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= Biomotors =

Viral DNA Packaging Motor for Single Pore Sensing
Viral genomes are enclosed in a protein shell called capsid. During last step of replication and morphogenesis, double-stranded DNA viruses such, as bacteriophage phi29 (Dwight Anderson AND Bernard Reilly) that infects bacteria Bacillus subtilis, uses a nanomotor to package its genome into a preformed procapsid, or proheads for bacteriophages (Lee Taejin AND Peixuan Guo, the books). After synthesis by separate machinery, viral structural proteins and the genome must interact with each other to form a complete virion through a process referred to as DNA or RNA packaging. However, all linear double-stranded (ds)DNA or dsRNA viruses, including dsDNA bacteriophages (Guo, 1994), adenoviruses (Zhang and Imperiale, 2000), poxviruses (Koonin et al., 1993), human cytomegaloviruses (HCMV) (Scheffczik et al., 2002), herpes simplex viruses (HSV) (Salmon and Baines, 1998) and dsRNA bacteriophages (Olkkonen et al., 1990), possess a common feature in that their genome is packaged into a preformed procapsid. This entropically unfavourable process is accomplished by an ATP-driven packaging motor (Earnshaw and Casjens, 1980).

The phi29 DNA-packaging nanomotor (Fig. 1, Guo et al, Science 1987, PNAS ) is one of the strongest biological motors known. A key challenge is to contextualize to human systems our understanding of the unique phi29 nanomotor and its components, and to manipulate this nanomachine in an artificial environment. Our center will continue our studies of the phi29 motor to characterize its physical properties, and we will also rebuild the motor for therapeutic purposes, possibly for drug delivery. We plan to re-engineer the motor to function in lipid bilayers and other polymers, continue our detailed mechanistic studies of the re-engineered motor, and develop arrays of motors for single molecule sensing of chemicals, biomarkers, diseased cells, pathogens as well as for single pore sequencing of DNA.

Structure and Function of the motor channel, the connector, as nanopore

 * phi29 connector: This phi29 connector was the first portal protein with the atomic structure solved. The connector consists of 12 protein subunits that form a ring-like truncated cone structure. The molecular weight for each subunit is 36 kDa. The end diameters of the cone structure are 6.6 and 13.8 nm, with the wider and narrower ends termed the C- and N-terminals, respectively. The area of the narrowest part of the inner channel is 10 nm2, corresponding to a diameter of ~ 3.6 nm. In bacteriophage phi29, the C-terminal is located in the procapsid, and the translocation of dsDNA during packaging is unidirectional from the N-terminal to C-terminal (Haque et al., 2015). The clip region in the phi29 connector can bind with pRNA to help with the DNA packaging. In addition, the negatively charged residues are necessary for phi29 DNA packaging, as these residues, including the aspartate and glutamate in the channel, have the potential to keep the DNA in the center of the channel. Moreover, the phi29 nanochannel includes positively charged residues, such as the lysine and arginine, which can attract the DNA and hence prevent the reverting of DNA.
 * SPP1 connector: This connector has four domains (Ignatiou et al., 2019, Serrano et al., 2020). They are the clip, stem, wing, and crown domain. The SPP1 connector has 13 protein subunits that assemble to form a cone structure similar to that of the phi29 connector, with a total molecular weight of 745 kDa. The overall diameter of the SPP1 nanochannel is ~16.5 nm, with a height of ~11 nm. The most constricted region in the tunnel is ~ 2.7 nm (Lebedev et al., 2007). The negatively and positively charged residues of the SPP1 channel are also required for DNA packaging akin to those in the phi29 connector.
 * T3 connector: This connector consists of a mixture of (Streff et al., 2020)12 and 13 subunits of gp8, based on the protein expression conditions and other factors (Donate et al., 1988, Valpuesta et al., 2000). The height, width, and diameter for the T3 connector with 12 units are 8.5, 14.9, and 3.7 nm respectively for the internal channel.
 * T4 connector: The 12 subunits in T4 connector form a dodecameric ring with a length of ~12 nm. The portal complex has a molecular weight of ~ 660 kDa, while its external diameter varies from 8 to 17 nm (Ali et al., 2019, Attai and Brown, 2019, Maghsoodi et al., 2019, Park et al., 2019, Shi et al., 2019). The thinnest part in the T4 inner channel is ~ 2.8 nm. The clip region in the T4 nanopore can bind to the C-terminal of the terminase for DNA packaging. Similar to the phi29 and SPP1, the negatively and positively charged residues are also in T4 nanochannel to help the DNA packaging (Buerger et al., 2019, Hodyra-Stefaniak et al., 2019, Joiner et al., 2019, Streff et al., 2020, Zhang et al., 2020). In addition, the positively charged residues in the T4 connector loop can help stabilize the DNA after packaging.
 * P22 connector: The P22 portal has 11 or 12 subunits (gp1) with molecular weight ~ 940 kDa (Cingolani et al., 2002, Motwani and Teschke, 2019, Zheng et al., 2008). The pore diameter for P22 ranges from 2.5 to 4 nm. It has a unique domain called alpha-helical barrel domain, which functions to prevent the tangling hence, facilitating the DNA translocation. The P22 connector clip region can bind to the prohead to cause a conformational change to assist in the DNA packaging (Asija and Teschke, 2019a, Gonzalez-Davis et al., 2020, Kim et al., 2019a, Uddin et al., 2019, Wang et al., 2019). For the portal ring, the positively and negatively charged residues attract each subunit to strengthen the stability of the formed ring nanochannel (Zheng et al., 2008)
 * T7 connector: Twelve protein subunits form this connector with a molecular weight of 59 kDa for each subunit (Alexyuk et al., 2019, Foster et al., 2019, Liu et al., 2020, Singh et al., 2019). The channel length is 13.1 nm, with external diameters ranging from 5.9 to 17.3 nm. The most restricted region in the T7 interior channel is 3.9 nm, which is relatively wide compared to the other types of bacteriophage connectors. (Agirrezabala et al., 2005, Dedeo et al., 2019). The lysine residue in the stem domain of T7 nanochannel is highly favored to interact with the phosphate backbone to help with DNA translocation.
 * HK97 connector: It consists of 13 subunits of gp6 of the long-tailed phages family (Cardarelli et al., 2010a). The inner and outer diameter for the ring-shaped HK97 connector is ~ 3.7 on average and 11.4 nm, respectively. The height is ~ 4 nm from the bottom ring to the C-terminus of the structure. The gp6 monomer includes four long α-helices and two β-strands.
 * Bacteriophage λ connector: The λ connector comprises rings of proteins gpW and gpFII (Cardarelli et al., 2010b, delToro et al., 2019). The gpW is necessary for the DNA stabilization within the head as well as the gpFII addition.

