Translation of the Long-Term Fundamental Studies on Viral DNA

Translation of the long-term fundamental studies on viral DNA packaging motors into nanotechnology and nanomedicine Chenxi Liang, Tao Weitao, Lixia Zhou and Peixuan Guo Citation: SCIENCE CHINA Life Sciences 63, 1103 (2020); doi: 10.1007/s11427-020-1752-1

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SPECIAL TOPIC: Nanobiology August 2020 Vol.63 No.8: 1103–1129 •REVIEW• https://doi.org/10.1007/s11427-020-1752-1

Translation of the long-term fundamental studies on viral DNA packaging motors into nanotechnology and nanomedicine Chenxi Liang1, Tao Weitao2, Lixia Zhou1 & Peixuan Guo1*

1Center for RNA Nanobiotechnology and Nanomedicine; College of Pharmacy; College of Medicine; Dorothy M. Davis Heart and Lung Research Institute; and Comprehensive Cancer Center. The Ohio State University, Columbus, Ohio 43210, USA; 2Biology Department, College of Science and Mathematics, Southwest Baptist University, Bolivar, Missouri 65613, USA

Received May 8, 2020; accepted June 4, 2020; published online June 29, 2020

Many years of fundamental studies on viral genome packaging motors have led to fruitful applications. The double-stranded DNA (dsDNA) viruses package their genomes into preformed protein shells via nanomotors including several elegant and meticulous coaxial modules. The motor is geared by the hexameric RNA ring. An open washer displayed as hexametric string of phi29 motor ATPase has been reported. The open washer linked into a filament as a queue with left-handed chirality along the dsDNA chain. It was found that a free 5′- and 3′-dsDNA end is not required for one gp16 dimer and four monomers to assemble into the hexametric washer on dsDNA. The above studies have inspired several applications in nanotechnology and nanomedicine. These applications include: (i) studies on the precision motor channels have led to their application in the single pore sensing; (ii) investigations into the hand-in-hand integration of the hexametric pRNA ring have resulted in the emergence of the new field of RNA nanotechnology; and (iii) the studies on the motor stoichiometry of homologous multi-subunits that subse- quently have inspired the discovery of new methods in highly potent drug development. This review focuses on the structure and function of the viral DNA packaging motors and describes how fundamental studies inspired various applications. Given these advantages, more nanotechnological and biomedical applications using bacteriophage motor components are expected. nanopores, packaging motor, phi29 pRNA, single pore sensing, chemical detection, peptide differentiation and sensing, nanotechnology, nanobiotechnology

Citation: Liang, C., Weitao, T., Zhou, L., and Guo, P. (2020). Translation of the long-term fundamental studies on viral DNA packaging motors into nanotechnology and nanomedicine. Sci China Life Sci 63, 1103–1129. https://doi.org/10.1007/s11427-020-1752-1

Introduction al., 2001). It has a very compact structure, for example, the prohead of the phi29 only consists of three kinds of proteins Among the viral assembly process, one of the most crucial (Bjornsti et al., 1985; Bjornsti et al., 1981). In 1981, Bjornsti machinery is the DNA packaging motor. These motors are et al. recorded that “all of the viral structural proteins have nanoscale machines that convert a primary energy source to been identified, and assembly occurs by a single pathway. mechanical motion between active and framework compo- Several true intermediates in the pathway have been identi- nents (Guo and Lee, 2007; Petrov and Harvey, 2008). Phi29 fied and their protein compositions have been defined.” In is a subset of bacteriophages which affects Bacillus subtilis the late 1980s, it was discovered that the phi29 DNA (Anderson et al., 1966; Reilly and Spizizen, 1965). Bacter- packaging motor was, in fact, a protein-RNA complex (Guo iophage phi29 has been a crucial model organism in studying et al., 1987b). With the development of structural biology viral morphogenesis, since the late 20th century (Meijer et technologies, such as single-molecule methods and biotechnological approaches, we now have a better under- *Corresponding author (email: [email protected]) standing of the molecular basis of structure in biological

© Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature 2020 life.scichina.com link.springer.com http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 1104 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8 movement. The elegant and delicate structures of the viral hexameric models, and the finding is strongly against the dsDNA packaging motors have opened a new chapter in the pentameric model. Besides, only the high architecture of the field of nanotechnology. The connector of the viral DNA hexameric revolving model can provide enough coordination packaging motor is a protein channel that can be used for required by the genomic DNA. These findings together single-molecule sensing. The discovery of the motor strongly support the hexametric revolving model of phi29 packaging RNA (pRNA) with a three-way junction (3WJ) DNA packaging motor. structure generates new ideas in the field of RNA nanotechnology. The RNA nanoparticles derived from RNA na- Structure of the viral DNA packaging motors notechnology are good candidates for the next generation of molecular medicine (Chandler and Afonin, 2019; Jang et al., Biological motors are nanoscale energy-transforming ma- 2016; Paredes et al., 2011; Parlea et al., 2016). This paper chines. They transform chemical energy into mechanical will introduce the study and application of viral DNA motion between framework and active components. Bio- packaging motors in single pore sensing and development of motors are essential to biological systems since they support RNA nanotechnology and nanomedicine. the transportation of cellular components (Chen and Guo, 1997; DeRosier, 1998; Noji and Yoshida, 2001). Thanks to the progress in biotechnology, structural biology, and single- Structures and functions of the biomotors for molecule methods (Ishii et al., 2001), our understanding of translocation of viral genomes the molecular basis of the motor structure and function in biological movements of the motor has inspired us to make The structure and mechanism of the DNA packaging motors possible applications of biomotors in nanotechnology (Hess of various bacteriophages such as phi29 have been a major and Vogel, 2001). topic for many years. For long, scientists have different Bio-nanomotors perform vital functions in the double- opinions about the structure and mechanism of the DNA stranded DNA bacteriophages, such as Bacillus phi29 (An- packaging motor of phi29 (Bourassa and Major, 2002; Guo derson et al., 1966; Reilly and Spizizen, 1965) and T7 (Csáky et al., 2014b). Since 1978, a number of scientists have be- et al., 1950) in the Podoviridae family of tailed phages lieved that the DNA-packaging motors of dsDNA viruses are (Daudén et al., 2013). During the replication and morpho- pentameric machines, while the proheads of viruses have a genesis, these viruses encapsulate and compress their geno- six-fold structure (Bourassa and Major, 2002). This five- mic DNA into the preformed protein shells, the procapsids, fold-six-fold mismatch is the mechanism to drive the motor with remarkable velocity. This DNA packing process is ac- following a nut-and-bolt rotatory manner (Hendrix, 1978). In complished by the viral DNA packaging motors, powered by this model, the motor and the DNA are like a nut and bolt ATP hydrolysis (Black, 1989; Casjens et al., 1988; Cue and system, such that the motor works as nuts, and the DNA is Feiss, 2001; Guo, 1994; Moore and Prevelige Jr., 2002). One the bolt. However, subsequent studies on the DNA packa- ATP molecule is spent on the packaging of two base pairs of ging motor of bacteriophage phi29 system have revealed that DNA (Guo et al., 1987c). The DNA translocation ATPase the motor has a dodecameric connector (Jiménez et al., 1986) activity is dependent on prohead proteins and DNA. geared by a hexameric RNA-protein ring (Guo et al., 1987b; The viral DNA packaging motor is comprised of a few Guo et al., 1998). This finding is against the rotatory me- portions (Figure 1A). The motor structure frame includes chanism, and several following studies point out a revolving ATPase for force generation. The gearing components work DNA translocation mechanism when motors transport their in conjunction with the portal connector complex. The con- dsDNA substrate (Schwartz et al., 2013b; Zhao et al., 2013). nector has a dodecameric structure (Bazinet and King, 1985; Even when only a few of the subunits are able to bind ATP, Black, 1988, 1989; Casjens, 1985) forming a central channel ATPase function is still retained (Guo et al., 2019; Martin et (30–60 Å) through which viral DNA is translocated into the al., 2005). Accompanying this nut-and-bolt model is a fer- capsid and exits during infection (Li et al., 2013). While the vent debate on whether the structure of the viral DNA portal proteins from various viruses share little sequence packaging motor is pentamer or hexamer, and whether the homology and display great variations in molecular weight, motion mechanism is rotation or revolution has continued for the portal connector complexes demonstrate significant more than 20 years. With the development of the technique, morphological similarities (Bazinet and King, 1985). Inter- such as Cryo-Electron Microscopy (Cyro-EM), ever in- estingly, the phi29 connector is associated with both pRNA creasing evidence about the structure and mechanism of the and ATPase gp16 (Chen and Guo, 1997; Garver and Guo, motor has been revealed. A recent study using the high- 2000; Guo et al., 1987c; Lee and Guo, 2006; Xiao et al., resolution Cryo-EM structures clearly shows six copies of 2005). Recruitment of ATPase to the packaging motor re- the complex (Yang et al., 2020). The results from Yang et al. quires pRNA, because pRNA has the central domain for compared the assembly process of both pentameric and connector binding and the 5′/3′ paired helical region for gp16 http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8 1105

