''Bacillus virus Φ29'' (bacteriophage Φ29) is a

double-stranded DNA Deoxyribonucleic acid (; DNA) is a polymer composed of two polynucleotide chains that coil around each other to form a double helix. The polymer carries genetic instructions for the development, functioning, growth and reproduction of ...

(dsDNA)

bacteriophage A bacteriophage (), also known informally as a phage (), is a virus that infects and replicates within bacteria. The term is derived . Bacteriophages are composed of proteins that Capsid, encapsulate a DNA or RNA genome, and may have structu ...

with a

prolate A spheroid, also known as an ellipsoid of revolution or rotational ellipsoid, is a quadric surface obtained by rotating an ellipse about one of its principal axes; in other words, an ellipsoid with two equal semi-diameters. A spheroid has circu ...

icosahedral In geometry, an icosahedron ( or ) is a polyhedron with 20 faces. The name comes . The plural can be either "icosahedra" () or "icosahedrons". There are infinitely many non- similar shapes of icosahedra, some of them being more symmetrical tha ...

head and a short tail that belongs to the genus '' Salasvirus'', order ''

Caudovirales ''Caudoviricetes'' is a class of viruses known as tailed viruses and head-tail viruses (''cauda'' is Latin for "tail"). It is the sole representative of its own phylum, ''Uroviricota'' (from ''ouros'' (ουρος), a Greek word for "tailed" + ...

'', and family ''Salasmaviridae''. They are in the same order as phages PZA, Φ15, BS32, B103, M2Y (M2), Nf, and GA-1. First discovered in 1965, the Φ29 phage is the smallest ''

Bacillus ''Bacillus'', from Latin "bacillus", meaning "little staff, wand", is a genus of Gram-positive, rod-shaped bacteria, a member of the phylum ''Bacillota'', with 266 named species. The term is also used to describe the shape (rod) of other so-sh ...

'' phage isolated to date and is among the smallest known dsDNA phages. Φ29 has a unique DNA packaging motor structure that employs prohead packaging RNA (pRNA) to guide the translocation of the phage

genome A genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding genes, other functional regions of the genome such as ...

during replication. This novel structure system has inspired ongoing research in

nanotechnology Nanotechnology is the manipulation of matter with at least one dimension sized from 1 to 100 nanometers (nm). At this scale, commonly known as the nanoscale, surface area and quantum mechanical effects become important in describing propertie ...

drug delivery Drug delivery involves various methods and technologies designed to transport pharmaceutical compounds to their target sites helping therapeutic effect. It involves principles related to drug preparation, route of administration, site-specif ...

, and

therapeutics A therapy or medical treatment is the attempted remediation of a health problem, usually following a medical diagnosis. Both words, ''treatment'' and ''therapy'', are often abbreviated tx, Tx, or Tx. As a rule, each therapy has indications an ...

. In nature, the Φ29 phage infects ''

Bacillus subtilis ''Bacillus subtilis'' (), known also as the hay bacillus or grass bacillus, is a gram-positive, catalase-positive bacterium, found in soil and the gastrointestinal tract of ruminants, humans and marine sponges. As a member of the genus ''Bacill ...

'', a species of

gram-positive In bacteriology, gram-positive bacteria are bacteria that give a positive result in the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their type of cell wall. The Gram stain is ...

, endospore-forming bacteria that is found in

soil Soil, also commonly referred to as earth, is a mixture of organic matter, minerals, gases, water, and organisms that together support the life of plants and soil organisms. Some scientific definitions distinguish dirt from ''soil'' by re ...

, as well as the

gastrointestinal tract The gastrointestinal tract (GI tract, digestive tract, alimentary canal) is the tract or passageway of the Digestion, digestive system that leads from the mouth to the anus. The tract is the largest of the body's systems, after the cardiovascula ...

s of various marine and terrestrial organisms, including

human being Humans (''Homo sapiens'') or modern humans are the most common and widespread species of primate, and the last surviving species of the genus ''Homo''. They are Hominidae, great apes characterized by their Prehistory of nakedness and clothing ...

History

In 1965, American

microbiologist A microbiologist (from Greek ) is a scientist who studies microscopic life forms and processes. This includes study of the growth, interactions and characteristics of microscopic organisms such as bacteria, algae, fungi, and some types of par ...

