Importantly piPolB holds a great promise for developing

Importantly, piPolB holds a great promise for developing novel biotechnological applications. For instance, in vitro activities of piPolB, namely, strand displacement and faithful, processive DNA polymerization, can be harnessed for efficient primer-independent whole-genome amplification, whereas the TLS can be useful for amplification of damaged or ancient DNA templates. Given that piPolBs do not display strong sequence requirement for replication initiation, replication origins may be selected in a random manner, a property useful for whole-genome amplification. In this framework it is worth mentioning that a recently developed method, dubbed TruePrime, using a combination of an AEP primase, TthPrimPol, and the Φ29DNAP, has been proposed for whole-genome amplification from single AZD-9291 mesylate (Picher et al., 2016). Similarly, piPolB could become a single-enzyme solution to achieve the same goal in single-cell genomic applications. Further structural and functional characterization of piPolBs and the pipolins that encode them will help to understand the details of this unique replication mechanism and to harness the potential of these enzymes for versatile biotechnological applications.
Experimental Procedures
Author Contributions
Acknowledgments We thank Laurentino Villar for protein purifications, Dr. Miguel de Vega for valuable suggestions and critical reading of the manuscript, and Professor Luis Blanco for helpful discussions and the [γ-32P]ATP-(dGMP)n ladder. M.S.’s lab is funded by the Spanish Ministry of Economy and Competitiveness (BFU2014-52656P). M.R.-R. was supported by a ComFuturo Grant (NewPols4Biotech) from Fundación General CSIC. C.D.O. was holder of a “Plan de Empleo Juvenil” contract from Madrid Regional Government (funded by YEI program from European Social Fund, EC). An institutional grant from Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa is also acknowledged. P.F. was funded by the European Research Council under the European Union’s Seventh Framework Program (FP/2007-2013)/Project EVOMOBIL - ERC Grant Agreement 340440.
Introduction Water-soluble fluorescent metal nanomaterials (NMs), such as nanoparticles (NPs) or nanoclusters (NCs), with small size about several nanometers containing few atoms have attracted much attention due to their unique size-dependent properties such as fluorescence and catalysis in the past years. Among them, noble metal NCs with sizes less than 2 nm, approaching the Fermi wavelength of the electron, have been widely utilized as emerging fluorophores on account of their lower toxicity and better photostability compared to traditional organic dyes and quantum dots (Berti and Burley, 2008, Guo et al., 2010, Gwinn et al., 2008, Houlton et al., 2009, Kennedy et al., 2012, Li et al., 2011). Compared with noble metals such as gold and silver, fluorescent copper NMs (CuNMs), including copper NPs (CuNPs) and copper NCs (CuNCs), show more extensive prospects in science and industry due to the lower cost and richer earth stock of metallic Cu, attracting lots of scientists to explore their synthesis and application (Anzlovar et al., 2007, Cho et al., 2001, Chrimes et al., 2013, de Oliveira et al., 2007; Du et al., 2005; Godovski, 1995; Hall et al., 2000; Helmy et al., 2013; Kang et al., 2016; Kharenko et al., 2005). To achieve the practical use of fluorescent CuNMs (Brouwer, 2011, Cao et al., 2014, Chen et al., 2014), various template ligands usually containing sulfur or nitrogen functional groups that can interact with copper ions (Chrimes et al., 2013, Cummings et al., 1980, Eaton, 1988, Han et al., 2017, Helmy et al., 2013, Kang et al., 2016, Liu et al., 2013a, Qing et al., 2013a, Rotaru et al., 2010, Vilar-Vidal et al., 2010, Wang et al., 2017a) have been explored to improve the stability of fluorescent CuNMs. Among all the capping ligands, DNA due to its diversity and ability to recognize molecules has been widely utilized as the efficient templates for the formation of CuNMs. The first report on the selective formation of fluorescent CuNPs (Rotaru et al., 2010) was based on the double-stranded DNA (dsDNA) and serials of biosensing platform including metal ions, small molecules, proteins, DNA and RNA. Subsequently, Liu et al. and Qing et al. found that single-stranded DNA (ssDNA) could also be used to synthesize CuNPs (Liu et al., 2013a, Qing et al., 2013a), which further extended application of DNA templated CuNMs (DNA-CuNMs). From then on, more and more DNA templates with different structures were well-designed and investigated for the probes for the sensing (Peng et al., 2018, Qing et al., 2017, Wang et al., 2017a, Wang et al., 2016c). For example, grafting the T-loop to the ds-DNA forming hairpin structure or utilizing crowded effect to improve the fluorescence of DNA-CuNMs were demonstrated for high-performance sensing (Qing et al., 2017, Wang et al., 2017a, Wang et al., 2016c). In the initial stage, the optical feature of DNA-CuNMs was often investigated as the single readout and lots of enzyme reaction and DNA based hybridization chain reaction have been introduced to broaden the application. Recently, to achieve the accurate results, coupling different metal NCs and other DNA fluorescent switcher (Chen et al., 2017a, Chen et al., 2016, Wu et al., 2016) with DNA-CuNMs provided a new strategy for sensing. Meanwhile, with the development of in situ eletrochemical reduction for the formation of DNA-CuNMs, the electrochemical-related features of DNA-CuNM have been excavated including the larger electrical resistance, the electrochemical stripping signal, the electrochemiluminescence (ECL) response of DNA-CuNMs and the Cu(II) based catalyzed oxidation of eletrochemical probes (Hu et al., 2017, Liao et al., 2018, Wang et al., 2015e, Zhou et al., 2018). Moreover, the chemical calalytical properties of the DNA-CuNMs, such as the enzyme-mimic properties of Cu(II) generated by acid treatment of DNA-CuNMs were also exploited (Borghei et al., 2018, Mao et al., 2016). And the applications have been extended from sensing to the logic gate construction, and staining.