Figure 1 Structure and function of phi29 DNA packaging motor (Schwartz et al., 2013b; Shu et al., 2013). A, Structure of hexameric pRNA and the connector showing a 30° tilt. B and C, dsDNA showing the shift of 30° angle between two adjacent connector subunits. Reprinted with permission from Schwartz et al., 2013b. Copyright 2013 Elsevier. D, AFM images of hexameric pRNA with 7-nucleotide loops. Adapted with permission from Shu et al., 2013. Copyright 2013 RNA Society. E, The mechanisms for the revolving motor. Reprinted with permission from Guo et al., 2019, further permissions related to the material excerpted should be directed to the American Chemical Society. F, Direct observation of ATPase complexes moving along dsDNA. Gp16 linked with Cy3 were incubated with (a, b, e) and without (d) dsDNA. Adapted from Schwartz et al., 2013b with permission from Elsevier. G, gp16 hexamer functions as an open filament with a different chirality from the rotatory motor RecA.H, The motor subunits in dimeric form can gather around the circular dsDNA first and then form a hexamer complex. Adapted from Guo et al., 2014b. Copyright 2014 Elsevier Inc. recruitment (Hoeprich and Guo, 2002; Lee and Guo, 2006). smaller-size proteins for DNA binding and processing. It These structural features are not found in the T7 packaging includes gp16 of T4, gp18 of T3/T7, gp1 of phi21, and motor. gpNu1 of λ (Guo et al., 1987c). Moreover, the structure of Of the motor parts, ATPase and portal protein of the group the phi29 portal protein has been characterized at atomic of dsDNA viruses have been studied extensively. The results resolution (Guasch et al., 2002). The connector forms a ring- about ATPase reveal that all DNA packaging motors known like structure by 12 α-helical subunits. The three long helices so far contain two nonstructural proteins for DNA packaging of each subunit create the central channel. The phi29 con- (Guo et al., 1987c). These proteins can be further classified nector takes a truncated cone-shape with the external dia- into two categories according to their roles and sizes (Guo et meters of the ring 138 Å at its wide and 66 Å at the narrow al., 1987c). One category has larger sizes binds procapsids end. The internal diameters are 60 Å at the top and 36 Å at and serves as ATPase. It includes gp16 of phi29 (Guo et al., the bottom. The wider end of the connector is anchored into 1987a; Guo et al., 1987b), gp17 of T4, gp19 of T3/T7 (Endy the prohead, whereas its narrow end partially sticks out of the et al., 1997; Morita et al., 1993; Sun et al., 1999), gpA of λ capsid. The connector is embedded in the pentagonal portal (Becker and Gold, 1988; Hang et al., 2001), and gp12 of vertex of the procapsid (Bazinet and King, 1985; Guasch et phi21 (Feiss et al., 1985). The second category includes the al., 2002; Jiménez et al., 1986). This causes a symmetry http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 1106 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8 mismatch between capsid and portal (Bazinet and King, mechanism for the DNA packaging motors, the revolving 1985; Hendrix, 1978). The connector acts as a nucleation mechanism of DNA translocation was proposed and first point for assembly of procapsid and the binding to pRNA discovered in the dsDNA packaging motor of phi29 (Lee and Guo, 1995; Guo et al., 1991). (Schwartz et al., 2013b; Zhao et al., 2013). The revolving model was originally supported by the evidence from The revolving biomotors for packaging of the viral dsDNA structural studies of the motor channel that has a diameter genomes larger than that of dsDNA and that has the left-chirality of the channel wall in contrast to the right chirality of the rotation Biomotors were previously classified into two categories: motor (De-Donatis et al., 2014). The low resolution Cryo- linear and rotational motors. Biotechnological and biophy- EM images (De-Donatis et al., 2014) exhibited both a toroid sical studies revealed that the connector portal does not rotate of dsDNA around the portal region (Sherratt et al., 2010; during DNA packaging (Baumann et al., 2006; Hugel et al., Tang et al., 2008) and a tilting of the dsDNA moving through 2007; Schwartz et al., 2013a). Tethering of the DNA termi- the connector channel (Guo et al., 2013). Further evidence nus to beads demonstrated that DNA translocation by the stemmed from the functional analysis. When the T4 con- motor was still active while no detectable rotation of the bead nector was fused to the procapsid protein, the connector or bead clusters was observed (Chang et al., 2008). These could not rotate, yet the motor was able to translocate the results led to a puzzle concerning how the spiral-shape motor DNA substrate (Baumann et al., 2006). The connector ex- nut can drive the helical dsDNA bolt without rotation of hibits no sign of rotation as examined by the single-molecule either the bolt or nut. This puzzle has been solved by the force spectroscopy combined with polarization spectroscopy recent discovery of a third type of biomotor mechanism of studies (Hugel et al., 2007). When the bead clusters were revolution without rotation (Schwartz et al., 2013a; Zhao et tethered to both ends of the DNA substrate, no sign of al., 2013). By analogy, rotation is the turning of the object complete rotation was observed during the active packaging along its own axis, resembles the Earth rotating one cycle of dsDNA (Chang et al., 2008). This is because the dsDNA every 24 hours; while revolution is the turning of the object substrate twists approximately 1.5°/base pair (bp) instead of along a second object, resembles the Earth revolving around a 360°/complete helical turn as it is translocated. If one he- the Sun, one cycle per 365 days (Guo, 2014) (Figure 2). This lical turn of dsDNA contains ~10.5 bases, then 1.5°/bp×10.5 revolving model requires a sequential conformational change bp/turn=15.7°, far smaller than 360°. This confirms a non- to utilize energy from ATP to drive DNA packaging. The rotation mechanism of the revolving motors. authors reported two distinct conformations of pUL28 with ATP bounded or apo state, which indicates the existence of Translocation of dsDNA by the substrate revolving may be ATP-driven conformational change. This structural evidence a common mechanism during biomotor evolution provides solid support of the basic mechanism of the revolving model agreeing with earlier findings that viral DNA The revolving mechanism exists in both prokaryotic and packaging protein is the procapsid and DNA-dependent eukaryotic systems. The results from many studies exhibit a ATPase (Guo et al., 1987a). revolving mechanism by which the dsDNA substrate re- The viral motors package the viral genomes resisting an volves through the channel of the biomotors in one direction accumulating internal pressure in an entropically-unfavor- (Figure 1). The dsDNA translocation is unidirectionally co- able fashion. The biomotors overcome this entropic barrier ordinated by several motor components as discussed below. by consuming energy for genome packaging (Guo et al., The revolution mechanism is commonly used by the dsDNA 1987a; Hwang et al., 1996; Sabanayagam et al., 2007). Al- packaging motors of all the dsDNA bacteriophages identi- though the rotational motion was long thought as a common fied so far. During viral replication, the dsDNA packaging

Figure 2 Illustration of two different types of motors. A, Rotation motors are like a wheel and like the Earth rotating on its own axis. Reprinted with permission from Guo et al., 2016. Copyright 2016 American Society for Microbiology; and adapted with permission from De-Donatis et al., 2014. Copyright 2014 Springer Nature. B, Revolution motors resemble the Earth revolving around the Sun without self-rotation. Reprinted with permission from Guo, 2014. Copyright 2014 Elsevier. http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8 1107 motors translocate the viral genomes into procapsids by the and prokaryotic systems (Ligat et al., 2018). The revolving same mechanism (Guo and Lee, 2007; Serwer, 2010; Zhang mechanism possibly represents a convergent evolution of et al., 2012) in an entropically-unfavorable process driven by dsDNA biomotors among the eukaryotic and bacterial ATP hydrolysis (Guo et al., 1987c; Hwang et al., 1996; Sa- viruses. This hypothesis is further grounded on the works banayagam et al., 2007). The following revolution mechan- about the structure of the Herpes Simplex virus DNA- ism is shared by the bacteriophage packaging motors of packaging terminase (Przech et al., 2003; Selvarajan Siga- phi29, HK97, SPP1, P22, T4, and T7. An anti-parallel ar- mani et al., 2013). rangement of the portal with the right-handed helix of the dsDNA results from the 30° left-handed twist of the channel Revolving mechanisms are defined by channel sizes of wall, leading to the one-orientation trend (Figure 1B and C). biomotors The same twist is seen in the motor channels of many dsDNA The nut-and-bolt rotation model requires a relatively small viruses, such as phi29, HK97, SPP1, P22, T4, and T7. While size of the connector channel relevant to the dsDNA helix. the primary amino acid sequences of the channel proteins are For dsDNA to rotate through the center of the motor channel, non-conserved, the higher structures of the swivel are clearly the diameter of the channel ought to be similar to that of conserved and aligned. One-direction flow loops within the dsDNA, which is about 2 nm (Figure 3A) (Besprozvannaya channel of SPP1 and phi29 facilitate a one-directional pro- and Burton, 2014; Guo et al., 2014a; Lebedev et al., 2007; cessing for the unidirectional translocation of dsDNA. In- Mancini et al., 2004; Massey et al., 2006). Accordingly, the teraction of the electropositive-lysine layers existing in the diameter of the channel should be about 2 nm for dsDNA or viral channels with one of the electronegative-dsDNA 1 nm for ssDNA. If the channel of the nut would have been phosphate backbones causes a relaying contact and transi- larger than the bolt, turning would not have occurred since tional pausing during dsDNA translocation. Apparently, the two layers will lose contact. Thus, most rotation motors translocation by revolving the dsDNA substrates appears a contain a channel that is smaller than 2 nm in diameter. common mechanism among many bacterial dsDNA viruses. However, if dsDNA revolves through the motor channel, This suggests that the revolving mechanism represents a dsDNA travels by touching the channel wall instead of parallel evolution of dsDNA biomotors of bacteriophages. proceeding through the channel center. Therefore, the dia- The revolving mechanism also is found in the eukaryotic meter of the channel should be much greater than that of system. In eukaryotic cells, the post-catalytic MCM forms an dsDNA to ensure adequate room for revolution. The crystal inactive double hexameric channel for dsDNA. The loading structures of the motor hexamers including those of T7 and of the MCM helicase motor onto dsDNA requires ATP hy- phi29 support that the channel diameters of the revolving drolysis and licenses the origins. The Cryo-EM structures of motors are higher than 3.5 nm, but those of rotation motors DNA-bound MCM show that as MCM pore loops touch both smaller than 2 nm. DNA strands, they constrain dsDNA in a bent configuration Extensive studies reveal that the diameter of the channel of possibly to facilitate DNA untwisting and melting at the many connectors (portal vertexes) of procapsids or the as- onset of replication (Abid Ali et al., 2017). While the re- sembly intermediates in dsDNA bacteriophages are larger volving mechanism remains to be determined in the MCM than the diameter of the dsDNA (Guo et al., 2019; Guo et al., model, the results from the most recent investigation on the 2014a; Schwartz et al., 2013b; Zhao et al., 2013). The con- architecture of the herpesvirus genome packaging complex nector channels of phi29 SPP1, T4, T7, HK97, and FtsK suggest a sequential revolution model for DNA transloca- share common parameters: the width of dsDNA helix is tion. Cryo-EM of a herpesvirus terminase complex exhibits a 2 nm, but the diameter of the narrowest regions of the do- hexameric ring structure. Each subunit of the hexameric ring decameric portals are 3–5 nm (Besprozvannaya and Burton, is made of a heterotrimer of three proteins pUL15, pUL28 2014; Massey et al., 2006). In the herpesvirus motor, it is and pUL33 (Yang et al., 2020). The hexameric ATPase/ter- reported that the channel size is about 50% larger than that of minase pUL15 forms a central channel with an internal dsDNA (Yang et al., 2020). Yang et al. recently reported a diameter of 3.9 nm, wider than that of dsDNA (Yang et al., high-resolution structural and mechanism of the ATP-driven 2020). The channel contains the conserved basic patches DNA packaging motor of the double-stranded herpesvirus, conducive to DNA binding. It has the transacting arginine which is a hexamer exercising the revolution mechanism fingers required for ATP hydrolysis. Surprisingly, the (Figure 3B). The elucidation of the structural ends the 20- dsDNA motor of the human virus shares the structural year fervent debate on the stoichiometry and mechanism of properties with that of the bacterial virus. Such similarities in the genome packaging motor for dsDNA viruses. All the the hexameric structure of ATPases with the transacting ar- above features support that these DNA packaging motors, ginine fingers support a notion of sequential DNA translo- including phi29 DNA packaging motor, belongs to the re- cation via revolving motion, which may be a common volving biomotor family, which have a much larger channel mechanism during viral motor evolution in both eukaryotic than most rotatory motor channels. Channel size correlation http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 1108 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8

