Generation of biologically scalable nanorods

Abstract

Disclosed herein is a nanorod generation system (NPS) useful for the generation of biologically scalable functionalized nanorods (BSF nanorods). The nanorods generated are derived from the filamentous phage Ff (f1, M13, or fd). The NPS disclosed herein allows for the efficient biological generation of non-infectious, thermostable, homotypic protein nanorods that contain modifications that orthogonally enable site-specific recombination, chemical and enzymatic attachment of peptide and non-peptide functionalities. Also disclosed are methods of generating and using these nanorods, such as in methods for detecting target molecules.

Description

[Technical Field]

[0001] The present invention generally relates to a system for generating biologically scalable functionalized nanorods (BSF nano) derived from the filamentous phage Ff (f1, M13, or fd). This system allows for the efficient biological generation of non-infectious, thermostable, homotypic protein nanorods containing modifications that orthogonally enable site-specific recombinant, chemical, and enzymatic attachment of peptide and non-peptide functionalities. [Background technology]

[0002] Numerous medical and nanotechnology applications require the use of particles that can be orthogonally functionalized with peptide (protein) or non-protein functionalities (Sarikaya et al., 2003). Nonbiological nanoparticles have been used in a variety of diagnostic and nanotechnology applications, but they pose several challenges, including the toxicity of the particles themselves and sustainability issues due to the use of toxic chemicals in their production (Wang and Tang, 2020). Their toxicity makes them unsuitable for medical therapeutic applications that require direct introduction into patients. Furthermore, the generation of isomorphic, orthogonally modifiable nonbiological nanorods is extremely challenging (Corrigan et al., 2021).

[0003] To date, a limited number of biological nanoparticles (including nanorods) have been used in nanotechnology and biomedical applications. The most famous of these biological nanoparticles is the filamentous bacteriophage Ff, a bacterial virus of Escherichia coli K12. The Ff bacteriophage is central to phage display technology and is used as a suitable biological particle for the attachment of functional groups. Numerous medical and nanotechnology applications are known that use whole phages or phage-derived long filaments containing complete plasmids called phagemids (Barbas III et al., 2001).

[0004] Ff filamentous bacteriophages (including f1, fd, and M13 species) contain DNA sequences necessary for replication and packaging in intergenic (IG) sequences (Model and Russell, 1988; Rakonjac et al., 2017). Ff phage replicate one strand at a time using the rolling circle mode. The Ff phage genome is a single-stranded, circular (positive, +) strand ssDNA. The second (negative, -) strand is synthesized from the (-) ori by host enzymes, resulting in the double-stranded, circular DNA replicative form (RF) of the genome. The RF serves as a template for the transcription and translation of phage proteins required for the replication and assembly of progeny phages. Rolling circle replication from the positive (+) strand replication origin (ori) using the RF as a template requires the phage-encoded replication protein pII, resulting in the generation of the single-stranded, circular DNA (ssDNA) that constitutes the filamentous phage genome.

[0005] The long hairpin structure of this ssDNA genome functions as a packaging signal required for the assembly of filamentous virus particles. During the early stages of the infection cycle, ssDNA is replicated from the (-) nucleotide sequence, increasing the RF copy number (up to 50 copies per cell). This contrasts with the later stages of infection, when the ssDNA is coated with the protein pV to form the "packaging matrix" required for virus particle assembly. The ssDNA within the packaging matrix forms a Watson-Crick helical structure, with each strand interacting with one subunit of the pV dimer. The exception is the packaging signal, a true DNA helix not covered by pV. This complex, called the "packaging matrix," targets the trans-envelope assembly and secretion machinery for virus particle assembly.

[0006] The (+) strand contains the site where replication protein pII cleaves the (+) strand, allowing replication to begin from the 3' OH end, which serves as a primer. As the new (+) strand is synthesized, the "old" (+) strand is replaced. Once replication of the (+) strand is complete, pII cleaves it at the same site as the initiation, sealing both the "old" ssDNA (+) strand and the new strand. The "old" strand serves as a template for (-) strand replication, generating more dsDNA, which can then become templates for a new round of (+) strand replication or be coated by pV to form packaging substrates for assembly of progeny virus particles.

[0007] Ff-derived phagemid particles resemble Ff phage, but their genome corresponds to a plasmid (called a phagemid), which contains a plasmid replication origin, an antibiotic resistance gene as a selectable marker, an Ff replication origin, and one of the Ff genes that usually encodes a virion coat protein (Barbas et al., 1991). A problem that arises when using Ff filamentous phage and derived phagemid particles in medical and diagnostic applications is that these phages and phage-derived particles are generally only available as long filaments under most conditions. In particular, the high length-to-diameter ratio of Ff phage or phagemid particles hampers applications that rely on diffusion, such as lateral flow diagnostics or analyte detection devices.

[0008] It has been reported that replication of a small portion of the phage genome containing an IG sequence, which occurs at low frequency in the phage population, results in the replication of the (+) strand ssDNA from the first (+) ori to the second (replicated) (+) ori, generating two types of virus-like particles: short (short interfering particles) and long (original phage genome) ( Enea et al., 1977 ; Ravetch et al., 1979 ).

[0009] The (+)ori consists of an essential part (termed A or I) and a non-essential part (termed B or II). An intact origin is required for 100% activity by the wild-type replication protein pII, but the essential part replicates with 1% efficiency relative to the intact origin unless specific mutants of the replication protein pII with increased affinity for (+)ori A are used (Dotto et al., 1984b).

[0010] Extensive studies mapping (+) origin function have shown that a truncated (+)ori A domain, in which the 3'-terminal 29 residues (△29) are deleted, can be cleaved by pII (the replication protein), at least when the intact (+)ori A domain is present on the same plasmid upstream of the mutated (+)ori (Dot et al., 1982, 1984a). In this configuration, the intact (+)ori functions as the initiator of (+) strand replication, and the (+)ori △29 functions as the terminator. Placing these two (+)ori sequences side by side generates a short circular ssDNA between the initiator and terminator cleavage sites, allowing the assembly of very short Ff-derived nanorods (50 nm in length), provided all the necessary Ff proteins are provided by a helper phage.

[0011] In this system, both short ssDNA and full-length helper phage DNA were replicated and packaged into two types of particles: short (50 nm) nanorods and full-length (900 nm) filamentous viruses (Specthrie et al., 1992). The resulting short nanorods were further functionalized by constructing a protein fusion between pIII, a minor coat protein within the helper phage, and the high-affinity fibronectin-binding domain (fibronectin-binding repeats, FnB) of the Streptococcus pyogenes protein serum opacity factor serotype 22 (Sof22), enabling FnB to be displayed on the nanorod surface. Purified 50 nm particles displaying FnB were used in a lateral flow (dipstick) assay to detect fibronectin and were shown to show a cleaner signal than 900 nm full-length phage particles displaying FnB with the same coat protein composition (Satta et al., 2015).

[0012] However, nanorods generated as described above are difficult to purify from the full-length helper phage, resulting in nanorod preparations containing nanorods of various sizes, including high levels of full-length viral particle contamination. Furthermore, the steps required to remove full-length helper phage (the majority of the generated particles) result in a low final yield of nanorods and significant production and purification costs. Furthermore, in the above system, the total number of circular ssDNA copies generated per cell is limited, as is replication efficiency.

[0013] Another problem that arises when using Ff phage and phagemid vectors to generate filaments, rods, and / or particles for use in diagnostic and / or medical applications is that the antibiotic resistance genes used as selection markers for transformed cells containing these expression vectors are retained in the filaments, rods, and / or particles.

[0014] Specifically, the complete template plasmid is replicated and recombined. Typical purified nanorod samples may be contaminated with longer particles containing antibiotic resistance genes (frequency 1 in 10 6 ). The number of particles used in a typical vaccination procedure (10 per mouse) 12 Considering the potential for antibiotic resistance, this level of contamination with antibiotic resistance coding gene sequences is unacceptable. R 10 infectious particles containing the gene 6 This is because there is a possibility that

[0015] Based on known facts about the filamentous phage infection process, antibiotic resistance genes contained in Ff phages and phagemid particles can be transmitted to other bacteria in the gut or the environment, potentially spreading antibiotic resistance genes (Russel et al., 1988). Furthermore, it has been shown that DNA from phage or phagemid filaments can be taken up by mammalian cells (Burg et al., 2002; Larocca and Baird, 2001), resulting in the expression of genes encoded by the DNA, including antibiotic resistance.

[0016] It is therefore an object of the present invention to at least partially address the deficiencies of the prior art highlighted above by providing a system for producing scalable biological nanorods for use in various medical and diagnostic methods, including medical applications requiring the direct introduction of nanorods into a subject, wherein scalable nanorods can be produced from Ff phage particles and / or Ff phage-derived particles with relatively high yield and / or relatively low contamination from longer Ff phage or Ff phage-derived filaments, and / or wherein the produced nanorods are free or substantially free of antibiotic resistance genes, and / or at least provide a useful option for the general public.

[0017] External sources of information, including patent specifications and other documents, are typically referenced herein to provide background for describing features of the invention. Unless otherwise expressly stated, reference to such external documents should not be construed as an admission that such documents or such sources are prior art or form part of the common general knowledge in the art in any jurisdiction. Summary of the Invention

[0018] Disclosed herein is a virus-free nanorod generation system (NPS). The disclosed NPS is a single-plasmid or two-plasmid system that directs the expression and assembly of short, scalable DNA-protein nanorods derived from the Ff bacteriophage. Nanorods produced by the disclosed NPS are not phages. Nanorods produced by the described NPS have a minimum length of 40 nm (Figure 1), are non-infectious, do not carry antibiotic resistance genes, and do not encode phage proteins required for replication and virion assembly; therefore, they cannot replicate in susceptible hosts. Furthermore, the disclosed NPS is designed to control the quantity and length of nanorods produced, allowing skilled workers to generate a variety of nanorod variants through specific orthogonal recombination, enzymatic, and chemical modifications.

[0019] Thus, in a first aspect, the present invention relates to a nanorod generating system (NPS) comprising a single nucleic acid expression construct, said construct comprising: a BSF nanoreplication assembly cassette; at least one auxotrophic marker; at least one inducible promoter operably linked to a nucleic acid sequence encoding at least one Ff phage protein; and at least one plasmid origin of replication that is not present in the BSF nanoreplication assembly cassette.

