Could an abiotic material mimic restriction enzymes?

da | Giu 9, 2019 | Biologia Molecolare

Design an abiotic material, such as chiral modified CdTe semiconductor nanoparticles, able to mimic a restriction enzyme could be a very interesting way to improve gene editing techniques

Restriction enzymes catalyse the DNA cleavage breaking the phosphodiester bond between bases on the DNA. This class of enzymes have four subclasses and their main applications are cloning, diagnosis of polymorphisms. In their work published in Nature Chemistry last August, Hua Kuang and collaborators [1] designed and tested the activity of chiral cysteine-modified CdTe semiconductor nanoparticles (NPs) to recognize and, following photonic excitation, to cut at the restriction site GAT’ATC on double-stranded DNA exceeding 90 bp in length, miming a restriction enzyme. Relying on previous works [2, 3] these researches synthesized this NPs with truncated tetraedral shape and chiral centres on superficial Cd’s atoms bound to Cysteine, Oxygen, Te-Cd and the rest of the nanoparticle. They obtained two different types of NPs because of the presence of Cysteine’s enantiomeric forms.

A double strand of 1839 bp DNA from Salmon sperm was used as substrate for the reaction, in which circularly polarized light (CPL) stimulates the chiral centres on the NPs and allows the DNA cleavage. In addition, during the reaction they discovered several changes in the NPs conformation and properties, like the NPs trasformation into nanorods, the increase of the elemental Te concentration and a fluorescence redshift. These changes were well investigated with different experiments and different techniques. These authors have obtained good results analysing the NPs’ site-selective scission activity of DNA. In particular, the cysteines on the NP’s surface and the DNA length (over 90 bp) play a key role for the site-selective DNA cleavage between T and A in the restriction site. A good practice of the researchers was to perform lots of control experiments to evaluate their results, using various fragments of DNA, with different lengths and mutations, and using CdTe NPs with different modification other than cysteine.

Analysing the bond between the 90 bp DNA and NPs with ITC and Transient Absorption Spettroscopy they found a strong and specific connection between them, demonstrating the presence of an electron transport necessary for the reaction. The authors also found an increasing in ROS concentrations and,  as a good point to begin the investigation about the mechanism of the reaction, they tried to explain the presence of ROS and their role in the DNA scission. They verified the production of ROS, in particular hydroxyl radicals, and that this production is clearly affected by the different CPL because of the differential absorption efficiency of the two enantiomeric chiral NPs. Therefore, they concluded that the NPs, as electron donors, induced ROS generation, while the DNA was the receptor, leading to phosphodiester bond cleavage.

A very interesting way was followed by Hua Kuang and collaborators to have further confirms of the site-selective cleavage mechanism using quantum chemically calculated interaction energies and density functional theory (DFT). The free energy landscape and the analysis on Helmholtz’s free energy demonstrated very well the high affinity of the amino group of the restriction site for the grooves made by cysteines along the edges of the NPs. On the other hand, the simulation of the reaction mechanism was not well defined by the authors, because they did not explain all the reaction but just a part . Also, they did not describe properly the role of the ROS in the cutting system but just how the oxygen, from which hydroxyl radicals are generated, can interact with the DNA backbone allowing its break. Other in silico analysis might be done to understand the cleavage mechanism and the specific role of ROS in the reaction.

Lastly Hua Kuang and collaborators designed a method to monitor the cutting reaction, using PEG-coated NPs, and using a DNA fragment labelled with a molecular fluorescence beacon [4], in vitro and in vivo. Transfecting Hela and neural stem cells in vitro with labelled DNA in presence of the Cys-modified CdTe NPs under CPL illumination for 2h, the reaction worked good and confocal microscopy confirms what was previously reported in the paper. A similar experiment was performed in vivo using nude mice with Hela cells, transfected subcutaneous, and using the labelled DNA. After intravenous injection of the NPs, authors showed how these NPs were accumulated on the tumour site and, after illumination under CPL the DNA was cleaved with high efficiency. In vitro experiments were convincing, meanwhile in vivo experiments leave us many doubts about the methods followed by the authors and the results analysis. New in vivo test could be done to understand how this NPs could accumulate in the tumour site without any driving system, and how this system can work in vivo with an high biocompatibility.

In conclusion, we can say, supported by this work, that abiotic materials as Cys-modified CdTe NPs can cut efficiently DNA in vitro. Differently from the Authors’ conclusion we are not sure, looking at these results, about the possible application of this system in vivo without implementation of the technique. Of course, in-depth studies could open up new application for these abiotic materials as novel tools for gene analysis, and gene manipulations.

References

  1. Hua Kuang et al., Site-selective photoinduced cleavage and profiling of DNA by chiral semiconductor nanoparticles, Nature Chemistry10, 821–830 (2018).
  2. Nicholas A. Katov et al., Similar Topological Origin of Chiral Centers in Organic and Nanoscale Inorganic Structures: Effect of Stabilizer Chirality on Optical Isomerism and Growth of CdTe Nanocrystals, J. Am. Chem. Soc.2010, 132, 17, 6006-6013
  3. Alexander Eychmuller et al., Colloidal semiconductor nanocrystals: the aqueous Approach, Chem. Soc. Rev., 2013, 42, 2905-2929

Simone Turella

Master Industrial Biotechnology student

Alice Zoppo

Master Industrial Biotechnology student