Figure 3 The structure of hexametric ATPase motor. A, Channel size different between rotating and revolving mechanism. Rotating motors have channel sizes ≤2.0 nm in diameter to ensure full contact between DNA and channel wall similar to the nut driving the bolt, while revolving motors have channel sizes ≥3 nm to have room to accommodate the revolving motion. Adapted with permission from Guo et al., 2014a and Besprozvannaya and Burton, 2014. Copyright 2014 Springer Nature. B, The structure for DNA packaging motor of the double-stranded DNA herpesvirus (left), and the elucidation of the revolving mechanism in DNA translocation (middle and right). Adapted in part with permission from: Creative Commons Attribution 4.0 International in Architecture of the herpesvirus genome-packaging complex and implications for DNA translocation (Yang et al., 2020). with the motor mechanism has been summarized (Guo et al., genome at uneven speeds as reported in both phi29 and T4. 2014a). The pauses in translocation speed cause stepwise translocation of dsDNA. They include four steps of dsDNA motion as The revolution mechanisms are distinguished by their described in DNA packaging motor of phi29 by laser trap chirality experiments. While these four steps may arise from four The anti-parallel arrangement between the subunits of phi29 states of the ATPase associated with ATP hydrolysis, a more connector and the helices of dsDNA facilitates revolution of plausible explanation is that the electrostatic interactions dsDNA in one direction (Schwartz et al., 2013b; Zhao et al., between the negatively-charged phosphates in dsDNA and 2013) (Figure 4). All 12 subunits of the connector portal the positively-charged amino acids within the motor channel protein of phi29 tilting at a 30° left-handed angle relative to of phi29 (Zhao et al., 2013). Such interactions between the the vertical axis of the channel form the motor channel that phosphate backbone of the dsDNA and lysine layers of the takes an anti-parallel configuration to the right-hand dsDNA connector are responsible for the uneven steps in dsDNA helix during genome packaging (Zhao et al., 2013). This translocation. Particularly, the uneven steps are due to mis- structural arrangement of the biomotors considerably facil- match between 10.5 bases per DNA helical pitch and the 12- itates the controlled motion of DNA translocation; thus, this subunit connector as reviewed recently (Guo et al., 2014b). is structural evidence for dsDNA revolving rather than ro- Crystal structure analysis of the connector unveiled that on tating through the connector channel of the revolving motor. the interior channel surface of the negatively-charged con- The revolving motion minimizes contact of dsDNA with nector are 48 positively-charged lysine residues (Guasch et each of the 12 connector subunits in 12 discrete steps of 30° al., 2002), which are organized as four lysine rings sur- transitions for each helical pitch, thus free of coiling or tor- rounding the 12 subunits of the connector. When dsDNA sion force (Schwartz et al., 2013b). revolves in a 360° motion across these 12 subunits, the negatively-charged phosphate backbone of dsDNA is attracted Stepwise translocation of dsDNA results from electrostatic to the same positively-charged lysine layer (Schwartz et al., interaction 2013a; Schwartz et al., 2013b; Zhao et al., 2013), resulting in Many dsDNA bacteriophages translocate the viral dsDNA an uneven speed of dsDNA translocation (Schwartz et al., http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8 1109