[0020] In a second aspect, the present invention provides a nanorod generation system (NPS) comprising: i) a nucleic acid replication construct, BSF nanoreplication assembly cassette, at least one auxotrophic marker, and the nucleic acid replication construct comprising at least one plasmid origin of replication that is not present in the BSF nanoreplication assembly cassette; ii) a helper nucleic acid expression construct, at least one selectable marker and and a helper nucleic acid expression construct comprising at least one inducible promoter operably linked to a nucleic acid sequence encoding at least one modified Ff phage-encoded protein.

[0021] Various embodiments of the different aspects of the invention described above are also set forth in the following detailed description, without the invention being limited thereto. Other aspects of the invention will become apparent from the following description, given by way of example only, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0022] The present invention will now be described, by way of example only, with reference to the following drawings. [Figure 1] Transmission electron micrograph (TEM) of negatively stained BSF nanos (biologically scalable functionalized Ff-derived nanorods). [Figure 2] Schematic diagram of the BSF nanorod generation system (NPS). [Figure 3] Ff (f1, fd, or M13) phage-encoded proteins and their functions. [Figure 4] Schematic diagram of the BSFp (A) and BSFpn (B) replica assembly cassettes and their replication. The minimum size of circular ssDNA obtained from each replica assembly cassette and the approximate yield of the corresponding nanorods are indicated. [Figure 5]Schematic diagram of the secondary structure of the Ff ori and BSF nanoreplication assembly cassette. Wild-type Ff ori is also referred to as the "intergenic sequence" (A). BSFpn (B) and BSFp (C), BSF nanoreplication assembly cassettes. Secondary structure and coordinates corresponding to the f1 genome sequence are shown. "Scaffold" indicates the sequence that is replicated to generate the (+)-strand circular ssDNA that forms the backbone of the nanorod. "Filler" indicates the position where DNA can be inserted to determine the size of the nanorod or to express the desired function (i.e., pVII and pIX). [Figure 6] Map of the BSF nanoreplication assembly cassette. A. BSFpn replication assembly cassette containing (+) and (-) Ff replication origins and packaging signals (PS). B. BSFpn cassette containing gVII and gIX expressed from within the BSF replication assembly cassette. C. BSFp replication cassette containing only the Ff(+) origin and PS. The replication assembly cassette, scaffold, filler sequences, and sequences required for nanorod ssDNA replication and assembly are shown on the map. [Figure 7]Map of the pPopUp plasmid series. The pPopUp plasmids contain variations of six functional blocks: i) a BSF nanoreplication assembly cassette, ii) a promoter upstream of gII, iii) genes encoding the Ff replication function [gII(gX)] and packaging substrate formation function (gV), iv) virion protein-encoding genes (gVII, gIX, gVIII, gIII, gVI) that allow display of peptides or attachment handles, v) genes encoding assembly functions (gIV and gI) and the remainder of the plasmid, including the plasmid replication origin, and vi) a marker. Examples of BSF nanoreplication assembly cassettes (i): BSFp (152 nt, 221 nt), BSFpn (289 nt, 313 nt, 395 nt, 529 nt). Examples of promoters (ii): FfpA, lacUV5. Examples of replication and ssDNA-binding protein (iii) mutants: gIIIR1B (Thr182Ile); examples of virion protein (iv) mutants: gVIII-20am; gVIII2am; gVIII-20amnAAGG, △P6S17LA27S; gVIII-20amnGGGGA1G; gVIII-20amY21M, encoded by engineered alleles. gIII wild-type; gIII::MCS; gIIIC::MCS3Cys; FnB-gIII; C121-FnB (C121 is a SARS-CoV-2 spike-specific scFv); N3-gIII (N3 is a SARS-CoV-2 nucleocapsid (NC) protein-specific camel antibody VHH). Examples of markers (vi): KmR, nadC. The arrow and small oval in the upper left corner of the image represent the excised BSF nanoreplication cassette. T: transcription terminator. Pz: phage promoter driving expression of the gIII-gVI-gI(gXI)-gIV operon. The circular ssDNA product of the BSF nanocassette is the backbone of the nanorod. AhdI, XhoI, PstI, SalI, HpaI, SnaBI, BamHI, AfeI, and PacI restriction sites are used to construct plasmid variants. MCS: multiple cloning site for inserting sequences encoding fusion peptides to be displayed using the minor coat protein pIII as a platform. [Figure 8]This is a map of the pHP helper plasmid series. pHP plasmids contain variations of five functional blocks: i) a promoter upstream of gII, ii) genes encoding Ff replication [gII(gX)] and packaging substrate formation function (gV), iii) virion protein-encoding genes (gVII, gIX, gVIII, gIII, gVI) that allow display of peptides or attachment handles, iv) genes encoding assembly functions (gIV and gI) and the remainder of the plasmid, including the plasmid replication origin, and v) a marker. Examples of promoters (i): FfpA, lacUV5. An example of a replication and ssDNA-binding protein (ii) mutant is gIIIR1B (Thr182Ile); an example of a virion protein (iii) mutant is encoded by engineered alleles: gVIII-20am; gVIII2am; gVIII-20amA9MS17LM28L; gVIII2amnGGGGS17L; gVIII-20amnAAGG, △P6S17LA27S; gVIII-20amnGGGGA1G; gVIII-20amY21M; gIII wild-type; gIII::MCS; gIIIC::MCS3Cys; FnB-gIII; C121-gIII (C121 is a SARS-CoV-2 spike-specific scFv); N3-gIII (N3 is a SARS-CoV-2 nucleocapsid protein-specific heavy chain-only antibody VHH); an example of a marker (v): KmR. T: transcription terminator. Pz: phage promoter driving expression of the gIII-gVI-gI(gXI)-gIV operon. AhdI, XhoI, PstI, SalI, HpaI, SnaBI, BamHI, AfeI, PacI, restriction sites used for constructing plasmid variants. MCS: multiple cloning site for inserting sequences encoding fusion peptides to be displayed using the minor coat protein pIII as a platform. [Figure 9]This is a map of the pBSF nanoplasmid series, which contains nanorod replication assembly cassettes. The pBSF nanoplasmid contains three functional blocks: i) the BSF nanoreplication cassette, ii) a marker, and iii) a replication origin. Examples of BSF nanoreplication cassettes (i): BSFp(152, 221), BSFpn(289, 313, 395, 529, 711, 728, 79a, 79lac, 1400). The small circle indicates the circular ssDNA replicated from the BSF replication assembly cassette (BSF nano). This circular ssDNA is assembled into BSF nanorods in the presence of assembly / secretion proteins and virion proteins. Examples of markers (ii): AmpR, CmR, or nadC. [Figure 10] Nanorods were generated using the single-plasmid generation system pBSFpn, containing both positive and negative origin replication assembly cassettes (pPOPUP529YM), or pBSFp, containing only the positive strand origin replication assembly cassette (pPOPUP221YM). DNA from SDS-digested nanorods was separated by agarose gel electrophoresis and visualized by EtBr staining. Lanes: Ladder, 1 kb plus ladder (a double-stranded linear DNA standard used as a migration guide due to the lack of a suitable circular ssDNA standard. Numbers indicate the size of the standard band in base pairs). Lane: 1: Nanorods obtained from cells transformed with the plasmid pPOPUP529Y. 2: pPOPUP221YM. Nanorods were concentrated 1000-fold by PEG precipitation from the supernatant of cultures obtained from pooled cells transformed with pPOPUP529YM or pPOPUP221YM (containing the BSFpn529 or BSFp221 replication assembly cassette, respectively). The nanorods were further purified by CsCl gradient and ion exchange chromatography. [Figure 11]Comparison of BSF nanorods produced using BSF plasmids with and without genes gVII and gIX. CsCl-purified nanorods are analyzed by agarose gel electrophoresis. A. DNA from SDS-digested nanorods is visualized by EtBr staining. B. Native nanorods visualized by EtBr after in situ NaOH-mediated removal of viral particle proteins. Lanes: Helper plasmid pHP2 combined with: 1: pBSFnano711; 2: pBSFnano79a (707 nt); 3: pBSFnano79lac (748 nt), IPTG-induced; 4: pBSFnano79Lac, uninduced. L, 1 kb plus ladder (double-stranded linear DNA standard used as a migration marker due to the lack of a suitable circular ssDNA standard. Numbers indicate the size of the standard bands in base pairs). Nanorods were PEG-precipitated from the supernatant of a double-transformed cell pool containing the helper and pBSF plasmids and further purified by CsCl gradient centrifugation. The single, double, triple, and quadruple asterisks correspond to single, double, triple, and quadruple length nanorods. [Figure 12]Inducible expression of protein pII increases the production of BSF nanorods. DNA from SDS-digested nanorods (A) or native nanorods treated in situ with NaOH to expose the DNA and stained with ethidium bromide (B) was generated using a single-plasmid (pPOPUP) system containing a 529-nt replication assembly cassette (BSFpn529). Expression of replication protein pII is driven by the constitutive f1 phage promoter pA (plasmid pPOPUP529YM) or the inducible promoter placUV5 (plasmid pPOPUP529LacYM). Lanes: L, ladder, 1 kb plus ladder (double-stranded linear DNA standard used as a migration marker due to the lack of a suitable circular ssDNA standard. Numbers indicate the size of the standard band in base pairs). 1: Particles obtained from plasmid pPOPUP529YM. 2: pPOPUP529LacYM without IPTG induction. 3: pPOPOPA529LacYM plasmid in the presence of placUV5 promoter inducer (IPTG; 0.1 mM). Nanorods were concentrated 1000-fold by PEG precipitation from the supernatant of a 1 L culture of pPOPOPA529YM or pooled cells transformed with pPOPOPA529LacYM. When applicable, IPTG was added to the pooled transformed cell culture at an OD600 of 0.1. [Figure 13]Comparison of BSF529 nanorods generated by a single-plasmid-driven nanorod generation system and a two-plasmid-driven nanorod generation system. A. Purified nanorod circular ssDNA; B. Native whole particles of PEG-precipitated nanorods. Lanes: L, 1 kb plus (double-stranded linear DNA standard used as a migration landmark due to the lack of a suitable circular ssDNA standard. Numbers indicate the size of the standard band in base pairs). 