Dr. Bernard Reilly discovered the Φ29 phage in Dr. John Spizizen's lab at the

University of Minnesota The University of Minnesota Twin Cities (historically known as University of Minnesota) is a public university, public Land-grant university, land-grant research university in the Minneapolis–Saint Paul, Twin Cities of Minneapolis and Saint ...

. Due to its small size and complex morphology, it has become an ideal model for the study of many processes in

molecular biology Molecular biology is a branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interactio ...

, such as

morphogenesis Morphogenesis (from the Greek ''morphê'' shape and ''genesis'' creation, literally "the generation of form") is the biological process that causes a cell, tissue or organism to develop its shape. It is one of three fundamental aspects of deve ...

, viral

DNA packaging A chromosome is a package of DNA containing part or all of the genetic material of an organism. In most chromosomes, the very long thin DNA fibers are coated with nucleosome-forming packaging proteins; in eukaryotic cells, the most importa ...

, viral replication, and transcription.

Structure

The structure of Φ29 is composed of seven main

protein Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residue (biochemistry), residues. Proteins perform a vast array of functions within organisms, including Enzyme catalysis, catalysing metab ...

s: the terminal protein (p3), the head or capsid protein (p8), the head or capsid fiber protein (p8.5), the distal tail knob (p9), the portal or connector protein (p10), the tail tube or lower collar proteins (p11), and the tail fibers or appendage proteins (p12*). The main difference between Φ29's structure and that of other phages is its use of pRNA in its DNA packaging motor.

DNA packaging motor

The Φ29 DNA packaging motor packages the phage genome into the procapsid during viral replication. The Φ29 packaging motor is structurally composed of the procapsid and the connector proteins, which interact with the pRNA, the packaging

enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

(gp16), and the packaging

substrate Substrate may refer to: Physical layers *Substrate (biology), the natural environment in which an organism lives, or the surface or medium on which an organism grows or is attached ** Substrate (aquatic environment), the earthy material that exi ...

(genomic DNA-gp3). Because the process of genome packaging is

energy Energy () is the physical quantity, quantitative physical property, property that is transferred to a physical body, body or to a physical system, recognizable in the performance of Work (thermodynamics), work and in the form of heat and l ...

-intensive, it must be facilitated by an ATP-powered motor that converts

chemical energy Chemical energy is the energy of chemical substances that is released when the substances undergo a chemical reaction and transform into other substances. Some examples of storage media of chemical energy include batteries, Schmidt-Rohr, K. (20 ...

mechanical energy In physical sciences, mechanical energy is the sum of macroscopic potential and kinetic energies. The principle of conservation of mechanical energy states that if an isolated system is subject only to conservative forces, then the mechanical ...

through

ATP hydrolysis ATP hydrolysis is the catabolic reaction process by which chemical energy that has been stored in the high-energy phosphoanhydride bonds in adenosine triphosphate (ATP) is released after splitting these bonds, for example in muscles, by produ ...

. The Φ29 packaging motor is able to generate approximately 57 piconewtons (pN) of

force In physics, a force is an influence that can cause an Physical object, object to change its velocity unless counterbalanced by other forces. In mechanics, force makes ideas like 'pushing' or 'pulling' mathematically precise. Because the Magnitu ...

, making it one of the most powerful biomotors studied to date.

pRNA

The Φ29 pRNA is a highly versatile

molecule A molecule is a group of two or more atoms that are held together by Force, attractive forces known as chemical bonds; depending on context, the term may or may not include ions that satisfy this criterion. In quantum physics, organic chemi ...

that can

polymerize In polymer chemistry, polymerization (American English), or polymerisation (British English), is a process of reacting monomer molecules together in a chemical reaction to form polymer chains or three-dimensional networks. There are many form ...

into dimers, trimers,

tetramer A tetramer () (''tetra-'', "four" + '' -mer'', "parts") is an oligomer formed from four monomers or subunits. The associated property is called ''tetramery''. An example from inorganic chemistry is titanium methoxide with the empirical formula ...

pentamer A pentamer is an entity composed of five subunits. In chemistry, it applies to molecules made of five monomers. In biochemistry, it applies to macromolecules, particularly pentameric proteins, made of five protein sub-units. In microbiology, a ...