Principle of single pore sensing
Single Pore Sensing can be readily described by its name, insertion of a singular biological nanopore into a membrane for the purpose of sensing polynucleotides or proteins, all with or without chemical modifications. Since its conception in the 1980s with the first demonstration of its potential by Kasianowicz et al., the researchers were able to definitively show that ssRNA and ssDNA (ss – Single Stranded) could be translocated or moved through an α-Hemolysin nanopore [1]. During the past 2 decades scientists have improved upon insertion techniques and the variety of pores to be inserted. Recent interest has been devoted to using bacteriophage nanopores to improve upon the limitations from α-Hemolysin. However, some features have remained constant throughout the development process such as usage of a Lipid Bilayer Membrane and electrical currents to drive the analytes through the inserted nanopore. Some of the most prominently used nanopores pores have been α-Hemolysin, Aerolysin, and Phi29[2]. The crucial feature in the use of all these nanopores has been the diameter of their inner channel, Aerolysin with 2.6 nm, α-Hemolysin with 1.2 nm and Phi29 with 3.6 nm [3,4,5]. The Phi29 and Aerolysin connectors with an inner channel diameter above 2nm, the diameter of dsDNA, make them more amenable to a variety of uses in Single Pore Sensing.


 * Once an electrical current is applied in a system with an inserted nanopore a variety of molecules from DNA, RNA to chemicals and peptides can be translocated. This translocation of any molecule will in turn generate a distinct fingerprint in the baseline electrical current. (Cao et al., 2019, Lu et al., 2019, Niu et al., 2019, Sun et al., 2019). The fingerprint electrical signal left by these can be described by their current blockage size and dwell time. (Haque et al., 2013, Varongchayakul et al., 2018, Wendell et al., 2009).
 * The general mechanism of biological nanopore sensing is based on the resistive pulse technique (Figure 5) (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 translocated through the membrane, ion flow is affected, resulting in a change to the current and creates fingerprinting signals. Commonly used electronic signatures that help identify different analytes include the current blockage and dwell time. The current blockage represents the percentage of the blocked current relative to the open current of the nanopore channel, while the dwell time measures the length of time the current blockage lasts.
 * As analytes are driven to translocate or interact with the connector by electric force, many things affect the current blockage and dwell time (Varongchayakul et al., 2018), including the intrinsic properties of the targets such as their shape, length, charge, hydrophobicity or hydrophilicity, as well as the type and modification of the connector (Gu et al., 2015, Lv et al., 2014, Qiu et al., 2016, Rajeev et al., 2019). Based on the size difference of analytes, there are two situations. First, analytes that are smaller than the nanopore diameter, translocation through the channel generates a large variety of electric signals. For example, analytes with longer chains will have a larger current blockage and longer dwell time than smaller analytes (Geng et al., 2013, Haque et al., 2013, Ji and Guo, 2019, Meller et al., 2001). Second, analytes that are larger than the diameter of the nanopore interact with the connector on one side and induce conformational changes to the connector, thus generating distinct electric signals without translocation (Haque et al., 2012a). Since only specific types of analytes can trigger conformational changes to the connector and cause current blockages with different dwell times, this is also a technique for analyte determination (Wang et al., 2013b). In both cases, the type and modification of the connector can greatly affect the electric signals (Haque et al., 2012a). A larger nanopore channel can allow larger analytes to translocate through the pore, while smaller nanopore channels do not.