1: pPOPUP529YM; 2: pHP1AevLac+pBSF529, no IPTG; 3: pHP1AevLac+pBSF529, 0.1 mM IPTG; 4: pHP1AevLac+pBSF529, 1 mM IPTG; 5: pPOPUP529LacAev, no IPTG; 6: pPOPUP529LacAev, 0.1 mM IPTG. Nanorods are concentrated 1000-fold by PEG precipitation from the supernatant of 1 L of culture obtained from cells transformed with pPOPA or cells double-transformed with the pHP1 helper plasmid variant and the pBSFpn529 nanorod replication assembly plasmid. [Figure 14] These are 395 nt (70 nm) nanorods generated from an inducible single-plasmid system. The BSF nanoreplication assembly cassette contains both positive and negative oligonucleotides (pPOPUP529LacYM and pPOPUP395LacYM). A: DNA from SDS-digested nanorods separated by agarose gel electrophoresis and visualized by EtBr staining. B: Native nanorods separated by agarose gel electrophoresis. The gel is immersed in 0.2 M NaOH to strip viral particle proteins in situ, and the bands corresponding to native / intact particles are visualized after staining the gel with EtBr. Lanes: L, ladder, 1 Kb plus (double-stranded linear DNA standard used as a migration marker due to the lack of a suitable circular ssDNA standard. Numbers indicate the size of the standard band in base pairs). 1: BSFpn529YM particles generated by the pPOPUP529LacYM plasmid; 2: BSFpn395 particles generated by the pPOPUP395LacYM plasmid. [Figure 15]These are 152 nt (40 nm) nanorods generated from an inducible single-plasmid system. The BSF-nanor replication-assembly cassette contains only positive oligonucleotides (pPOPUP221LacYM and pPOPUP152LacYM). A: DNA from SDS-digested nanorods was separated by agarose gel electrophoresis and visualized by EtBr staining. B: Native nanorods separated by agarose gel electrophoresis. After immersing the gel in 0.2 M NaOH to strip the viral particle proteins in situ and staining the gel with EtBr, bands corresponding to native / intact particles were visualized. Lane: L, ladder, 1 Kb plus (double-stranded linear DNA standard used as a migration marker due to the lack of a suitable circular ssDNA standard. Numbers indicate the size of the standard band in base pairs). 1: BSFp221YM particles generated by the pPOPUP221LacYM plasmid. 2: BSFp152 particles generated by the pPOPUP152LacYM plasmid. The upper band in A is not DNA; it is removed after nanorod DNA purification (not shown). It may represent EtBr-stained detergent-associated membrane or peptidoglycan fragments. [Figure 16] BSF nanorods generated using a template plasmid containing the auxotrophic marker NadC. Lanes: L, 1 kb, plus ladder (double-stranded linear DNA standard used as a migration guide due to the lack of a suitable circular ssDNA standard. Numbers indicate the size of the standard band in base pairs). 1: BSF529 nanorods obtained from the pBSFpn529N plasmid encoding the auxotrophic marker NadC. Nanorods were concentrated from the supernatant of pooled double-transformed cells by pressure-mediated ultrafiltration and further purified by CsCl gradient centrifugation. [Figure 17] TEM analysis of purified BSF Nano 221YM 2221nt nanorods. A. Negatively stained electron micrograph of 221nt nanorods. B. Histogram of nanorod length distribution plotted from length measurements of 100 well-separated particles using ImageJ software. [Figure 18]TEM of spontaneously formed BSF Nano 221.2 liquid crystal. [Figure 19] TEM analysis of purified BSF Nano 529YM2 529nt nanorods. A, B: Electron micrographs of negatively stained 529nt nanorods. C: Histogram of nanorod length distribution plotted from length measurements of 300 well-separated particles using ImageJ software. [Figure 20] Nanorod-based lateral flow assay for detecting fibronectin. A: Detection of FnB-pIII fusion in BSF nanorods by SDS-PAGE and Western blotting. Left panel: All proteins in the gel were detected by Coomassie blue staining. Right panel: FnB-pIII fusion was detected by Western blotting using a pIII-specific antibody (Rakonjac and Model, 1998). Lanes: M: molecular weight standard (kDa); 1: control particles BSF nano711G82 containing no fusion protein; 2: BSF nano711G8FnB2, particles exhibiting the FnB-pIII fusion. B. A series of fibronectin dilutions analyzed using a BSF nano-based dipstick assay. Each assay contained 10 nanorods in a total volume of 100 μL. Fn concentrations are indicated below each stick. C. Fibronectin dipstick assay using fluorescently labeled BSF nano711G8FnB2 nanorods. The signal is detected using a fluorescent imaging device (Azurec600). A total of 10 nanorods are mixed with 1 μg of fibronectin diluted in PBS (final volume 100 μL) or PBS alone and incubated at room temperature for 30 minutes. A test strip is immersed in the mixture and allowed to flow laterally for 15 minutes. Particles carrying Fn bound to collagen are visualized in the lower (test) line. The control line captures all BSF nanoparticles (BSF nano711G82 and BSF nano711G8FnB2). Stick 1: Assay in the presence of analyte (Fn); Stick 2: Assay in the absence of Fn. [Figure 21]Enzymatic modification of Ff phage-derived nanorods with Streptococcus pyogenes sortase A (SrtA Sp). Mechanism of the sortase reaction between the feature (gray circle) and the pVIII subunit within the nanorod. The feature (small molecule or protein) contains a C-terminal LPETA sortase recognition motif, while the pVIII subunit within the nanorod contains an N-terminal double alanine. The active site of Streptococcus sortase A cleaves the LPETA motif between the threonine and alanine to form an acyl intermediate. An Ala-Ala nucleophile attacks the acyl intermediate bond, liberating the sortase and forming an amide bond between the two target proteins. [Figure 22] Agarose gel electrophoresis of enzymatically FITC-labeled nanorods. A: Image without staining. Only FITC-labeled nanorods are detected. B: Image after removal of the protein coat by NaOH treatment and staining of the exposed DNA with ethidium bromide. Lanes: L, ladder, 1 kb plus ladder NEB (double-stranded liner DNA standard used as a migration landmark due to the lack of a suitable circular ssDNA standard. Numbers indicate the size of the standard bands in base pairs). 1: FITC-labeled nanorods displaying a SARS-CoV-2 spike-specific scFv (BSF nano728Aev1C121-FITC). 2: Unlabeled nanorod control (BSF nano728Aev1) displaying no antibody. 3: Unlabeled nanorods displaying a spike-specific scFv (BSF nano728Aev1C121). [Figure 23] TEM immunogold analysis of enzymatic biotin conjugation to BSF Nano728Aev1 nanorods. Nanorods were generated using the pHP1AevLac helper plasmid and the pBSFpn728 nanorod replication assembly plasmid and enzymatically conjugated to LPETA-biotin using Sortase A from Streptococcus pyogenes (SrtA Sp). Images show BSF Nano728LacAev1 nanorods reacted with 20 μM biotin-KLPETAA and 50 μM sortase A (AF) and 20 μM biotin-KLPETAA without sortase A (GI). Black dots indicate the presence of streptavidin-gold nanoparticles. The scale bar represents 200 nm. [Figure 24] Enzymatic modification of BSF nanorods. A. Biotinylated and non-biotinylated nanorods analyzed by native particle agarose gel electrophoresis and Western blotting. 1: Biotinylated BSF nano728Aev1C121; 2: Non-biotinylated BSF nano728Aev1C121. Each lance was loaded with 10 particles. Signal was generated by BCIP / NBT substrate after incubation with streptavidin-alkaline phosphatase. Nanorods (5 nM or 3 x 10 particles) were biotinylated in a reaction containing 200 μM biotin-KLPETAA and 50 μM S. pyogenes sortase A (lane 1). B. A reporter tagged with the LPETG peptide (E. coli β-glucuronidase, UidA, or GUS) was enzymatically attached to GGGGG-tagged nanorods using Staphylococcus aureus sortase. 1: GUS-modified BSF nano728Gly5, 2: Unmodified BSF nano728G8. Modified and unmodified nanorods were analyzed by native particle agarose gel electrophoresis. The gel was directly stained with the GUS reporter reaction in the presence of a chromogenic substrate (100 mM NaPO4 pH 7.0, 1 mM MMX-GLUC:Na, 200 μM NBT). 10 particles were loaded in each lane. [Figure 25] Dot blot SARS-CoV-2 nanorod-based antigen (spike) detection. A. Experimental schematic: Dilutions of spike protein are spotted onto a nitrocellulose filter. Spike protein is detected by enzymatically biotinylated nanorods (10 nanorods per assay), followed by reaction with an avidin-alkaline phosphatase conjugate and a chromogenic substrate. B. Experimental results showing detection of 1 ng of spike protein. The positive control (+) is directly immobilized nanorods. The negative control (-) is buffer. [Figure 26]This is a sandwich ELISA assay using SARS-CoV-2-specific detection particles. The assay is performed as detailed in Materials and Methods. The antibody CR3022 specific for the SARS-CoV spike protein extracellular domain (ECD) is used to capture serially diluted ECD (A, B). A. The captured ECD is detected by nanorods displaying the SARS-CoV-2 ECD-specific antibody C121 fused to pIII (BSF Nano728Aev1C121). Bound nanorods are visualized with the nanorod-specific antibody and a secondary HRP-conjugated antibody. B. Enzymatically biotinylated BSF Nano728Aev1C121 nanorods were used to detect plate-bound ECD via the capture antibody. Streptavidin-HRP conjugate is used to visualize bound nanorods. C. SARS-CoV-2 nucleocapsid (NC) protein sandwich ELISA. An aminated aptamer (ssDNA molecule) specific for the SARS-CoV nucleocapsid (NC) protein was immobilized on an ELISA plate and used to capture the SARS-CoV-2 nucleocapsid (NC) protein. Enzyme-biotinylated nanorods displaying NC-specific VHH (BSF Nano728Aev1N3) were used to detect the aptamer-bound SARS-CoV-2 nucleocapsid (NC). Streptavidin-HRP conjugates were used to visualize the bound nanorods. [Figure 27]Lateral flow assay using SARS-CoV-2-specific nanorods. SARS-CoV antigen-specific capture molecules (antibodies or aptamers) are immobilized on the test line (B) or test dot (C), and nanorod-specific capture molecules are immobilized on the control line (B) or control dot (C). + and - indicate the presence or absence of antigen. B. The extracellular domain (ECD) of the spike protein is detected by biotinylated nanorods displaying the SARS-CoV-2 ECD-specific antibody C121 (which binds to a different epitope than the capture antibody) fused to pIII (BSF Nano728Aev1C121). C. The SARS-CoV-2 nucleocapsid (NC) protein bound to the aptamer is detected using enzymatically biotinylated nanorods displaying a VHH (BSF Nano728Aev1N3) specific for NC. Visualization of bound nanorods uses a streptavidin-alkaline phosphatase conjugate as described in Materials and Methods. [Figure 28-1] 1 is a list of the nucleic acid and amino acid sequences disclosed herein. [Figure 28-2] Same as above. [Figure 29] 1 is the wild-type pII amino acid sequence and the corresponding gIICDS nucleic acid sequence. [Figure 30] Amino acid and corresponding nucleotide sequences of the IR1B mutant of gII used in the nanorod generation system (pHP and pPOPUP plasmids). [Figure 31] pV, pVII, and pIX wild type are used in the nanorod generation system. [Figure 32] Figure 1 shows the sequences of wild-type and modified pVIII / gVIII. The vertical arrow indicates the signal sequence cleavage site. Bold underlined residues indicate mutations and amino acid changes. [Figure 33]