s, and

hexamer In chemistry and biochemistry, an oligomer () is a molecule that consists of a few repeating units which could be derived, actually or conceptually, from smaller molecules, monomer, monomers.Quote: ''Oligomer molecule: A molecule of intermediate ...

s. Early studies such as Anderson (1990) and Trottier (1998) hypothesized that pRNA formed intermolecular hexamers, but these studies had a solely genetic basis rather than a

microscopy Microscopy is the technical field of using microscopes to view subjects too small to be seen with the naked eye (objects that are not within the resolution range of the normal eye). There are three well-known branches of microscopy: optical mic ...

based approach. In the year 2000, a study by Simpson et al. employed

cryo-electron microscopy Cryogenic electron microscopy (cryo-EM) is a transmission electron microscopy technique applied to samples cooled to cryogenic temperatures. For biological specimens, the structure is preserved by embedding in an environment of vitreous ice. An ...

to determine that, ''

in vivo Studies that are ''in vivo'' (Latin for "within the living"; often not italicized in English) are those in which the effects of various biological entities are tested on whole, living organisms or cells, usually animals, including humans, an ...

'', only a pentamer or smaller polymer could spatially fit in the virus. Ultimately, single isomorphous replacement with anomalous scattering (SIRAS)

crystallography Crystallography is the branch of science devoted to the study of molecular and crystalline structure and properties. The word ''crystallography'' is derived from the Ancient Greek word (; "clear ice, rock-crystal"), and (; "to write"). In J ...

was used to determine that the ''in vivo'' structure is a tetramer ring. This discovery aligned with what was known about the structural geometry and necessary

flexibility Stiffness is the extent to which an object resists deformation in response to an applied force. The complementary concept is flexibility or pliability: the more flexible an object is, the less stiff it is. Calculations The stiffness, k, of a ...

of the packaging motor's three-way junction. When pRNA is in this tetramer ring form, it works as a part of the DNA packaging motor to transport DNA molecules to their destination location within the prohead capsule. Specifically, the functional domains of pRNA bind to the gp16 packaging enzyme and the structural connector molecule to aid in the translocation of DNA through the prohead channel. After DNA packaging is complete, the pRNA dissociates and is degraded.

Genome and replication

The Φ29 phage has a linear dsDNA genome consisting of 19,285 bases. Both 5’ ends of the genome are capped with a

covalently bonded A covalent bond is a chemical bond that involves the sharing of electrons to form electron pairs between atoms. These electron pairs are known as shared pairs or bonding pairs. The stable balance of attractive and repulsive forces between atoms ...

terminal protein (p3) that complexes with

DNA polymerase A DNA polymerase is a member of a family of enzymes that catalyze the synthesis of DNA molecules from nucleoside triphosphates, the molecular precursors of DNA. These enzymes are essential for DNA replication and usually work in groups to create t ...

during replication. Φ29 is one of many phages with a

that has a different structure and function compared to standard DNA

polymerase In biochemistry, a polymerase is an enzyme (Enzyme Commission number, EC 2.7.7.6/7/19/48/49) that synthesizes long chains of polymers or nucleic acids. DNA polymerase and RNA polymerase are used to assemble DNA and RNA molecules, respectively, by ...

s in other organisms. Φ29 forms a replication complex involving the p3 terminal protein, the dAMP nucleotide, and its own DNA polymerase to synthesize DNA in a 5’ to 3’ direction. This replication process also employs a sliding-back mechanism towards the 3’ end of the genome that uses a repeating TTT motif to move the replication complex backward without altering the template sequence. This allows the initiation of

DNA replication In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. DNA replication occurs in all life, living organisms, acting as the most essential part of heredity, biolog ...

to be more accurate by having the polymerase complex check a specific sequence before beginning the elongation process.

Applications

Targeting_of_TNBC_molecules_by_bacteriophage_phi29_pRNA

Nanoparticle assembly

Versatility in

RNA Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself (non-coding RNA) or by forming a template for the production of proteins (messenger RNA). RNA and deoxyrib ...

structure and function provides the ability to assemble

nanoparticle A nanoparticle or ultrafine particle is a particle of matter 1 to 100 nanometres (nm) in diameter. The term is sometimes used for larger particles, up to 500 nm, or fibers and tubes that are less than 100 nm in only two directions. At ...

s for nanomedicinal

. The pRNA in bacteriophage Φ29 can use its three-way junction in order to self-assemble into nanoparticles. One major challenge of using pRNA-derived nanoparticles is large-scale production, as most industries are currently unequipped to handle industrial pRNA synthesis. This is primarily because RNA nanotechnology is still an emerging field that lacks industrial application and manufacturing optimization of small RNAs.