package the genomic RNA into the previously assembled procapsid (Collier et al., 2016; Hu and Liu, 2017; Roy, 2017; Sato et al., 2018; Zhang et al., 2015). The Cystoviridae family of bacteriophage, phi6-phi14, has been used widely to study the genome packaging process of the dsRNA virus (Borodavka et al., 2018). These dsRNA bacteriophages of the Cystoviridae family first package the positive-sense(+) single-strand RNA into the assembled procapsid (Poranen et al., 2001). This step might be alternated by assembling the capsid around the RNA molecules and RNA-protein complexes (Borodavka et al., 2012). Then the viral +RNA will be converted by the RNA polymerases located inside the procapsid. Figure 4 Different chiralities of rotating and revolving motors. Rotating The packaging process of the +RNA is precise and serially biomotors exhibit right-handed chirality to drive the right-handed dsDNA similar to the nut driving the bolt or the screw driver turning the screw, dependent. The genome of the phi6, one member of the whereas revolving biomotors exhibit left-handed chirality within the Cystoviridae family, can be divided into three segments, S, channel (De-Donatis et al., 2014). Crystal structure analysis of viral DNA M and L (Frilander et al., 1995; Juuti and Bamford, 1995; packaging motors reveals that this class of biomotors package DNA using the revolving mechanism. Reprinted with permission from Guo et al., Mindich, 2012). The segment S will first bind to the pro- 2014a. Copyright 2014 Springer Nature. Reprinted in part with permission capsid. While segment S is being packaged by the ATPase from De-Donatis et al., 2014. Copyright 2014 Springer Nature. motor, the binding site of the procapsid will change from segment S binding sites to segment M binding sites. Then, 2013a; Zhao et al., 2013). As the channel is largely nega- when segment M is fully packaged, segment L can bind to tively charged, the same charged dsDNA is repulsed during the procapsid (Guo and Lee, 2007). All of the three segments translocation. Yet for dsDNA revolution to occur, the posi- have the same sequence, pac, on the 5′ ends, and the pac tively charged lysine residues in the channel pull dsDNA to sequence functions as the packaging signal (Iba et al., 1982). the wall repeatedly to maintain the revolving motion of One of the studies showed that RNA chaperone molecules dsDNA through the channel. might be essential for the segment assortment and packaging (Borodavka et al., 2015). This packaging process is com- A model has been proposed that the ATPase gp16 hexamer pleted by the phi6 packaging motor which contains four functions as an open washer linked into a filament with a different kinds of protein, P1, P2, P4, and P7 (Bamford et al., left-handed chirality 1993; Borodavka et al., 2012; de Haas et al., 1999). P1 is the It has been reported that an earlier step in phi29 DNA core scaffold of the procapsid formed by 60 dimers. P2 packaging is the binding of multiple gp16 in a queue along functions as an RNA-dependent RNA polymerase (de Haas the dsDNA (Figure 1F) (Schwartz et al., 2013b). A string of et al., 1999). P4 might be a purine-specific NTPase that has a multiple Cy3-gp16 complexes have been observed on the similar structure and function to the hexameric helicase dsDNA chains. This suggests that the queuing of ATPase (Kainov et al., 2004). This protein is essential for all the gp16 along the DNA is the earlier step in phi29 DNA packaging processes of the members of the Cystoviridae packaging. It is possible that gp16 forms a dimer before the family. P7 functions as a packaging and assembly factor, binding to the DNA. It has been noted that both ends of the which is important for the function of P4 (Poranen et al., dsDNA are tethered to the beads (Schwartz et al., 2013b). 2001). This observation proves that a free 5′ or 3′ dsDNA end is not required for the ATPase gp16 to bind dsDNA, and that as- Special aspects of the revolving motors sembly of the hexametric gp16 ring occurs only upon binding to DNA (Figure 1H). EM images also reveal multiple Force generation and energy conversion gp16 complexes bound to phi29 genome (Schwartz et al., In all biomotors known so far, cycles of nucleotide binding 2013b). A mechanism of open washer ATPase gp16 string and hydrolysis are coupled to conformational entropy re- formation on the dsDNA has been reported (Guo et al., arrangements of the substrate-binding subunits of the bio- 2014b; Schwartz et al., 2013b). motors (de la Peña et al., 2018; Harrison and Muench, 2018; Ueno et al., 2018; Watanabe et al., 2018; Wiegand et al., Packaging of the viral dsRNA genomes 2019). Three primary chemo-mechanical coupling mechanisms have been described as biomotors. By the sequential The genome packaging process of dsRNA viruses is some- mechanism, individual events of ATP binding and hydrolysis how similar to dsDNA viruses, which uses a motor to proceed sequentially. In the concerted mechanism, all active http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 1110 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8 sites on the motors hydrolyze ATP simultaneously. With the fact, one dimer complex and four monomers in the ring form stochastic mechanism, the ATPase sites hydrolyze ATP an asymmetrical hexameric structure (Dong et al., 2019; randomly (Khataee and Liew, 2015). For all the three me- Lyubimov et al., 2012; Puchades et al., 2017; Soultanas and chanisms, ATP binding to the disordered subunits of ATPase Wigley, 2001; Su et al., 2017; Sun et al., 2017; Zehr et al., stimulates conformational changes of the enzyme with the 2017). Such communication promotes an asymmetrical entropy alteration (De-Donatis et al., 2014). This secures the hexameric organization, which is consistent with the asym- bound ATPase at a less random configuration than the un- metrical conformation in other hexameric ATPase systems bound ATPase. Such a new conformation facilitates the such as V1-ATPase, TRIP13, and ClpX (Arai et al., 2013; binding of DNA to the ATPase subunits and priming of ATP Stinson et al., 2015; Ye et al., 2015; Zhao et al., 2016). hydrolysis. ATP hydrolysis by the ATPase initiates the sub- Therefore, the arginine finger functions as a bridge over two sequent entropic and conformational alterations of the en- subunits of ATPase forming a transient dimer. Explicitly, the zyme. These changes render the enzyme a low affinity for the arginine finger located at the interface of two consecutive dsDNA substrates so that dsDNA leaves to the next ATP- subunits of gp16 spreads into the ATP binding pocket of the bound subunit of a high affinity with dsDNA. By these re- downstream subunit of ATPase. The asymmetry of one dimer current actions, dsDNA revolves around the motor subunits and four monomers in high-resolution structural complexes through the interior channel of the motor. is evidence for the mechanisms by which the arginine finger promotes inter-subunit interactions and sequential actions of Unidirectional dsDNA translocation individual subunits of ATPase. The arginine finger has an The directional translocation of dsDNA is controlled by important role in regulation of energy transduction and motor several factors in the phi29 motor (Figure 1). ATPase un- function (Figure 1E) (Kötting et al., 2008; Liu et al., 1996; Yi dergoes cycles of entropy transitions and conformational et al., 2016). These findings about the arginine finger changes during ATP and dsDNA binding: ATP hydrolysis strongly support the revolving mechanism driven by the leads to the second change in entropy and conformation of conformational change of the packaging motor. the ATPase, which involves a low affinity for dsDNA to push dsDNA away and to enable DNA to revolve inside the The prohead RNA plays a role in motor conformation channel. Additionally, the 30° angle of each subunit of the dynamics dodecameric connector goes anti-parallel to dsDNA to match The phi29 pRNA functions in maintenance of the structural with the 12 subunits of the connector channel (360°/12=30°) integrity of the entire phi29 motor, especially the protein as revealed by crystallography (Figures 1 and 3). Moreover, components of the connector and ATPase. As a conserved the unidirectional flowing property of the internal channel and non-coding RNA component, pRNA is essential for loops functions as a ratchet valve to prevent dsDNA reversal. translocation and packaging of the phi29 genome (Guo, Furthermore, the 5′–3′ single-directional revolution of one 2002; Guo et al., 1987b). The 5′/3′ secondary structure of strand of dsDNA proceeds through the connector channel pRNA is generated by folding of 117 nucleotides (nt) into a wall. Lastly, the electrostatic force from the relaying inter- complex structure containing two major domains. An in- action of the electropositive-lysine layers with the electro- terlocking domain with stems and loops serves as the negative-DNA phosphate backbone affects unidirectional connector-binding domain (Reid et al., 1994a; Reid et al., dsDNA translocation (Zhao et al., 2013). 1994b; Reid et al., 1994c; Zhang et al., 1995a; Zhang et al., 1995b), whereas a helical domain with an open 5′/3′ end Communications/interactions between motor subunits for acts as the gp16-binding domain (Lee and Guo, 2006). A sequential action motif of the thermostable 3WJ (Haque et al., 2012b; Shu et A sequential action of the ATPase subunits of the phi29 al., 2011a) is situated in the center holding the connector- motor was originally described (Chen and Guo, 1997) and binding domain of the right-hand loops, left-hand loops and confirmed. Briefly, these subunits act both sequentially and gp16-binding domain. Dimeric, tetrameric, and even hex- cooperatively (De-Donatis et al., 2014; Schwartz et al., americ rings can be generated through intermolecular in- 2013b). Communication and interaction among the ATPase teractions that these loops generate (Shu et al., 2013) subunits are mediated by the arginine finger motif that (Figure 5). This pRNA 3WJ has remarkable thermo- bridges the adjacent subunits into a dimers (Zhao et al., dynamic stability to sustain the packaging motor (Haque et 2016) (Figure 1E). Upon the ATP binding, the R346 of the al., 2012b; Shu et al., 2011a; Zhang et al., 2013). pRNA arginine finger in one subunit inserts into the ATP binding affects the conformation of the phi29 motor through the pocket of another nearby subunit. This R346 will then in- ATPase-binding domain interacting with gp16 in response teract with the γ-phosphate. The two adjacent subunits may to ATP; therefore, the conformation change of the pRNA- form a more compact dimer configuration, and hence could gp16 complex has a critical role in translocation and not be distinguished in low-resolution Cryo-EM images. In packaging process of dsDNA. http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8 1111

Figure 5 Structure of a pRNA hexamer ring and docking of the hexameric pRNA model with the dodecameric connector ring of phi29. A, A 3D model of hexameric pRNA based on the crystal structure of 3WJ with views from three mutually perpendicular angles. B, AFM images of hexameric re-engineered pRNA rings show strong correlation in size and shape with the 3D computer models for the intact, full-length pRNA hexamers. C, Histogram of RNA particles in AFM images with discernible stoichiometry. D, Top view of the published 3D computer model of hexameric pRNA constructed based on biochemical data (PDB 1L4O) (Hoeprich and Guo, 2002). E, Model of a pRNA hexamer complexed with a dodecameric connector ring based on the 3WJ and connector (PDB 1H5W) (Guasch et al., 2002) crystal structures. F, A close-up view of proper anchoring of the connector N-terminal helices for optimal interactions with pRNA. G, Three mutually perpendicular views of the hexameric pRNA-connector assembly. H, A side-view of the 3D model of the hexameric pRNA-connector assembly based on biochemical data (PDB 1L4P) (Hoeprich and Guo, 2002). Adapted from Zhang et al., 2013.

The application of the revolving biomotors in the electrical force is applied to the sensing system, different single-pore sensing molecules of DNA, RNA, chemicals, peptide and proteins will generate fingerprinting electrical signals (Cao et al., As mentioned above, dsDNA viruses package their genome 2019; Lu et al., 2019; Niu et al., 2019; Sun et al., 2019). via a revolving nanomotor. The connector of the nanomotor These signals include distinctive current blockages and dwell is a dodecameric channel that can be applied to single-mo- time by translocating through or inducing conformational lecule sensing with high selectivity and sensitivity (Feng et changes of the nanochannel (Haque et al., 2013; Var- al., 2015; Gu and Shim, 2010; Thakur and Movileanu, 2019; ongchayakul et al., 2018; Wendell et al., 2009). Through Wang et al., 2013a). The connector with the left-handed single-pore sensing, we can detect and differentiate various chirality functions as a “push-through the one-way valve” analytes such as proteins, DNA, and RNA. (Geng et al., system. This special structure allows analytes to have a 2013; Haque et al., 2012a; McIntyre et al., 2019; Tan et al., single directional movement. These connectors of the re- 2018). The technology can be applied to medical, nano- volving motors have a size larger than 3 nm in diameter, technological, and biotechnological applications (Kono and which makes them more amenable for the platform in single- Arakawa, 2019). Here, we discuss the application of phage molecule sensing, in comparison to that of the common DNA packaging motor for analytes differentiation based on membrane pores with a size of 1.2 nm in general. When unique current blockage and dwell time signatures. The http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 1112 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8 technique can differentiate peptides with difference in length, charge, and location (Ji and Guo, 2019; Ji et al., 2018).