[0039] Figure 1. Sequences of modified pVIII / gVIII variants. Vertical arrows indicate signal sequence cleavage sites. Bold underlined sequences indicate mutations or amino acid changes. [Figure 34]Wild-type pIII and gIII. The vertical arrow indicates the signal sequence cleavage site. The underlined sequence is a BamHI site. [Figure 35] Full-length modified pIII and gIII. pIII::MCS, pIII showing the inserted peptide encoded by the multiple cloning site (corresponding to the highlighted displayed sequence of the gIII::MCS nucleotide sequence). The vertical arrow indicates the signal sequence cleavage site. The underline indicates the BamHI recognition site. [Figure 36] Modified pIII and gIII, C-terminal domain, f1 wild-type pVI and gVI. pIIIC::MCS, pIII showing the inserted peptide encoded by the multiple cloning site (corresponding to the highlighted sequence of the gIIIC::MCS nucleotide sequence). The vertical arrow indicates the signal sequence cleavage site. The underlined BamHI recognition site. [Figure 37] 1 shows the amino acid and nucleic acid sequences of pIII-displayed FnB-pIII fusions. The shaded sequence corresponds to FnB, the fibronectin-binding repeat of Streptococcus pyogenes M22 serum opacity factor, strain D734 (Rakonjac et al., 1995). [Figure 38] The amino acid and nucleic acid sequences of the scFv C121-pIII fusion are shown. The top is the amino acid sequence, and the bottom is the nucleic acid sequence. The shaded sequence corresponds to scFv C121, a single-chain variable domain of an antibody against the SARS-CoV-2 spike protein (Robbiani et al., 2020). [Figure 39] Sequences of the BSFpn728 and BSFpn711 replication assembly cassettes and scaffolds. The arrow indicates the pII cleavage site. The circular ssDNA within the nanorod corresponds to the sequence between the two pII cleavage sites. [Figure 40] Sequences of components within the BSFpn728 and BSFpn711 replication assembly cassettes. The arrow indicates the pII cleavage site. The circular ssDNA within the nanorod corresponds to the sequence between the two pII cleavage sites. [Figure 41]Sequences of the BSFpn79a and BSFpn79lac replication assembly cassettes. The arrow indicates the pII cleavage site. The circular ssDNA within the nanorod corresponds to the sequence between the two pII cleavage sites. [Figure 42] Sequences of components within the BSFpn79a and BSFpn79lac replication assembly cassettes. The arrow indicates the pII cleavage site. The circular ssDNA within the nanorod (scaffold) corresponds to the sequence between the two pII cleavage sites. [Figure 43] Sequences of the BSFpn529 and BSFpn395 replication assembly cassettes and scaffolds. The arrow indicates the pII cleavage site. The circular ssDNA within the nanorod corresponds to the sequence between the two pII cleavage sites. [Figure 44] Sequences of components within the BSFpn529 and BSFpn395 replication assembly cassettes. The arrow indicates the pII cleavage site. The circular ssDNA within the nanorod corresponds to the sequence between the two pII cleavage sites. [Figure 45] Sequences of the replication assembly cassette and scaffold for BSFpn313 and BSFpn289. The arrow indicates the pII cleavage site. The circular ssDNA within the nanorod corresponds to the sequence between the two pII cleavage sites. [Figure 46] Sequences of components within the replication assembly cassettes of BSFpn313 and BSFpn289. The arrow indicates the pII cleavage site. The circular ssDNA within the nanorod corresponds to the sequence between the two pII cleavage sites. [Figure 47] Sequences of the replication assembly cassette and scaffold for BSFpn221 and BSFpn152. The arrow indicates the pII cleavage site. The circular ssDNA within the nanorod corresponds to the sequence between the two pII cleavage sites. [Figure 48] Sequences of components within the BSFp221 and BSFp152 replication assembly cassettes. The arrow indicates the pII cleavage site. The circular ssDNA within the nanorod corresponds to the sequence between the two pII cleavage sites. [Figure 49]The sequences of the pII recognition sequence and promoter driving gII(gX)-gV-gVII-gIX-gVIII expression in the pHP1 and pPopUp plasmid series are shown. The top row shows the pII recognition sequence, and the vertical arrow indicates the nick site. The middle row shows the wild-type Ff promoter pA, and the bottom row shows placUV5 in the pPopUpLac and pHP1Lac plasmid series. The highlighted ATG in the middle and bottom rows indicates the first codon of gIIorf. [Figure 50] This is the auxotrophic marker NadC. The top line is the amino acid sequence of NadC. The middle and bottom lines are the nucleic acid sequences of the marker block. The underlined lines indicate the pairs of restriction sites on either side of the marker block used for cloning: BamHI and SnaBI for pBSF Nano, and AhdI and XhoI for pPopUp Plasmid. [Figure 51-1]

[0039] Figure 10 is the sequence (FASTA format) of a representative ppopup plasmid, ppopupBSFpnLac529YM. [Figure 51-2] Same as above. [Figure 52-1] The sequence (FASTA format) of a representative pHP helper plasmid series pHP1Lac. [Figure 52-2] Same as above. [Figure 53] The sequence (FASTA format) of a representative pBSF Nano replicative assembly plasmid series, pBSF Nano 529N. [Figure 54]

[0033] Figure 1. Sequence of modified pVIII / gVIII variant to display the GGGGG tag. Vertical arrow indicates the signal sequence cleavage site. Bold underlined sequence indicates mutations or amino acid changes. [Figure 55] The amino acid and nucleic acid sequences of the VHHN3-pIII fusion are shown. The top and bottom are the amino acid and nucleic acid sequences, respectively. The shaded sequence corresponds to VHHN3, a single-chain variable domain of an antibody against the SARS-CoV-2 nucleocapsid (NC) protein (Sherwood and Hayhurst, 2021). [Figure 56]Sequence of the BSFpn1400 replication assembly cassette and scaffold. The arrow indicates the pII cleavage site. The circular ssDNA within the nanorod corresponds to the sequence between the two pII cleavage sites. [Figure 57] Sequence of components within the BSFpn1400 replica assembly cassette. The arrow indicates the pII cleavage site. The ssDNA within the nanorod corresponds to the sequence between the two pII cleavage sites. DETAILED DESCRIPTION OF THE INVENTION

[0023] (Description of the embodiment)

[0024] Definition: The following definitions are presented to more clearly define the present invention and as a guide to those skilled in the art in practicing the present invention.

[0025] Unless otherwise specified, all technical and scientific terms used herein are understood to have the same meaning as understood by one of ordinary skill in the relevant art to which this disclosure pertains. Examples of definitions of common terms in microbiology, molecular biology, pharmacology, and biochemistry can be found in (Lederberg, 2000; Lewin et al., 2011; Madigan et al., 2009; Meyers, 1995; Reddy, 2007; Singleton and Sainsbury, 2006).

[0026] The practice of the present invention is believed to be carried out using standard microbiological, molecular biology, pharmacological and biochemical protocols and procedures known in the art and described in, for example, (Burtis et al., 2015; Lewin et al., 2011; Reddy, 2007; Sambrook and Russell, 2001; Whitby and Whitby, 1993) and other publicly available reference works relevant to the technical fields to which this disclosure pertains, all of which are incorporated herein by reference in their entirety.

[0027] The term "comprises" as used in this specification and claims means "consisting at least in part of." That is, when interpreting statements in this specification and claims that include "comprises," all features preceded by this term in each statement must be present, although other features may also be present. Related terms such as "comprise" and "comprised" are to be interpreted in the same manner.

[0028] As used herein, the term "consisting essentially of" means the specified materials or steps and those materials or steps that do not materially affect the basic and novel characteristics of the claimed invention.

[0029] As used herein, the term "consisting of" means the specified materials or steps of the claimed invention, and excludes any element, step, or ingredient not specified in the claim.

[0030] As used herein, the term "BSF nanoreplication assembly cassette" refers to a nucleic acid sequence that includes at least one positive strand replication origin (+) ori.

[0031] As used herein, the term "(+) origin" refers to a nucleic acid sequence that functions as an origin of positive strand DNA replication.

[0032] As used herein, the term "(-)ori" refers to a nucleic acid sequence that functions as a negative DNA strand replication origin.

[0033] In one embodiment, the BSF nanoreplication assembly cassette comprises at least one (+) oligonucleotide and at least one (-) oligonucleotide. In one embodiment, the BSF nanoreplication assembly cassette comprises at least two (+) oligonucleotides. In one embodiment, at least one (+) oligonucleotide is a replication initiator. In one embodiment, at least one (+) oligonucleotide is a replication terminator.

