too much. In our system, the PCR efficiency dropped drastically when more than 10 mmol/L Mg²⁺ was added (Fig. S6B in Supporting information).

We then investigated the PCR performance of the encapsulated nanoparticles. The PCR regents were encapsulated into the Cu²⁺nanoparticles. After centrifugation and drying, the formed nanoparticles were treated with EDTA and dispersed in buffer and different concentrations of the target template DNA were added to perform PCR. As shown in Figs. 4A-D, the PCR products increased as the concentration of DNA template changed from 0 to 100 nmol/L, and the detection limit was 17 pmol/L, which was similar to the normal PCR (10 pmol/L), confirming good performance of the encapsulated nanoparticles (Table S1 in Supporting information).

Figure 4

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Fig. 4. PCR products of (A, B) the encapsulated nanoparticles compared with (C, D) the normal PCR in the presence of different concentrations of the target template DNA. F represents the fluorescence of each point, and Ff means the fluorescence when DNA concentration was 100 nmol/L. The PCR efficiency of (E) the proposed system and (F) the conventional PCR method after one-week storage.

To investigate the stability of the formed nanoparticles, we dried the nanoparticles, rehydrated them with buffer and then run the PCR. After drying, the nanoparticles deposited at the bottom of the tube and had obvious green color indicative of Cu²⁺ coordination, while the normal PCR regents showed no color (Fig. S4B in Supporting information). After running gel, the yield of PCR products was slightly lower compared with the normal PCR.

We also measured the PCR efficiency of the proposed system after long-time storage compared with the conventional PCR method. The PCR efficiency for the proposed system after a week was measured to be 90% of the original system, while for the normal PCR, the efficiency decreased to be 60% (Figs. 4E and F), suggesting better storage stability of the proposed system.

In conclusion, we screened a few transition metal ions to form coordination nanomaterials with dNTP and DNA primers and encapsulate DNA polymerase, forming an integrated PCR reaction system. For the detection, users only need to dissolve the coordination nanomaterials with buffer and EDTA and add a template DNA for PCR amplification. Cu²⁺ was found to be the most effective metal ion for this purpose, and the encapsulation efficiency reached close to 100% for the primers and DNA polymerase, although only around 10% of dNTP was incorporated. This is the first work to use both dNTP and DNA oligonucleotides to form coordination polymers and it is an interesting system to simplify PCR reactions.

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.

Funding for this work was from the Natural Sciences and Engineering Research Council of Canada (NSERC), the National Natural Science Foundation of China (Nos. 31901776 and 32072181), and Agricultural Science and Technology Innovation Program (No. CAAS-ASTIP-2021-IFST-SN2021-05). C. Lu received a China Scholarship Council (CSC) Scholarship to visit the University of Waterloo.

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2023.108808.