The mechanism of the single-pore sensing

The single-pore sensing technique has built up a powerful platform for numerous applications, including the detection of DNAs, peptides, and chemicals (Lin et al., 2020a; Sheng et al., 2020; Si et al., 2020; Zhao et al., 2019). The general mechanism of biological nanopore sensing is based on the resistive pulse technique (Figure 6) (Deamer and Branton, 2002; Jing et al., 2010). Briefly, the purified connector is inserted into the lipid bilayer membrane to form the nanopore channel. When voltage is applied across the membrane, ions pass through the channel freely. When analytes are Figure 6 Schematic diagram of the single pore sensing of a membrane- embedded connector with applied voltage in vitro (Jing et al., 2010). translocated through the membrane, ion flow is affected, Reprinted with permission from Jing et al., 2010, further permissions re- resulting in a change to the current and creates fingerprinting lated to the material excerpted should be directed to the American Che- signals. Commonly used electronic signatures that help mical Society. identify different analytes include the current blockage and dwell time. The current blockage represents the percentage ligands into the nanopore, can significantly influence the of the blocked current relative to the open current of the interaction between the analytes and the nanopore, resulting nanopore channel, while the dwell time measures the length in different electric signals (Haque et al., 2012a; Rajeev et of time the current blockage lasts. al., 2019; Wang et al., 2013b). For example, introducing As analytes are driven to translocate or interact with the more hydrophobic groups into the nanopore can increase the connector by electric force, many things affect the current dwell time for samples with many hydrophobic groups blockage and dwell time (Varongchayakul et al., 2018), in- (Rokitskaya et al., 2017). cluding the intrinsic properties of the targets such as their In conclusion, biological nanopore sensing is based on shape, length, charge, hydrophobicity or hydrophilicity, as distinct electric signals generated when analytes pass well as the type and modification of the connector (Gu et al., through or interact with the nanopore (Varongchayakul et al., 2015; Lv et al., 2014; Qiu et al., 2016; Rajeev et al., 2019). 2018). The intrinsic properties of both analytes and the na- Based on the size difference of analytes, there are two si- nopore determine the fingerprinting signals, which allows for tuations. First, for analytes that are smaller than the nanopore the differentiation and identification of unique analytes (Li et diameter, translocation through the channel generates a large al., 2019; Ling and Ling, 2013; Wei et al., 2019). variety of electric signals. For example, analytes with longer chains will have a larger current blockage and longer dwell The connectors in the single-pore sensing system time than smaller analytes (Geng et al., 2013; Haque et al., 2013; Ji and Guo, 2019; Meller et al., 2001). In addition, Nanomotors are ubiquitous and play significant roles in stronger interactions between analytes and the connector can many biological processes such as the viral genome packa- produce longer dwell times. Second, analytes that are larger ging (Catalano, 2000; Hilbert et al., 2015; Lee et al., 2008; than the diameter of the nanopore interact with the connector Němeček et al., 2007; Wolfe et al., 2014; Yang et al., 2009). on one side and induce conformational changes to the con- The DNA packaging motors of bacterial viruses, such as nector, thus generating distinct electric signals without phi29, SPP1, T4, and T7, package their DNAs into the translocation (Haque et al., 2012a). Since only specific types procapsid, a preformed protein shell, during genome re- of analytes can trigger conformational changes to the con- plication (Ji et al., 2018; Wang et al., 2017; Yu and Schaefer, nector and cause current blockages with different dwell 2008). As mentioned above, in bacteriophage phi29, the re- times, this is also a technique for analyte determination volving nanomotor consists of a hexameric packaging RNA (Wang et al., 2013b). In both cases, the type and modification (pRNA), ATPase gp16, and a nanopore channel named as the of the connector can greatly affect the electric signals (Haque connector. Briefly, the ring-shaped ATPase present on the et al., 2012a). A larger nanopore channel can allow larger pRNA binds ATP to trigger the conformational changes of analytes to translocate through the pore, while smaller na- the ATPase subunit to revolve dsDNA, moving it forward. nopore channels do not. Moreover, modifications of nano- The phi29 gp10 connector has a large diameter and the pores, such as site-directed mutagenesis or introducing narrowest part in the channel is ~3.6 nm to allow the trans- http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8 1113 location of dsDNA into the procapsid (Guo et al., 2016; Hu such as the lysine and arginine, which can attract the DNA and Garen, 2001). The movement of the dsDNA around the and hence prevent the reverting of DNA. motor, similar to how the Earth revolves around the sun SPP1 connector: This connector has four domains (Igna- without self-rotation of the Earth, avoids the coiling and tiou et al., 2019; Serrano et al., 2020). They are the clip, stem, tangling. From this DNA packaging process, researchers wing, and crown domain. The recombinant SPP1 connector determined that biomotors drive DNA passage through the has 12 or 13 protein subunits that assemble to form a cone nanopore into the procapsid, and that this process is uni- structure similar to that of the phi29 connector, with a total directional (Guo et al., 2016). molecular weight of 745 kD. The overall diameter of the Single-pore sensing is derived from the natural phenom- SPP1 nanochannel is ~16.5 nm, with a height of ~11 nm. The enon of DNA packaging. Scientists have discovered that as most constricted region in the tunnel is ~2.7 nm (Lebedev et analytes are driven through the extracted nanopore, the al., 2007). The negatively and positively charged residues of translocation generates a unique electric signal with applied the SPP1 channel are also required for DNA packaging akin voltage (Varongchayakul et al., 2018). The proof of concept to those in the phi29 connector. about the nanopore sensing was initially demonstrated by the T3 connector: This connector consists of a mixture of inspection of DNA translocation through the α-hemolysin (Streff et al., 2020) 12 and 13 subunits of gp8, based on the nanopore in 1996 (Kasianowicz et al., 1996). In the original protein expression conditions and other factors (Donate et research, the qRT-PCR verified the successful translocation al., 1988; Valpuesta et al., 2000). The height, width, and of the single-stranded DNA that was electronically driven diameter for the T3 connector with 12 units are 8.5, 14.9, and through the α-hemolysin nanochannel (Kasianowicz et al., 3.7 nm respectively for the internal channel. 1996). To date, the nanopore sensing has been developed for T4 connector: The 12 subunits in T4 connector form a different analytes detection as it has many advantages as an dodecameric ring with a length of ~12 nm. The portal analytical tool (Wang et al., 2018). First, the detection is complex has a molecular weight of ~660 kD, while its ex- label- and amplification-free. Second, it can achieve single- ternal diameter varies from 8 to 17 nm (Ali et al., 2019; Attai molecule detection with high specificity and sensitivity and and Brown, 2019; Maghsoodi et al., 2019; Park et al., 2019; offer real-time identification. Third, it only requires small Shi et al., 2019). The thinnest part in the T4 inner channel is analyte sample volumes (µL range) of analytes and low ~2.8 nm. The clip region in the T4 nanopore can bind to the concentrations (nmol L–1 or pmol L–1 range) for analysis. C-terminal of the terminase for DNA packaging. Similar to The well-known biological connectors that are found in the phi29 and SPP1, the negatively and positively charged bacteriophage (Asija and Teschke, 2019b; Duda and residues are also in T4 nanochannel to help the DNA Teschke, 2019; Podgorski et al., 2020; Weiditch et al., 2020; packaging (Buerger et al., 2019; Hodyra-Stefaniak et al., Weiditch et al., 2019) include but are not limited to those of 2019; Joiner et al., 2019; Streff et al., 2020; Zhang et al., phi29, SPP1, T3, T4, P22, and T7 (Gao and Yang, 2020; 2020). In addition, the positively charged residues in the T4 Holtzman et al., 2020; Ma et al., 2020; Serwer et al., 2019). connector loop can help stabilize the DNA after packaging. The following is a list of the well-studied connectors with a P22 connector: The recombinant P22 portal has 11 or 12 detailed description. subunits (gp1) with a molecular weight ~940 kD (Cingolani Phi29 connector: This phi29 connector portal protein et al., 2002; Motwani and Teschke, 2019; Zheng et al., 2008). atomic structure has been solved. The connector consists of The pore diameter for P22 ranges from 2.5 to 4 nm. It has a 12 protein subunits that form a ring-like truncated cone unique domain called alpha-helical barrel domain, which structure. The molecular weight for each subunit is 36 kD. functions to prevent the tangling, hence facilitating the DNA The end diameters of the cone structure are 6.6 and 13.8 nm, translocation. The P22 connector clip region can bind to the with the wider and narrower ends termed the C- and N- prohead to cause a conformational change to assist in the terminals, respectively. The area of the narrowest part of the DNA packaging (Asija and Teschke, 2019a; González-Davis inner channel is 10 nm2, corresponding to a diameter of et al., 2020; Kim et al., 2019; Uddin et al., 2019; Wang et al., ~3.6 nm. In bacteriophage phi29, the C-terminal is located in 2019). For the portal ring, the positively and negatively the procapsid, and the translocation of dsDNA during charged residues attract each subunit to strengthen the sta- packaging is unidirectional from the N-terminal to C-term- bility of the formed ring nanochannel (Zheng et al., 2008). inal (Haque et al., 2015). The clip region in the phi29 con- T7 connector: Twelve protein subunits form this connector nector can bind with pRNA to help with the DNA packaging. with a molecular weight of 59 kD for each subunit (Alexyuk In addition, the negatively charged residues are necessary for et al., 2019; Foster et al., 2019; Liu et al., 2020; Singh et al., phi29 DNA packaging, as these residues, including the as- 2019). The channel length is 13.1 nm, with external dia- partate and glutamate in the channel, have the potential to meters ranging from 5.9 to 17.3 nm. The most restricted keep the DNA in the center of the channel. Moreover, the region in the T7 interior channel is 3.9 nm, which is rela- phi29 nanochannel includes positively charged residues, tively wide compared to the other types of bacteriophage http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 1114 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8 connectors (Agirrezabala et al., 2005; Dedeo et al., 2019). Application of the biological nanopore sensing system in The lysine residue in the stem domain of T7 nanochannel is DNA, RNA, and protein analysis highly favored to interact with the phosphate backbone to As mentioned above, different analytes can generate dis- help with DNA translocation. tinctive electric signals to be distinguished from the back- HK97 connector: It consists of 13 subunits of gp6 of the ground. The following will address several experimental long-tailed phages family (Cardarelli et al., 2010a). The in- examples to elucidate the single-pore sensing technique ner and outer diameter for the ring-shaped HK97 connector better. is ~3.7 on average and 11.4 nm, respectively. The height is The single-pore sensing of DNA is based on the fact that ~4 nm from the bottom ring to the C-terminus of the struc- different sizes and conformations of DNA can cause various ture. The gp6 monomer includes four long α-helices and two current blockage events (Haque et al., 2013; Wendell et al., β-strands. 2009). The phi29 gp10 connector has been shown capable of Bacteriophage λ connector: The λ connector comprises the differentiation of dsDNA with diverse conformations by rings of proteins gpW and gpFII (Cardarelli et al., 2010b; Haque and coworkers (Haque et al., 2015). In the experi- delToro et al., 2019). The gpW is necessary for the DNA ment, Haque et al. examined the translocation and analyzed stabilization within the head as well as the gpFII addition. the current blockage of the folded 5 kbp dsDNA. Figure 7 Epsilon15 connector: This connector has a 6-fold sym- shows the current signatures for dsDNA with different con- metry, with its height and width of ~20 and 18 nm, respec- formations. It is found that different conformations caused tively (Jiang et al., 2006). distinct blocking events. The straight dsDNA caused a single Over the years, the nanopore-based sensing technique has level blockage of ~32%, while the folded part of the dsDNA been developed for the detection of various molecules and that is depicted in Type I, II, and III resulted in a double level has proven its potential for the diagnosis of diseases (Wang et blockage of ~64%. Moreover, the dwell time for the folded al., 2013b). The following sections discuss the mechanisms dsDNA was ~3 times longer than that for the unfolded DNA and provide examples of single-molecule sensing using na- with an applied voltage at 40 mV. Besides, the dsDNA and nopores from bacteriophages. tetra-stranded DNA (tsDNA) showed a difference in their