[0034] In one embodiment, the BSF nanoreplication assembly cassette comprises at least one (-) oligonucleotide.

[0035] As used herein, the term "fusion gene" refers to a gene encoding a translational fusion between a peptide and a major (pVIII) and minor (pIII, pVI, pVII, and pIX) coat protein or portion thereof of a filamentous bacteriophage, preferably an Ff phage coat protein or portion thereof. The fusion proteins described herein are encoded by fusion genes.

[0036] As used herein, the term "polynucleotide" in its broadest sense refers to single- or double-stranded deoxyribonucleotide or ribonucleotide polymers of any length, including, but not limited to, coding and non-coding sequences of genes, sense and antisense sequences, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polynucleotides, isolated and purified natural DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers, fragments, gene constructs, vectors, and modified polynucleotides. References to nucleic acids, nucleic acid molecules, nucleotide sequences, and polynucleotide sequences shall be understood similarly.

[0037] In some embodiments, the polynucleotides described herein are isolated.

[0038] Nucleic acids contemplated herein may be or may include (but are not limited to) deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), locked nucleic acid (including LNA, LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (diastereomer of LNA), 2′-amino-LNA having a 2′-amino functional group, and 2′-amino-α-LNA having a 2′-amino functional group), threose nucleic acid (TNA), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA), glycol nucleic acid (GNA), or chimeras or combinations thereof.

[0039] In some embodiments, the nucleic acids or polynucleotides described herein are messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a polypeptide of interest, such as those described herein, and that can be translated in vitro, in vivo, ex vivo, or in situ to produce the polypeptide.

[0040] The encoded polypeptide may be a naturally occurring, non-naturally occurring, or modified amino acid polymer. In a preferred embodiment, the encoded polypeptide is a non-naturally occurring polypeptide. As used herein, unless otherwise specified, the DNA polynucleotide sequences described herein describe thymine (T), but in the RNA polynucleotide sequences, thymine is replaced with uracil (U). Therefore, those skilled in the art will understand that a specifically recognized DNA (i.e., SEQ ID NO: 2) includes a corresponding RNA (e.g., mRNA) sequence in which each thymine in the DNA sequence is replaced with uracil (i.e., T>U substitution).

[0041] Those skilled in the art also understand that an mRNA that can be translated into a polypeptide of interest will also contain some or all of the following features: a 5' cap, a 5' untranslated region (UTR), at least one coding region, a 3' UTR, and a polyA tail.

[0042] The term "open reading frame" means a contiguous stretch of DNA that begins with a start codon (e.g., methionine (ATG)) and ends with a stop codon (e.g., TAA, TAG, or TGA). An open reading frame encodes a polypeptide.

[0043] The term "amber mutation" refers to a mutation that results in the premature termination of a polypeptide chain. Amber mutations are the result of a base substitution that converts a codon specifying an amino acid into a stop codon, such as UAG, that signals chain termination. Other mutations that convert amino acid codons into stop codons are known as ochre (UAA) and opal (UGA).

[0044] The term "3' untranslated region" (3'UTR) is used herein as understood by those of skill in the art and refers to the region of an mRNA that is immediately downstream (i.e., 3') of the stop codon (i.e., the codon in an mRNA transcript that signals the end of translation). The 3'UTR does not contain an open reading frame and / or is not translated into a polypeptide.

[0045] The term "5' untranslated region" (5'UTR) is used herein as understood by those of skill in the art and refers to the region of an mRNA that is immediately upstream (i.e., 5') from the start codon (i.e., the first codon of an mRNA transcript that is translated by a ribosome). The 5'UTR does not contain an open reading frame and / or is not translated into a polypeptide.

[0046] As used herein, the term "poly(A) tail" refers to a region of an mRNA downstream (i.e., 3') of the 3' UTR and containing multiple consecutive adenosine monophosphate (A) residues. As recognized in the art, the function of the poly(A) tail is to protect the mRNA from enzymatic degradation and to facilitate both transcription termination and export of the mRNA from the nucleus. The number of consecutive A residues in a "poly(A) tail" may vary, for example, from 10 to 300. By way of example only, a poly(A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 A residues.

[0047] As used herein, the term "vector" refers to, but is not limited to, any type of polynucleotide molecule that can be used to manipulate genetic material so that it can be amplified, replicated, manipulated, partially replicated, modified, and / or expressed. In some embodiments, a vector may be used to transport the polynucleotide contained in the vector into a cell or organism. In some embodiments, the vector is selected from the group consisting of a plasmid, a bacterial artificial chromosome (BAC), a P1-derived artificial chromosome (PAC), a yeast artificial chromosome (YAC), a bacteriophage, a phagemid, and a cosmid. In a preferred embodiment, the vector is a plasmid.

[0048] In some embodiments, the nucleic acid expression constructs, nucleic acid replication constructs, and / or helper nucleic acid expression constructs described herein are vectors or are contained in vectors. In some embodiments, the nucleic acid expression constructs, nucleic acid replication constructs, and / or helper nucleic acid expression constructs described herein are plasmids or are contained in plasmids. In some embodiments, a vector or plasmid may consist essentially of a nucleic acid expression construct, nucleic acid replication construct, and / or helper nucleic acid expression construct described herein. In some embodiments, a vector or plasmid may consist of a nucleic acid expression construct, nucleic acid replication construct, and / or helper nucleic acid expression construct described herein.

[0049] The term "coding region" or "open reading frame" (ORF) refers to the sense strand of a genomic DNA or cDNA sequence that is capable of producing a transcript and / or polypeptide under the control of appropriate regulatory sequences. A coding sequence is recognized by the presence of a 5' translation start codon and a 3' translation stop codon. When inserted into a gene construct or expression cassette, a "coding sequence" can be expressed when operably linked to promoter and terminator sequences and / or other regulatory elements.

[0050] "Operably linked" means that the sequences to be expressed are under the control of regulatory elements.

[0051] As used herein, "regulatory element" refers to any nucleic acid sequence element that controls or influences the expression of a polynucleotide insert from a vector, gene construct, or expression cassette, and includes promoters, transcriptional control sequences, translational control sequences, origins of replication, tissue-specific regulatory elements, temporal control elements, enhancers, polyadenylation signals, repressors, and terminators. Regulatory elements may be "homologous" or "heterologous" to the polynucleotide insert expressed from a gene construct, expression cassette, or vector described herein. When a nucleic acid expression construct, expression cassette, or vector described herein is present in a cell, the regulatory element may be "endogenous," "exogenous," "naturally occurring," and / or "non-naturally occurring" with respect to the cell.

[0052] The term "non-coding region" refers to the untranslated sequences upstream of the translation start site and downstream of the translation stop site. These sequences are also called the 5'UTR and 3'UTR, respectively. These regions contain elements necessary for transcription initiation and termination and for regulating translation efficiency.

[0053] Terminators are sequences that terminate transcription and are present at the 3' untranslated end of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and, in some cases, have been shown to have spatial regulatory functions.

[0054] The term "promoter" refers to a non-transcribed cis-regulatory element upstream of a coding region that regulates the transcription of a polynucleotide sequence. A promoter consists of a cis-initiation element and a conserved box that specify the transcription start site. In a non-limiting example, a bacterial promoter may consist of a "Pribnow box" (also called a -10 region) and other motifs that are bound by transcription factors and promote transcription. A promoter can be homologous or heterologous with respect to the polynucleotide sequence being expressed. When a polynucleotide sequence is expressed intracellularly, the promoter can be an endogenous or exogenous promoter. A promoter can be a constitutive promoter, an inducible promoter, or a regulatable promoter, as known in the art. In a preferred embodiment considered herein, the promoter is an inducible promoter.

[0055] As used herein, the term "polypeptide" is used broadly to include naturally occurring polypeptides, artificial polypeptides, synthetic polypeptides, gene products, homologs, orthologs, paralogs, variants, fragments, and other equivalents, as well as analogs thereof as understood by those skilled in the art. A polypeptide may be a single molecule or part of a molecular complex. Such complexes include, but are not limited to, dimers, trimers, tetramers, hexamers, and the like. A polypeptide may be composed of a single amino acid chain (i.e., a single polypeptide) or, in the case of a molecular complex, multiple amino acid chains (multiple polypeptides). Molecular complexes containing multiple polypeptides often contain disulfide bridges or bonds between specific amino acid residues. As used herein, the term "polypeptide" also refers to a polymer of amino acid residues containing at least one modified amino acid residue, including, by way of non-limiting example, an artificial chemical analog of the corresponding naturally occurring amino acid.

[0056] The term "naturally occurring" as used herein with respect to a polypeptide or polynucleotide refers to a polynucleotide or polypeptide sequence having a primary nucleic acid or amino acid sequence found in nature. A synthetic polynucleotide or polypeptide sequence identical to a wild-type polynucleotide sequence is considered a naturally occurring sequence for the purposes of this disclosure. What is important for a naturally occurring polynucleotide or polypeptide sequence is that the actual sequence of nucleotide bases or amino acid residues that make up the polynucleotide or polypeptide is as found in nature or known in nature, respectively.

[0057] The term "wild type" is used herein in the sense that is generally understood in the art. For example, a wild type polynucleotide sequence is a naturally occurring polynucleotide sequence. A naturally occurring polynucleotide sequence also refers to a mutant polynucleotide sequence found in nature that differs from the wild type. For example, but not limited to, allelic variants and naturally occurring recombinant polynucleotide sequences resulting from hybridization or horizontal gene transfer.

[0058] "Non-naturally occurring," as used herein with respect to a polypeptide or polynucleotide, refers to a polynucleotide or polypeptide having a primary nucleic acid or amino acid sequence that is not found in nature. Such peptides are also referred to herein as "artificial polypeptides" (and grammatical variations thereof).

[0059] Examples of non-natural polynucleotide and polypeptide sequences include artificially generated mutant and variant polynucleotide and polypeptide sequences. These sequences are created, for example, by point mutation, insertion or deletion, domain rearrangement, but are not limited to these. Non-natural polynucleotide and polypeptide sequences also include chemically evolved sequences. What is important about the non-natural polynucleotide or polypeptide sequences described herein is that the actual sequence of nucleotide bases or amino acid residues that make up the polynucleotide or polypeptide is not found in nature and is not known from nature.