Drug delivery

Φ29’s DNA packaging system, using pRNA, incorporates a motor for the delivery of therapeutic molecules like

ribozyme Ribozymes (ribonucleic acid enzymes) are RNA molecules that have the ability to Catalysis, catalyze specific biochemical reactions, including RNA splicing in gene expression, similar to the action of protein enzymes. The 1982 discovery of ribozy ...

s and

aptamer Aptamers are oligomers of artificial ssDNA, RNA, Xeno nucleic acid, XNA, or peptide that ligand, bind a specific target molecule, or family of target molecules. They exhibit a range of affinities (Dissociation constant, KD in the pM to μM rang ...

s. The small size of pRNA-derived nanoparticles also helps to deliver

drug A drug is any chemical substance other than a nutrient or an essential dietary ingredient, which, when administered to a living organism, produces a biological effect. Consumption of drugs can be via insufflation (medicine), inhalation, drug i ...

s in tight spaces like

blood vessel Blood vessels are the tubular structures of a circulatory system that transport blood throughout many Animal, animals’ bodies. Blood vessels transport blood cells, nutrients, and oxygen to most of the Tissue (biology), tissues of a Body (bi ...

s. The main difficulty in using aptamer-based drug delivery is sourcing unique aptamers and other multimers for specific treatments for

disease A disease is a particular abnormal condition that adversely affects the structure or function (biology), function of all or part of an organism and is not immediately due to any external injury. Diseases are often known to be medical condi ...

s that potentially degrade therapeutic multimers and nanoparticles ''in vivo.'' Nanoparticles need to be stabilized as delivery mechanisms in order to adapt to microenvironments that may result in loss of therapeutic cargo.

Triple-negative breast cancer treatment

Triple-negative breast cancer Triple-negative breast cancer (TNBC) is any breast cancer that either lacks or shows low levels of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) overexpression and/or gene amplification (i.e ...

(TNBC) is an aggressive form of

breast cancer Breast cancer is a cancer that develops from breast tissue. Signs of breast cancer may include a Breast lump, lump in the breast, a change in breast shape, dimpling of the skin, Milk-rejection sign, milk rejection, fluid coming from the nipp ...

that accounts for ten to fifteen percent of all breast cancer cases.

Chemotherapy Chemotherapy (often abbreviated chemo, sometimes CTX and CTx) is the type of cancer treatment that uses one or more anti-cancer drugs (list of chemotherapeutic agents, chemotherapeutic agents or alkylating agents) in a standard chemotherapy re ...

is the only viable current treatment for TNBC because the loss of target receptors inherent to the disease causes

cancer cell Cancer cells are cells that divide continually, forming solid tumors or flooding the blood or lymph with abnormal cells. Cell division is a normal process used by the body for growth and repair. A parent cell divides to form two daughter cells, an ...

s to resist therapeutic

pharmaceuticals Medication (also called medicament, medicine, pharmaceutical drug, medicinal product, medicinal drug or simply drug) is a drug used to diagnose, cure, treat, or prevent disease. Drug therapy ( pharmacotherapy) is an important part of the ...

. The three-way junction in the Φ29 DNA packaging motor can help sensitize TNBC cells to chemotherapy using a

siRNA Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded non-coding RNA molecules, typically 20–24 base pairs in length, similar to microRNA (miRNA), and operating within the RN ...

drug delivery mechanism to inhibit TNBC growth and volume. This treatment can also be combined with anti-cancer drugs like

Doxorubicin Doxorubicin, sold under the brand name Adriamycin among others, is a chemotherapy medication used to treat cancer. This includes breast cancer, bladder cancer, Kaposi's sarcoma, lymphoma, and acute lymphocytic leukemia. It is often used toge ...

to enhance therapeutic effects.

References

{{DEFAULTSORT:Phi 29 Phage Model organisms Podoviridae Bacillus phages