Figure 7 The translocation of different types of DNA in nanopore. A, The current trace signals of dsDNA. B, The distribution of 7,500 translocation events for different types of DNA from quantitative analysis. C, The illustration and corresponding current blockage signals for different types of DNAs. Reprinted with permission from Haque et al., 2015. http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8 1115 current blockage events. In short, the dsDNA mainly caused 32% current blockage events, whereas the tsDNA produced both the 32% and 64% blockage events (Haque et al., 2015). These experiments build up a foundation for future studies on the detection of biomarkers from the conformational changes. Different types of RNAs have been found to be able to serve as the biomarkers for disease diagnosis, demonstrating their importance to lives (Arantes et al., 2018; Meng et al., 2017; Rysz et al., 2017; Yang et al., 2014; Zhu et al., 2019). Currently, the sensing of RNAs with bacteriophage nanopores is at the beginning stage (Yang et al., 2018). Detection of single-stranded RNA (ssRNA) with a modified phi29 nanopore has advanced the application of bacteriophage Figure 8 A schematic explanation of chemicals binding with nanopore connectors in RNA detection (Geng et al., 2013). Geng et al. and the corresponding current signals. Reprinted with permission from determined that the removal of the internal loop segment of Haque et al., 2012a. Copyright 2012 American Chemical Society. the phi29 channel creates a modified channel with a cross- sectional area of about 40% less than that for the wild-type phi29 connector. The translocation of ssRNA was identified (Table 1). by the modified phi29 connector, which resulted in ~20% Even though the nanopore-based sensing of proteins or current blockage. peptides is a nascent technology, it can detect proteins or Biological nanopores are also proven to be capable of peptides at the single-molecule level presently (Ji and Guo, detecting chemicals after connector modifications (Haque et 2019; Ji et al., 2018). In the single-pore sensing of poly- al., 2012a). Presently, the detection of the chemicals is peptides, the short peptides can translocate through the na- mainly based on the physical blocking of current signals nopore channel to produce current blockage (Wang et al., when chemicals interact with the functional groups in the 2013b). However, due to the relatively small size of the connector. By mutating the lysine-234 to cysteine in the nanopore, the larger-sized polypeptides or proteins cannot be phi29 nanopore, the modified phi29 nanopore can distin- translocated through the channel. In this situation, a current guish among ethane, thymine, and benzene with thioester blockage may occur through nanopore conformational moieties (Haque et al., 2012a). Figure 8 shows that the changes caused by specific interactions between the larger- binding events between the connector and chemicals can sized proteins and the connector. Therefore, the dwell time cause current blockage events. There are covalent and tran- indicates either the time required for one complete translo- sient binding events of different chemicals from experi- cation event or the interaction time between the nanopore ments. The unique electric signals are generated by the and the analytes (Ouldali et al., 2020; Piguet et al., 2018; physical blockages when the chemicals reacted with the Restrepo-Pérez et al., 2019). As mentioned earlier, as pep- cysteine residues on the connector during the translocation tides are driven to translocate through or interact with the process (Haque et al., 2012a). From the analysis of the connector by the electric force, the intrinsic properties of the binding events among ethane, thymine, and benzene, the polypeptide such as length, charge, type, and modification of current blockage for the permanent binding events was found the connector can significantly influence the current block- to allow for the discrimination among the three chemicals age and dwell time (Varongchayakul et al., 2018). Re-

Table 1 Comparison of permanent current blockage events among thioestersa) Thioesters: permanent binding events Transient binding events Permanent binding events Ratio of permanent to transient events Thioesters (Cys-X) (Cys-X+Cys-X) P value (Cys-X+Cys-X)/(Cys-X) P value <0.001 (thymine), <0.001 (thymine), Ethane 16.4%±2.0% 33.5%±0.5% (N=63) 2.04±0.12 <0.001 (benzene) <0.065 (benzene) <0.001 (ethane), <0.001 (ethane), Thymine 18.9%±2.6% 36.3%±1.2% (N=44) 1.92±0.14 <0.001 (benzene) <0.437 (benzene) <0.001 (ethane), <0.065 (ethane), Benzene 19.5%±6.2% 38.4%±2.0% (N=66) 1.96±0.32 <0.001 (thymine) <0.437 (thymine) a) Reprinted with permission from Haque et al., 2012a. Copyright 2012 American Chemical Society.

http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 1116 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8 searchers have been able to experimentally discriminate connector channel to detect the EpCAM antibody (Wang et peptides of different lengths by the nanopore technique (Ji et al., 2013b). Upon the specific interaction between the Ep- al., 2018). For example, Ji and coworkers were able to dif- CAM peptide and the antibody, people were able to collect ferentiate among peptides of different numbers of arginine distinct current blockage signals that can be gathered from residues using T7 nanopore technology (Ji et al., 2018). Si- the background, even if many other nonspecific antibody or milar to other biomolecules, the longer-length peptides serum components were present. would generate larger blockage peaks and longer dwell time. Figure 9 compares the current blockage and dwell time to distinguish peptides that have the number of arginine re- Studies on the bacteriophage phi29 motor pRNA sidues ranging from 8 to 12. Moreover, the peptides of the lead to the emergence of RNA nanotechnology same length can be detected using the peptide digestion assay (Ji and Guo, 2019). Timeline of phi29 motor pRNA research in the The main idea for the detection is that specific enzyme development of RNA nanotechnology digestion creates peptides of various sizes (Ji and Guo, It was reported in 1987 that bacteriophage phi29 DNA 2019). For instance, after digestion with Lys-C enzyme, packaging motor was geared by a small pRNA (Guo et al., peptides with the same length were able to be separated from 1987b). This finding brought about a speculation that cells the digested peptides that have different lengths (Figure 10) might have many small RNA molecules with novel yet un- (Ji and Guo, 2019). Furthermore, scientists have developed a discovered role in cells, thus the authors named them method for analysis of fingerprinting signals from specific “sRNA” (small RNA) (Guo et al., 1987b). Binomial dis- protein-nanopore interactions to detect proteins that are too tribution studies applying “Yanghui Triangle” (“Pascal’s large to pass through a nanopore. For example, Wang and Triangle”) using truncated pRNA lead to the finding that the coworkers incorporated an Epithelial Cell Adhesion Mole- number of pRNA in one motor is between 5 and 6 (Trottier cule (EpCAM) peptide into the C-terminus of the phi29 and Guo, 1997). Subsequent studies revealed that the number

Figure 9 The differentiation of peptides with different number of arginine residues (R8, R9, R10, and R12). A, The electric profiles of the peptide mixture. B, The current trace of the peptide mixture. C, The scatter plot of the current blockage and dwell time for the peptide mixture. Reprinted with permission from Ji et al., 2018 from Elsevier. http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8 1117

Figure 10 The identification and differentiation among peptides with the same length but distinctive composition with protease Lys-C digestion. The ionic signatures of (A) RK2, (C) RK3, and (E) RK4 before Lys-C digestion. The ionic signatures of digested (B) RK2, (D) RK3, and (F) RK4 after Lys-C cleavage. G, Left: Current trace before the addition of Lys-C control reaction. Middle: Sample current trace during 10 min post addition of the Lys-C control reaction. Right: Sample current trace during 19 min post addition of excess Lys-C control reaction. Reprinted with permission from Ji and Guo, 2019 from Elsevier. of RNA in the motor is the common multiple of 2 and 3, al., 2017; Grabow and Jaeger, 2014; Guo, 2005; Guo et al., since the use of either two or three complementary pRNA 2012; Haque et al., 2018; Hill and Hall, 2020; Jasinski et al., mutants leads to the assembly of infectious phi29 visions 2017; Kim and Franco, 2020; Lin et al., 2020b; Mitchell et (Guo et al., 1998). A formation of the hexameric RNA ring al., 2019; Murthy and Delong, 2017). on the motor via hand-in-hand interaction was eventually elucidated (Chen et al., 1999), thus confirming that the Techniques for the construction and the applications of stoichiometry of the RNA is not five but six, since the RNA nanoparticles number of six agrees with the results of the common multiple of 2 and 3, and the number is not 12 but six since the Using RNA 3WJ structure as scaffolds “Yanghui Triangle” excludes the possibility of 12. These One of the most important modules used in RNA nano- intriguing discoveries on mathematical, geometrical, and technology is the 3WJ structure from pRNA. The pRNA engineering data together lead to the emergence of the field subunit forms dimers and hexamers via hand-in-hand inter- of RNA Nanotechnology (Guo, 2010; Khisamutdinov et al., actions between right- and left-interlocking loops (Guo et al., 2014a; Khisamutdinov et al., 2016; Sharma et al., 2015) 1998; Jasinski et al., 2014; Shu et al., 2011a; Shu et al., (Figure 11). Since the field of RNA nanotechnology has 2011b) (Figure 12). The 3WJ structure, together with the 2′- grown rapidly, due to space limitation, here we only describe modifications, allows the RNA nanoparticles to maintain one technique of using phi29 motor pRNA as an example for their secondary structures, resulting in a higher resistance the construction of RNA nanoparticles, and readers are di- towards serum degradation (Binzel et al., 2016; Jasinski et rected to the previous reviews for more details of each al., 2014; Li et al., 2016; Pi et al., 2016a; Shu et al., 2015a). technique (Afonin et al., 2014; Chan and Ng, 2015; Geary et The RNA with 3WJ or similar structure can be easily de- http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 1118 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8