[0060] The term "fused" as used herein with respect to "fused" polypeptides and portions of polypeptides (including other grammatical variations) means that amino acid sequences are covalently linked to one another by peptide bonds.

[0061] The "fusion polypeptides" disclosed in the present application are artificial polypeptides, i.e., the fusion polypeptides disclosed herein are non-naturally occurring. As described herein, fusion polypeptides or fusion proteins (these terms are used interchangeably and mean the same thing) are expressed from a fusion gene.

[0062] "Homologous" as used herein with respect to a polynucleotide or polypeptide or portion thereof means a polynucleotide or polypeptide or portion thereof that is a naturally occurring polynucleotide or polypeptide or portion thereof.

[0063] "Heterologous" as used herein with respect to a polynucleotide or polypeptide or portion thereof means a polynucleotide or polypeptide or portion thereof that is a non-naturally occurring polynucleotide or polypeptide or portion thereof.

[0064] A homologous polynucleotide or portion thereof may be operably linked to one or more different polynucleotides or portions thereof to form a single polynucleotide that can be expressed or translated in a cell to form a polypeptide of interest, preferably an antigenic polypeptide. In some embodiments, the different polynucleotides or portions thereof are homologous polynucleotides or portions thereof. In some embodiments, the different polynucleotides or portions thereof are heterologous polynucleotides or portions thereof.

[0065] Similarly, a heterologous polypeptide or portion thereof may be fused to one or more different polypeptides or portions thereof to form a single polypeptide of interest, preferably an antigenic polypeptide. In some embodiments, the different polypeptides or portions thereof are homologous polypeptides or portions thereof. In some embodiments, the different polypeptides or portions thereof are heterologous polypeptides or portions thereof.

[0066] The term "functional variant or fragment thereof" of a polypeptide refers to a subsequence of the polypeptide that performs a function necessary for the biological activity or binding of the polypeptide and / or provides the three-dimensional structure of the polypeptide. The term may also refer to a polypeptide, an aggregate of the polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or a functional polypeptide derivative thereof that performs the polypeptide activity.

[0067] "Isolated," as used herein with respect to a polynucleotide or polypeptide sequence, refers to a sequence that has been removed from its natural cellular environment or from the cellular environment in which it was synthesized or expressed. Isolated molecules may be obtained by any method or combination of methods known and used in the art, including biochemical, recombinant, and synthetic techniques. The polynucleotide or polypeptide sequence may be prepared by at least one purification step.

[0068] In some embodiments, the fusion polypeptides described herein are isolated. In some embodiments, the polynucleotides described herein are isolated.

[0069] As used herein, the term "variant" refers to a polynucleotide or polypeptide sequence that differs from a specifically recognized sequence by the deletion, substitution, or addition of one or more nucleotides or amino acid residues. Variants may be naturally occurring allelic variants or non-naturally occurring variants. Variants may be from the same or other species and may include homologs, paralogs, and orthologs. In certain embodiments, variants of the polynucleotides and polypeptides described herein have biological activity that is identical, similar, or substantially similar to that of the corresponding wild-type molecule, i.e., a naturally occurring polypeptide or polynucleotide. In certain embodiments, the similarity is similar activity and / or binding specificity.

[0070] In certain embodiments, variants of the polynucleotides and polypeptides described herein have a different biological activity than the corresponding wild-type molecule, hi certain embodiments, the difference is an altered activity and / or binding specificity.

[0071] The term "variant" with respect to polynucleotides and polypeptides encompasses all forms of polynucleotides and polypeptides defined herein.

[0072] Polynucleotide variants The variant polynucleotide sequence preferably has at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% identity to a sequence of the invention. Identity is found over a comparison window of at least 8 nucleotide positions, preferably at least 10 nucleotide positions, preferably at least 15 nucleotide positions, preferably at least 20 nucleotide positions, preferably at least 27 nucleotide positions, preferably at least 40 nucleotide positions, preferably at least 50 nucleotide positions, preferably at least 60 nucleotide positions, preferably at least 70 nucleotide positions, preferably at least 80 nucleotide positions, preferably over the entire length of the polynucleotides described herein.

[0073] Polynucleotide variants include those that exhibit similarity to one or more of the specifically identified sequences that are likely to maintain functional equivalence of those sequences and that would not reasonably be expected to occur by chance.

[0074] Polynucleotide sequence identity and similarity can be readily determined by one of ordinary skill in the art.

[0075] Variant polynucleotides also include polynucleotides that differ from the polynucleotide sequences described herein but, due to the degeneracy of the genetic code, encode polypeptides that have similar activity to the polypeptides encoded by the polynucleotides of the present invention. Sequence changes that do not change the amino acid sequence of a polypeptide are "silent mutations." With the exception of ATG (methionine) and TGG (tryptophan), other codons for the same amino acids may be changed using art-recognized techniques, for example, to optimize codon expression in a particular host organism.

[0076] Polynucleotide sequence changes that result in conservative substitutions of one or more amino acids in the encoded polypeptide sequence and do not significantly alter its biological activity are also included in the present invention. Skilled artisans know how to make phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).

[0077] In the context of this specification, a "functional variant or fragment" of a polynucleotide is one that contains nucleotide residues encoding non-essential amino acid residues, and / or additions, substitutions, and / or deletions in a non-essential amino acid sequence (e.g., SEQ ID NO:1), where "non-essential" means an amino acid residue or sequence that does not affect the functionality of the expressed protein. 1) Here, "non-essential" means an amino acid residue or sequence that does not affect the functionality of the expressed protein.

[0078] In some embodiments, a functional variant of a fusion polypeptide described herein is a fusion polypeptide that includes a particular peptide or polypeptide inserted between the signal sequence and the mature portion of the mutant fusion polypeptide.

[0079] In some embodiments, functional variants of the polynucleotides described herein are polynucleotides that contain short nucleotide sequences or single residue substitutions that allow for site-specific (targeted) chemical or enzymatic modification of the displayed polypeptide expressed from the polynucleotide variant.

[0080] Polypeptide variants The term "variant" with respect to a polypeptide includes naturally occurring, recombinantly produced, and synthetically produced polypeptides. The variant polypeptide sequence preferably has at least 35%, preferably at least 40%, preferably at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 71%, preferably at least 72%, preferably at least 73%, preferably at least 74%, preferably at least 75%, preferably at least 76%, preferably at least 77%, preferably at least 78%, preferably at least 79%, preferably at least 80%, preferably at least 81%, preferably at least 82%, preferably at least 83%, preferably at least 84%, preferably at least 85%, preferably at least 86%, preferably at least 87%, preferably at least 88%, preferably at least 89%, preferably at least 90%, preferably at least 91%, preferably at least 92%, preferably at least 93%, preferably at least 94%, preferably at least 95% or more, preferably 96% or more, preferably 97% or more, preferably 98% or more, preferably 99% or more identity to a sequence of the invention. Identity is found over a comparison window of at least two amino acid positions, preferably at least three amino acid positions, preferably at least four amino acid positions, preferably at least five amino acid positions, preferably at least seven amino acid positions, preferably at least ten amino acid positions, preferably at least 15 amino acid positions, preferably at least 20 amino acid positions, preferably over the entire length of a polypeptide described herein.

[0081] The terms "variant polypeptide," "polypeptide variant," and "altered polypeptide" (including grammatical variations thereof) are used interchangeably herein and have the same meaning.

[0082] Polypeptide variants also include those that exhibit similarity to one or more of the specifically identified sequences and that would not reasonably be expected to occur by chance, while still likely maintaining the functional equivalence of those sequences.

[0083] Polypeptide sequence identity and similarity can be readily determined by one of ordinary skill in the art.

[0084] Variant or modified polypeptides include polypeptides whose amino acid sequence differs from the polypeptides herein by one or more conservative amino acid or non-conservative substitutions, deletions, additions, or insertions that do not affect the biological activity of the peptide.

[0085] Conservative substitutions typically involve replacing one amino acid with another amino acid with similar properties, for example, substitutions with the group consisting of valine, glycine, glycine, alanine, valine, isoleucine, leucine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, lysine, arginine, phenylalanine, and tyrosine.

[0086] Analysis of evolved biological sequences has shown that not all sequence changes are equal, reflecting, at least in part, the difference between conservative and non-conservative substitutions at the biological level. For example, certain amino acid substitutions may occur frequently, while others are very rare. Evolutionary changes or substitutions of amino acid residues can be modeled using scoring matrices, also known as substitution matrices. Such matrices are used in bioinformatics analysis to recognize relationships between sequences and are known to skilled practitioners.

[0087] Other variants include peptides with modifications that affect the stability of the peptide. Such analogs may, for example, include one or more non-peptide bonds (replacing peptide bonds) in the peptide sequence. Also included are analogs containing residues other than naturally occurring L-amino acids, such as D-amino acids, or non-naturally occurring synthetic amino acids, such as beta or gamma amino acids, and cyclic analogs.

[0088] Substitutions, deletions, additions, or insertions may be made by mutagenesis techniques known in the art. A skilled practitioner will know how to make phenotypically silent amino acid substitutions. See, for example, (Bowie et al., 1990).

[0089] As used herein, a polypeptide can also refer to a polypeptide that has been modified during or after synthesis, for example, by biotinylation, benzylation, glycosylation, phosphorylation, amidation, derivatization with blocking / protecting groups, etc. Such modifications may increase the stability or activity of the polypeptide.

[0090] In the context of this specification, a "functional variant or fragment thereof" of a polypeptide, including a fusion polypeptide, is one that contains additions, substitutions and / or deletions of non-essential amino acid residues and / or non-essential amino acid sequences, where "non-essential" means an amino acid residue or sequence that does not affect the functionality of the expressed polypeptide.

[0091] An antibiotic resistance selectable marker, as used herein as known in the art, includes an antibiotic resistance gene that, in the polynucleotides described herein, is expressed from a nucleic acid expression construct to produce a polypeptide and confers on a host cell into which that polypeptide is transformed and expressed resistance to at least one antibiotic used in the culture medium to select for cells transformed with the polynucleotide.