Figure 11 The design and AFM images of RNA nanoparticles. Adapted and reproduced with permission from Shu et al., 2013, © 2013 RNA Society; Khisamutdinov et al., 2014a, © 2014 Oxford University Press; Sharma et al., 2015, © 2015 Elsevier; Li et al., 2016, © 2016 John Wiley & Sons, Inc.; Guo et al., 2012, © 2012, Mary Ann Liebert, Inc; and Khisamutdinov et al., 2016, © 2016 John Wiley & Sons, Inc. signed by solid-phase chemical synthesis, which means it is RNA. The 3WJ of pRNA can be assembled effectively from also possible for large-scale production and different che- three strands of RNA oligo without the existence of a metal mical modifications (Guo, 2010; Guo et al., 2012). The in- salt (Shu et al., 2011a). This special structure shows excep- herent flexibility and the diversity of structure of RNA tional thermodynamic and kinetic stability (Hao and Kieft, molecules have allowed RNA to become a promising na- 2016; Hill and Schroeder, 2017; Shu et al., 2011a). The self- nomaterial with a wide range of applications, which lead to assembled 3WJ can remain intact in 8M urea or at a low the emerging field of RNA nanotechnology. RNA nano- concentration of the 3WJ, both of which are strong condi- technology allows us to use the pRNA-3WJ as a scaffold, tions of denaturing (Shu et al., 2011a). The high stability of demonstrating that the resulting RNA nanoparticles harbor- the 3WJ RNA is dependent on the structure of the 3WJ core, ing different functional modules retained their folding and as each strand of the 3WJ can be linked with different kinds independent functionalities for specific cell binding, cell of molecules while maintaining the 3WJ core structure and entry, gene silencing, catalytic function, and cancer targeting function (Cui et al., 2015; Haque et al., 2012b; Lee et al., both in vitro and in animal trials (Haque et al., 2012b; Shu et 2015; Shu et al., 2015a; Shu et al., 2013). Hence, the 3WJ al., 2011a; Shu et al., 2013). Besides, the crystal structure of can be used as a scaffold or a linker when constructing the the pRNA-3WJ has been solved at 3.05 Å (Zhang et al., RNA polygons (Guo et al., 2018). The RNA polygons can be 2013), which will facilitate the designs of bi-specific and designed through some computational software, such as trispecific RNA antibodies. Because of the formation of a UCSF Chimera (Pettersen et al., 2004), Swiss PDB Viewer strong core, the 3WJ has been tested as a scaffold for gen- (http://www.expasy.org/spdbv/) or PyMOL Molecular Gra- erating a new class of RNA antibodies for binding to pep- phics System (https://www.pymol.org/) by a manual align- tides, cells, or other ligands. ment of RNA motifs which are connected with dsRNA 3WJ derived from the pRNA of the phi29 DNA packaging linkers, and the computational methods are a promising tool motor is one of the multi-way junction secondary structure of to predict the 3D structure of RNA molecules (Miao and http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8 1119

Figure 12 (Color online) Secondary structure of pRNA and hexamer and mechanism of assembly via hand-in-hand interaction. A, Sequence and predicted secondary structure of wild-type pRNA. B and C, The formation of the pRNA hexametric ring by upper (right hand, upper case letter) and lower loop (left hand, low case letter) interaction. D, E and F, Inter-pRNA interaction of two, three and six pairs of loops. Adapted from Guo et al., 1998. Copyright 1998 Cell Press. G, A picture showing the hand-in-hand interaction of the pRNA hexamer. Reprinted from Shu et al., 2011b. Copyright 2011 Elsevier Inc.

Westhof, 2017; Qiu et al., 2013; Sharan et al., 2017). The 2017; Mohammadniaei et al., 2019; Zhang et al., 2014). The RNA polygons can be divided into n+1 strands, where n is pRNA-3WJ nanoparticles can be used to deliver the RNA the number of nanoparticle edges. Take an equilateral tri- aptamer of EGFR and anti-miR-21 to inhibit the growth of angle that has three sides as an example, the RNA triangle the Triple Negative Breast Cancer (TNBC) (Obad et al., can be constructed from 3+1=4 strands. Three external 2011; Pi et al., 2018). When systemically injected to the strands from one half of the edge will combine with one orthotopic mice with TNBC, the EGFR aptamer functions as internal strand, comprising the other half of each edge to a targeting device to internalize the nanoparticle into the tumor cell through receptor-mediated endocytosis. Then the form a dsRNA complex (Khisamutdinov et al., 2014b). anti-miR-21 will down-regulate the oncogene miR-21, trig- Many functional molecules can be linked to the end of the gering a series of downstream pathways and inhibiting the external strands. This design method of RNA nanoparticles growth of the TNBC tumor in mice. allows the designer to change the shape of RNA nanoparticles easily by simply adding more external strands and extending the inner strand (Guo et al., 2020a; Guo et al., A brief summary of RNA nanotechnology 2020b; Jasinski et al., 2014). The 3WJ motif of the pRNA offers high chemical and thermodynamic stability to the RNA nanoparticles (Shu et Applications of RNA nanoparticles in RNA interference al., 2011a). The branched homogenous RNA nanoparticles (RNAi) therapy can harbor different functionalities while retaining their in- The 3WJ RNA nanoparticles can deliver miRNA molecules dependent structure and function in vitro and in vivo (Davis to silence the expression of the targeted gene (Doyle et al., et al., 2005; Huang and Lilley, 2016; Pi et al., 2016a). Fur- http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 1120 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8 thermore, chemical modifications such as 2′-Fluoro (2′-F) The nature of the poly-homo-subunit of the nucleic acid modifications to the pyrimidine increase the thermodynamic translocation motor in relation to the drug inhibition stability and resistance to RNase digestion of the 3WJ na- efficiency noparticles (Piao et al., 2018). All in all, RNA nanoparticles show great ability to break the barrier of the traditional The first milestone, in the invention for the development of methodology. They can also work as not only a drug delivery highly potent inhibitory drugs, was the discovery of one platform in different fields such as immunotherapy and mutant pRNA of the bacteriophage phi29 DNA packaging cancer therapy, but also a promising multifunctional platform motor completely inhibiting viral replication in vivo (Trottier (Hartshorn et al., 2018; Jeong et al., 2016). The RNA na- et al., 1996). noparticles are intact and stable in vivo with a high affinity of Trottier et al. found that adding 45% mutant pRNA re- tumors while have little accumulation in other organs. This duced the infectious bacteriophage phi29 production by four platform is able to deliver all kinds of RNA cargos, which orders of magnitude, which is rather effective. Generally, opens a new chapter of the biomedical research. drugs can reduce the production of viruses by one or two orders of magnitude. The complete inhibition in vivo by a competition mutant is very unusual. The experimental results Studies on the poly-homo-subunit of the nucleic implied that the mechanism leading to the high efficiency of acid translocation motor lead to the discovery of a inhibition was attributed to two features. First, there are two method for the development of highly potent in- essential domains of pRNA. One is responsible for procapsid hibitory drugs binding, and the other has a DNA-packaging role other than procapsid binding. Mutation of the DNA-packaging domain During the last several years, an invention of a method for resulted in a pRNA with no DNA-packaging activity. This the development of highly potent inhibitory drugs has been mutant pRNA still has the intact ability to bind the procapsid, reported (Pi et al., 2016c; Pi et al., 2016b; Shu et al., 2015b). making it a competitive inhibitor against the normal pRNA. The stoichiometry of the drug target in a biocomplex or Second, the genome packaging process needs multiple co- nanomachine is the key factor for the potency of the drug. pies of pRNA. This higher-order requirement of pRNA leads The mathematical formula of binomial distribution and to the higher-order effect of inhibition with mutant pRNA Yanghui Triangle was applied to the inhibition efficiency monomers. This study implies that targets with poly-homo- studies, which results from the study of the structure and subunits have the potential to increase the drug inhibition function of the viral DNA packaging motor. efficiency.

Extension of the finding in the inhibition efficiency of Use the mathematical formula of binomial distribution viral motors and Yanghui Triangle to investigate the inhibition efficiency The second milestone in the invention for the development of highly potent inhibitory drugs was the illustration of the Inhibition efficiency follows a power function of the stoi- stoichiometry of viral assembly components with binaural chiometry of the target biocomplex with Yanghui Triangle distribution. As mentioned above, the pRNA-gp16 ring of Z the bacteriophage phi29 DNA packaging motor has a hex- (p + q) Z = Z p Z M q M , where Z=stoichiometry, M =0( M ) ametric structure, and it was surprising to find that the viral M=drugged subunits per biocomplex, p and q are the fraction replication could be completely inhibited by only one mutant of drugged and non-drugged subunits in the population, K is pRNA (Trottier et al., 1996). The effective inhibition is a the required number of drugged subunits to block the func- promising tool against the viral infection. The study in 1997 tion of the whole biocomplex. When K=1, the ratio of by Trottier and Guo gives a clearer explanation of this phe- functional biocomplex equals qz, while any combination of p nomenon in a mathematical way, which bridges the gap and q with one or more than one copy of drugged subunit (p) between the theory and the application. Two methods were will be unfunctional. This demonstrates the significance of used for stoichiometry determination. One was to use stoichiometry on inhibition efficiency. Drug inhibition po- mathematic models to analyze the relationship between the tency depends on the stoichiometry of the targeted compo- mutant pRNA percentage versus the yield of virion assembly nents of the biocomplex or nanomachine. The higher efficiency in vitro. Both the probability of mutant and wild- stoichiometry the target complex has, the stronger inhibition type pRNA were predicted with an equal binding affinity in effect the drug will have. This theory suggests that devel- the binomial equation. The results showed that five or six oping drugs targeting biocomplexes with high stoichiometry copies of pRNA were required for one DNA-packaging will have great potency to improve the drug inhibition effect. event, which can be blocked by one mutant. The other http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8 1121 methods involved the analysis of slopes of curves of dilution drug targets (Z=6) (Shu et al., 2007), and drugging a single factors versus the yield of virions. The experimental curve pRNA subunit inhibits the entire biomotor function (K=1), was compared to a series of standards which calculated on resulting in strong inhibition (Figure 13D). In contrast, tra- components with known stoichiometries. The larger stoi- ditional drug development is analogous to parallel circuits chiometry will have a more dramatic influence of the dilution that are less effective, as a single bulb burning out has no factor on the reaction. A slope of one indicates that only one effect on the rest of the strand. Drugs that target viral motors copy of the component is needed in a single assembly event, following the high inhibition mechanism include rimanta- while a slope larger than one indicates multiple-copy in- dine for influenza (Jing et al., 2008), ALLINI for HIV volvement (Guo et al., 2003). The result showed that the (Garmann et al., 2015; Pettersen et al., 2004), and bedaqui- stoichiometry of gp11 in phi29 particles was approximately line for tuberculosis (Figure 14) (Lakshmanan and Xavier, 12. These two methods are useful to determine not only 2013). single molecules but also the stoichiometry of oligomers in The idea of targeting the viral DNA packaging motor is the viral assembly process (Trottier and Guo, 1997). also applied to the treatment of herpesvirus (De Clercq, 2013; Kornfeind and Visalli, 2018; Yang et al., 2019). As one The poly-homo-subunit of the nucleic acid translocation of the dsDNA viruses, the DNA packaging process of the motor herpesvirus is similar to the bacteriophage phi29 (King et al., 2018). The genome DNA is packaged into the procapsid The third milestone in the invention for the development of through a portal protein channel with a DNA packaging highly potent inhibitory drugs was the finding of the se- motor, and then released by the terminase. It is showed that quential actin of six viral-encoded DNA packaging RNAs the terminase and the portal protein might be a potential during phage phi29 genomic DNA translocation. In 1997, target of antiviral drugs (Kornfeind and Visalli, 2018; Yang Chen and collaborators used compensation and com- et al., 2019). Terminase is one of the essential components of plementation analysis to prove that the six units of pRNA the DNA packaging process of herpesviruses that could be worked as an integrated entity instead of working in- used as a target. The terminase of herpesvirus, for example, dependently, and the pRNA molecules followed a sequential pUL15 of HSV-1, consists of three subunits and has a hex- model instead of a random model. The sequential action is ametric structure (Higgs et al., 2008; Yang et al., 2007; Yang another reason for the high inhibition efficiency. A pRNA et al., 2020). The large subunit of the terminase is highly mutation can be amplified by six orders of magnitude due to conserved among the herpesvirus, and functions as an AT- the six consecutive steps in DNA packaging process. Hence, Pase and nuclease (Dasgupta and Wilson, 1999; Selvarajan a small portion of mutant can completely block the DNA Sigamani et al., 2013). The terminase’s large subunit also packaging (Chen and Guo, 1997). interacts with the portal protein which is the docking site of the terminase and also the tunnel for the viral genome DNA Development of highly potent drugs against multi-subunit to enter the procapsid (Lokareddy et al., 2017). ATPases analogous to a series circuit Molecules that inhibit the terminase and portal function can be used as antiviral drugs. There are numerous anti- The three milestones discussed above have led to the de- herpesvirus agents that are effective against the herpesvirus velopment of highly potent drugs against multi-subunit AT- (De Clercq, 2013). For example, one nucleoside analog Pases analogous to a series circuit. The idea of intervening called benzimidazole D-ribonucleoside (BDCRB) can in- targets that are analogous to a series circuit rather than a hibit the ATPase activity of the terminase’s small subunit parallel circuit is novel. In series circuits, like Christmas pUL56 of HCMV (Komazin et al., 2004). Another pUL56 lights, breaking one bulb prevents the entire series from inhibitor is letermovir, which shows great effect against lighting up. As such, a system for highly potent drug de- CMV infection during clinical trials (Frange and Leruez- velopment should consist of multiple homologous subunits, Ville, 2018; Marty et al., 2017). As for the portal protein, in which the inhibition of one subunit prevents the action of there are a series of small thiourea compounds inhibiting the the entire complex (Figure 13A). A drug targeting one se- HSV-1 portal vertex, pUL6 (van Zeijl et al., 2000). CL- quentially acting multi-subunit complex could block its 253824 shows the greatest antiviral effect against the HSV-1 function completely. The typical hexameric AAA+ ATPase infection (van Zeijl et al., 2000). There are also nonnucleo- subfamily meets this requirement to serve as the target for side thiourea compounds against other kinds of herpesvirus, potential drugs, since many ATPase motors contain multiple such as Varicella-Zoster Virus (VZV) (Di Grandi et al., 2004; homo-subunits that work sequentially. We found that in- Kornfeind and Visalli, 2018). These compounds block the activation of one subunit in the hexamer stops the function of encapsidation, resulting in many empty capsids during viral the entire phi29 DNA packaging motor. Many ATPase bio- replication (Visalli et al., 2003). This inhibition can also be motors are composed of hexamer subunits that can serve as achieved by synthetic peptides which share the conserved http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 1122 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8

Figure 13 The poly-homo-subunit of the nucleic acid translocation motor is a series circuit. A and B, Drug inhibition efficiency is correlated with the stoichiometry of the targeted biocomplex with a mechanism of series circuit, using the hexametric ATPase as an example. In the series circuit of Christmas lights, one broken bulb will turn off the whole chain. Adapted and reproduced with permission from Pi et al., 2016b. Copyright 2016 American Society for Microbiology. C, One key factor regarding drug potency is the stoichiometry of the homo-subunit serving as a target. The expansion of the binomial is derived from Yanghui Triangle. For example, if Z=3, then (p+q)Z=p3+3p2q+2pq2+q3=100%, where q3=probability of procapsid possessing three wild-type pRNA; 3pq2=probability of procapsid possessing one mutant and two wild-type pRNA. Suppose p=70% and q=30%, then the probability of procapsids possessing at least two wild-type pRNA is 3pq2+q3=21.6%. D, Assay to prove the potent drug development applying the series circuit model. The virus assembly inhibition effect by drugged components of DNA, pRNA, gp16, and ATP is compared with stoichiometry of 1, 6, 6, and 1,000, respectively. Inhibition to ATP is the strongest. Adapted with permission from Schwartz et al., 2013b. Copyright 2013 Elsevier.

Conclusions and perspectives

In living systems, transportation of dsDNA from one location to the other is a ubiquitous phenomenon critical for life process. These essential activities include cell mitosis, binary fission, chromosome segregation, DNA replication, genome repair, homologous recombination, RNA trafficking, viral infection, viral assembly, and genome packaging. Among Figure 14 Drugs that target ATPase have high stoichiometry. A, Be- these events, the studies on the structure and mechanism of daquiline for tuberculosis (TB). B, Rimantadine for influenza. C, ALLINI dsDNA translocation by the ATPase motors have led to the for HIV. Mycobacterium tuberculosis (causes TB) is one of the most resistant bacteria to chemical drugs due to its cell wall rich in lipids. The application of the motor channel for the sensing of proteins, recent FDA-approved drug bedaquiline targets the ATPase of TB. Adapted peptides, DNA, RNA, and other macromolecules. The dis- and reproduced with permission from Pi et al., 2016b. Copyright 2016 covery of the hand-in-hand interaction in the formation of the American Society for Microbiology. RNA hexamer ring has led to the opening of a new field of RNA nanotechnology. Studies on the sequential action of the interaction site of the portal and other scaffold proteins of the poly-homo-subunit of the motor have led to the finding of the virus (Yang et al., 2013). The synthetic scaffold protein can approach for the development of highly potent inhibitory interact with the portal protein and function as a competitor drugs. The elucidation of structure and mechanism of the of the capsid, interfering with the viral replication. The asymmetrical hexametric dsDNA transportation motor via a scaffold peptide constituting one of the HSV-1 portal binding revolving motion will provide a prototype for the construc- domains can decrease the production of infectious virus by tion of these types of the revolving motors or nanomotors 10,000-fold under certain conditions (Yang et al., 2013). The with high efficiency in energy conversion. The prototype method of targeting the viral DNA packaging components is will ultimately create an excellent target to develop a plat- rather effective, and it is one of the solutions to the drug- form for the designs of potent inhibitory drugs to treat viral resistant problem of the current drugs which target the DNA infections that can be extended to the treatment of cancer and polymerase (Chou, 2015; Xiong et al., 1997). The applica- other diseases. The revolving mechanism for the motion of tion of letermovir proves the principle of using small mole- objects along a helical chain provides a hint for the design of cules inhibiting the series circuit of the viral DNA packaging new moving machines along a track, such as that used by process, and small molecular inhibitors and analogs are roller coasters, trolley cars or rocket launchers to depart from promising candidates for antiviral drugs. a helical track without the need for the object to rotate. The http://engine.scichina.com/doi/10.1007/s11427-020-1752-1 Liang, C., et al. Sci China Life Sci August (2020) Vol.63 No.8 1123 answer to these unsolved questions can also bring new light Microbiol 93, 599–608. Binzel, D.W., Shu, Y., Li, H., Sun, M., Zhang, Q., Shu, D., Guo, B., and to nanotechnology, synthetic biology, and materials science. Guo, P. (2016). 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