[0092] The term "origin of replication" and grammatical variations thereof as used herein means an origin of replication of a nucleic acid as known and used in the art. As used herein, the term "Ff phage gene" and grammatical variations thereof refer to a polynucleotide or nucleic acid sequence that encodes the replication and coat proteins of Ff phage, as described herein. Ff phage genes can be organized into operons, as known in the art and described herein.

[0093] As used herein, the term "scaffold nucleic acid sequence" and grammatical variations thereof refer to the DNA sequence corresponding to the (+) strand circular ssDNA replicated from the BSF nanoreplication assembly cassette and subsequently packaged into nanorods.

[0094] The term "functionalized" and grammatical variations thereof, as used herein with respect to the nanorods described herein, refers to at least one polypeptide included in the nanorod, which polypeptide comprises a modifiable amino acid sequence at appropriate positions and / or contexts within the nanorod and the polypeptide itself, and which modifiable amino acid sequence is modifiable to allow for the attachment of chemical moieties to the nanorod.

[0095] In some embodiments, the chemical moiety is a small molecule, an antibody, a polypeptide, a polynucleotide, a small organic molecule such as biotin, a fluorescent dye such as FITC, various affinity tags, or an immunoadjuvant molecule such as alpha-galactoceramide (α-GalCer).

[0096] As used herein with respect to nanorods made using the NPSs described herein, the term "generate" (and grammatical variations thereof) refers to the expression, replication, and assembly of nanorods from the NPSs described herein.

[0097] The term "at least one" as used with respect to features described herein (including, but not limited to, "at least one inducible promoter," "at least one selectable marker," "at least one auxotrophic marker," etc.) means that at least one of the described feature is present. However, as used herein, this term also specifically contemplates the singular forms "the," "a," "an," and / or "one" (including other grammatical variations) as embodiments.

[0098] As used herein, the term "(+) strand DNA" and grammatical variations thereof means (+) strand circular single-stranded DNA (ssDNA).

[0099] Reference to a numerical range (e.g., 1 to 10) disclosed herein also includes all rational numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and any rational range within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7), and therefore all subranges of every range explicitly disclosed herein are expressly disclosed herein. These are merely examples of specific intent, and all possible combinations of numerical values ​​between the lowest and highest values ​​recited should be considered to be expressly set forth herein in the same manner.

[0100] Detailed Description of the Invention The present inventors have discovered that the problems outlined herein related to the efficient generation of biologically scalable functionalization-enabled nanorods (BSF nanos) can be overcome by providing a biological system that can efficiently provide relatively high yields of substantially pure short nanorods that do not contain antibiotic resistance genes in their DNA backbone.

[0101] Thus, we describe herein a platform for generating biologically scalable, functionalized nanorods (BSF nano) with dimensions of 6 nm x ≥ 40 nm. This platform is disclosed as two systems. The first system consists of a single plasmid, referred to as the "pop-up" plasmid, and includes single plasmid variants described herein (the "pop-up series"). The second system described herein consists of two plasmids: a helper plasmid (pHP) and a nanorod replication assembly plasmid (pBSF nano). The second system includes variants of the helper plasmid and nanorod replication assembly plasmid, pHP (the "pHP series") and pBSF nano (the "pBSF nano series"), respectively.

[0102] As mentioned above, each of these two systems contains variants that are suitable for specific applications. System variants are constructed by combining a series of interchangeable sequence units within each plasmid (Tables 2, 3, 6, 7; Figures 7–9, 29–57).

[0103] Bacterial cells containing the above-mentioned plasmids are used to produce nanorods. These cells belong to strains that contain specific mutations required for various aspects of coat production and vary according to the characteristics of the specific functional units suitable for specific applications (bacterial genotypes are listed in Table 1).

[0104] As outlined in this disclosure, the inventors have identified a surprising and unexpected technical solution that allows skilled workers to overcome the problems outlined herein, particularly by enabling the generation of biologically scalable nanorods derived from Ff phage without the simultaneous generation of longer, filamentous Ff phage particles.

[0105] As disclosed herein, we replace the helper phage with a helper plasmid that does not assemble into a phage particle but still provides all the Ff phage proteins necessary for replication of short nanorods from the nanorod replication assembly cassette. In this way, we eliminate the use of the helper phage itself, including all of its associated drawbacks (Figure 2A). Furthermore, we have confirmed that the same advantages associated with eliminating the use of the helper phage or helper plasmid itself can be achieved by using a single-plasmid system containing a single nucleic acid expression construct that contains all of the functions of the nucleic acid expression construct, including the replication assembly cassette and the helper construct described above, for the generation of short nanorods. This single-plasmid system is referred to herein as pPOP (Figure 2B).

[0106] As an additional technical advantage of the method described herein, we have expanded the replication assembly cassette for the generation of short nanorod backbones by including a (-) strand replication origin ("(-)Ori") and a complete (+)Ori as initiators (Figures 4B, 5B, 6A and B). We discovered that the p-popup and dual-plasmid system described herein improves the efficiency of generating nanorods with longer minimum lengths (70 nm, Figure 1C), which we term BSFpn (Biologically Scalable Ff Replication Assembly Cassette, Positive and Negative Origins). On the other hand, a p-popup system containing only the BSF nanoreplication assembly cassette, containing only the positive origin, as described herein, is termed BSFp (p stands for positive origin; Figures 1A, B, 4A, 5C, 6C).

[0107] As a further technical advantage of the system described herein, the inventors have discovered that biologically scalable nanorods can be generated without the use of antibiotic resistance markers in the BSF nanoreplication assembly cassette (single-plasmid system) or nanorod replication assembly plasmid (two-plasmid system). In this disclosure, positive transformants are selected using the auxotrophic marker nadC, which encodes an enzyme in the biosynthetic pathway of the essential metabolite NAD (nicotinamide dinucleotide). Thus, using the nanorod generation system (NPS) described herein, biologically scalable nanorods can be generated that are completely devoid of antibiotic resistance gene sequences. Although the inventors determined that the use of a helper plasmid can eliminate the generation of helper phage, the introduction of the nanorod replication assembly plasmid into E. coli results in an absolute number of transformed cells of approximately 10 per transformation. 7 To increase the number of cells that produce nanorods and thus the total yield of nanorods, inoculate the transformation reaction into fresh medium (e.g., 1 L) and culture for at least 13 generations until the cells reach the exponential growth phase (10 per L). 11 cells).

[0108] The regulatory circuitry controlling the production and function of the replication protein pII limits Ff (and by induction BSF nanoparticles) replication and the number of particles produced per cell to 10 per liter. 11 This gradually decreases over the 13 generations required to reach a cell density of 1000 kJ / s (Lerner and Model, 1981; Merriam, 1977).

[0109] To overcome these drawbacks, we have introduced yet another technical advantage of the system described herein. Specifically, we have introduced a constitutive promoter P upstream of gII. ABy replacing pBSF with the inducible lacUV5 promoter (Figures 7, 8, 49; SEQ ID NO: 89), we were able to induce expression of genes involved in Ff phage replication (Figures 7, 8, 49; SEQ ID NO: 90). This substitution is effective in both the single- and two-plasmid nanorod production systems described herein (e.g., the helper plasmid (pHP) in the two-plasmid system and the pPopUp single-plasmid system (Tables 7, 8)). This modification allows us to precisely control the timing of nanorod production as described herein, delaying the onset of replication, and therefore nanorod production, until the density of pBSF template plasmid-containing cells in the transformed culture reaches a desired value (Table 8). In one embodiment, the desired value of cells / mL corresponds to an exponentially growing culture (e.g., approximately 10 8 / mL or 10 11 / L).

[0110] Based on this overall concept, we have designed a series of embodiments that include elements within the plasmids of a two-plasmid system or within a single-plasmid p-pop-up system. These elements can be used to tailor nanorod production in response to recombination, desired enzymatic or chemical functionalization, and markers (antibiotics or auxotrophy) (Figures 7-9, Examples 1 and 2).

[0111] In some embodiments, at least one variant described herein is a variant of the major coat protein pVIII that has been modified to include a functional group suitable for chemical or enzymatic modification (SEQ ID NO: 13, SEQ ID: 15, SEQ ID: 17, SEQ ID: 19, SEQ ID: 21, SEQ ID: 23, SEQ ID: 25, SEQ ID: 27, SEQ ID: 97, and Figures 32, 33, 54).

[0112] In some embodiments, at least one variant described herein is a variant of a minor coat protein (e.g., but not limited to, pIII, pVI, pVII, or pIX) that has been modified to include a functional group suitable for chemical or enzymatic modification.

[0113] In one embodiment of a modification to include functional groups suitable for enzymatic modification, an AlaGlyGly was inserted at position 2 of mature pVIII and a Pro was deleted at position 6. This modification resulted in an N-terminal AlaAla motif, but resulted in very low nanorod production (Figure 32, SEQ ID NO:17, SEQ ID NO:18). To overcome this issue, this gVIII variant was introduced into Ff recombinant bacteriophage, resulting in poor replication, pinpoint plaques, and low-titer stocks. The virus was then "evolved" through three rounds of growth, resulting in a mutant that restored production, as evidenced by wild-type-like plaque size and titer (Figure 32, SEQ ID NO:19; Figure 33, SEQ ID NO:20, SEQ ID NO:21, and SEQ ID NO:22).

[0114] In certain embodiments, two evolved variants that produced the highest titers of phage are described herein. Both variants have a missense mutation in gVIII, resulting in an amino acid change in the mature portion of pVIII. One of the evolved variants has an Ala to Ser substitution at position 27 (Figure 32, SEQ ID NO:19 and Figure 33, SEQ ID NO:20), and the other has an Asp to Ala substitution at position 5 (Figure 33, SEQ ID NO:21). In certain embodiments, two evolved variants that produced the highest titers of phage are described herein. Both variants have a missense mutation in gVIII, resulting in an amino acid change in the mature portion of pVIII. 21, SEQ ID NO:22) are counted with wild-type mature pVIII. Reintroduction of the mutated gVIII sequence into the helper plasmid pHP1 or pPOPUp has been shown to restore the production of BSF nanorods (Figures 32-33; SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22).

[0115] Pop-up single-plasmid system One type of BSF nanorod generation system (NPS) consists of a single plasmid that is expressed in a suitable host cell (Figure 7). The pop-up plasmid typically consists of three main parts (A, B, and C).

[0116] A) BSF nanoreplication assembly cassette B) Ff phage genes (described in Figure 3) C) The origin of replication of the plasmid and a selection marker for maintaining the plasmid in E. coli cells. Each of these parts is made up of small interchangeable units or blocks that can be assembled together. By combining these two materials, specific properties can be imparted to BSF nanorods (Figure 7).

[0117] A) BSF nanoreplication assembly cassette (Block i) The BSF nanoreplication assembly cassette serves as a template for Ff rolling circle replication, generating multiple (+)stranded circular ssDNA fragments that serve as backbones for the assembly of short nanorods, referred to herein as BSF nanos (Figures 4-6, 39-48, 56-57, SEQ ID NOs: 41-52, 101-104). Those skilled in the art will appreciate that these backbone ssDNA fragments, also referred to herein as "scaffolds," mediate the assembly of Ff phage proteins into nanorods, as described herein.

[0118] In one example, the BSF nanoreplicative assembly cassette of the pPopUp plasmid series is a combination of the following units:

[0119] - Initiator (+)ori1, a functional plus-strand replication origin (+)ori that allows binding of replication protein pII (a DNA strand transferase) and cleavage of the (+)strand to form a primer (Figures 4-6, 39-48, 56-57, SEQ ID NO: 45, SEQ ID NO: 65, SEQ ID NO: 81, SEQ ID NO: 103). -The packaging signal (PS) required to target the (+)-strand circular ssDNA replicated from the BSF nanoreplication assembly cassette to the trans-envelope assembly machinery for nanorod assembly (Figures 4-6, 40, 42, 44, 46, 48, 57; SEQ ID NO:48, SEQ ID NO:68, SEQ ID NO:76, SEQ ID NO:83, SEQ ID NO:84).

[0120] -(-) Ori allows for negative strand replication using the short BSF nano (+) strand ssDNA as a template, increasing the copy number of the (+) strand circular ssDNA generated from the BSF nanoreplication assembly cassette (Figures 4-6, 40, 42, 44, 46, 57; SEQ ID NO: 50).

[0121] -terminator ((+)Ori2), which cleaves the template (+) strand where replication initiated at (+)Ori1 and ligates both ends of the (+) strand to generate a truncated (+)Ori mutant (△29) that can generate a (+) strand circular ssDNA that serves as the backbone for nanorod assembly described herein (Figures 4-6, 40, 42, 44, 46, 48, 57, SEQ ID NO:51, SEQ ID NO:69, SEQ ID NO:87).

[0122] -BSF Nanoreplication Assembly Cassette Characteristics and Variants: The initiator (+)Ori can be either only the minimal or core domain of the (+)Ori (A or I) (Figures 4-6, 46, 48, SEQ ID NO: 74, SEQ ID NO: 81), or the complete (+)Ori (both A and B domains) (Figures 4-6, 40, 42, 44, 57, SEQ ID NO: 45, SEQ ID NO: 65, SEQ ID NO: 103), the latter being more efficient at initiation than the former due to the presence of the complete pII-binding sequence.

[0123] The length of the nanorods generated is determined by the size of the scaffold nucleic acid sequence included in the BSF nanoreplication assembly cassette described herein. The scaffold nucleic acid sequence is located at the first pII nick site of (+)Ori1 and the second pII nick site of (+)Ori2 (gttcttt(AATA) (SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 101) in the BSF nanoreplication assembly cassette (Figures 4-6, 39-48, 56-57, SEQ ID NO: 88). BSFp replication assembly cassette For example, a BSF nanoreplication assembly cassette (here named BSFp) consisting of the initiator (+)Ori1 (containing only the (+)Ori core (or domain A)), the packaging signal, and the terminator (+)Ori2 corresponding to (+)OriΔ29 generates 152- or 221-nt circular (+)ssDNA and the assembly of nanorods 40 or 50 nm in length, respectively (Figures 1, 4-6, 47-48, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:77, SEQ ID NO:78). The 40-nm nanorods are the shortest Ff-derived nanorods generated to date.

[0124] BSFpn replication assembly cassette In another example, a replication assembly cassette, designated BSFpn, contains a combination of an initiator ((+)Ori1), a packaging signal (-)Ori, and a terminator ((+)Ori△29) corresponding to the complete (+)Ori (domain AB). In the presence of pII, this replication assembly cassette generates (+)-strand ssDNA of 395, 529, 707, 711, 728, and 748 nt and nanorods of 70, 80, 100, or 110 nm in length (Figures 1, 4-6, 39-44, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:61, SEQ ID NO:63). Inserting a DNA sequence between (+)Ori1 and PS can generate longer BSF nanorods.

[0125] Another variation of the BSFpn replication assembly cassette is to include only the core (+)Ori (Domain A) as the initiator in the (+)Ori1, as well as the (-)Ori (Figures 45 and 46, SEQ ID NO:70, SEQ ID NO:72; SEQ ID NO:74). The ssDNA generated from such BSFpn cassettes is 313 or 289 nt, resulting in nanorods with calculated lengths of 57 or 54 nm (approximately 55-60 nm), respectively.

[0126] Scalability of BSF nanorods In both the single- and two-plasmid NPSs described herein, a scaffold nucleic acid sequence is included between (+)Ori1 (initiator) and (+)Ori2 (terminator, Figures 1, 4-6, 39-48) at the pII cleavage site ((GTTCTTTT(AATA) (SEQ ID NO: 88, Figure 49)) within the BSF nanoreplication assembly cassette. One of skill in the art will recognize, based on the disclosure herein and as known in the art, that scaffold nucleic acid sequences of appropriate size to generate nanorods and / or multiple nanorods of the desired size can be readily selected for use in an NPS, as described herein.

[0127] As previously mentioned, the length of the (+)-strand circular ssDNA backbone (scaffold) generated by rolling circle replication of the BSF nanoreplication assembly cassette is determined by the number of nucleotides between the pII cleavage sites of (+)Ori1 (initiator) and (+)Ori2 (terminator). To reduce the size of the nanorods, the length of the scaffold nucleic acid sequence can be shortened. To achieve this, the (-)Ori is removed as was done in the BSFp replication assembly cassette, the filler sequence is completely removed, and the size of (+)Ori1 and (+)Ori2 in the BSFpn replication assembly cassette is reduced (Table 9, e.g., Figures 43 and 44, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66 vs SEQ ID NO:67; Figures 45 and 46, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74 vs SEQ ID NO:65; Figures 47 and 48, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:74). Conversely, the length of nanorods can be extended by inserting "filler" nucleic acid sequences between the initiator ((+)Ori1) and PS, between the PS and (-)Ori of BSFpn, or between the PS and (+)Ori2 of the BSFp replication assembly cassette (Figure 5B and C, Figure 6, Figures 56-57, SEQ ID NO: 104). Consequently, by designing filler nucleic acid sequences of appropriate length, nanorods can be extended to the desired length. Based on structural analysis of the Ff phage shaft, it can be accurately calculated that the length of nanorods increases by 0.133 nm for each nucleotide added to the ssDNA genome (Newman et al., 1977).

[0128] Protein-coding genes within the replicate assembly cassette It is also contemplated herein that the filler nucleic acid sequence can encode a second copy of gVIII to be used as a platform for the expression of pVIII fusions to longer peptides or proteins (Figure 7, block i). Alternatively, a second copy of gVIII can be encoded on a compatible plasmid, and this Ff phage protein can be incorporated into the generated nanorods, as is typically done with phage display technology. An example of the expression of Ff phage proteins from filler nucleic acid sequences contained in a BSF nanoreplication assembly cassette is provided by the expression of pVII and pIX from the BSF nanoreplication assembly cassette (SEQ ID NOs: 52-55), as shown in Figure 6B and Figure 41. In addition to Ff phage proteins expressed in E. coli, filler sequences can also be used to house eukaryotic gene expression cassettes for expression in eukaryotic cells.

[0129] B) Ff phage gene The Ff phage genes are organized into two operons, gII(gX)-gV-gVII-gIX-gVIII and gIII-gVI-gI-gIV, which encode all functions required for replication of the BSF nanoreplication assembly cassette and assembly of BSF nanorods. The Ff genes are functionally categorized into replication-encoding genes gII(gX) and gV (block iii of the p-popup plasmid, Figure 7), coat protein-encoding genes gVII, gIX, gVIII, gIII, and gVI (block iv of the p-popup plasmid, Figure 7), and assembly-encoding genes gI(gXI) and gIV (block v of the p-popup plasmid, Figure 7). In some embodiments, nucleic acid constructs containing these operons further contain multiple elements that serve to increase nanorod production or introduce functional groups in an orthogonal manner at specific nanorod locations and copy numbers, depending on the application for which the system is designed.

[0130] Promoter of gII(gX)-gV-gVII-gIX-gVIII operon A surprising technical advance provided by the present disclosure is the ability to regulate the production of the replication function encoded by gII (gX) and the packaging substrate formation function encoded by gV to direct replication of the BSF replication assembly cassette at sufficiently high cell densities. The NPS nucleic acid constructs described herein (e.g., pBSFp, pBSFpn, or pPopUp plasmids) can be grown at approximately 10 per liter of culture medium. 7 are introduced into E. coli with a transformation efficiency of 10 transformed cells (approximately 3 x 10 total cells per liter of culture). 12 (This is in contrast to a complete culture with 10 cells.) Therefore, the number of generations (cell divisions) from nanorod transformation to harvest is approximately 20. Based on quantitative monitoring and derived mathematical modeling (Smeal et al., 2017a, b), phage production declines to baseline after 107 E. coli cell divisions. When applied to a culture of transformed cells (starting number 10 per L), 7 ), and the 7th generation is only 10 per liter 9 This corresponds to cells, which corresponds to just 1 mL of overnight culture. Because each cell produces a finite number of nanorods, a low number of nanorod-producing cells reduces the overall yield of nanorods that can be produced from 1 liter of transformed cells.

[0131] The inventors surprisingly found that after the transformed cell culture reached higher cell densities, the culture was still in the exponential growth phase (approximately 10 cells per liter). 11 cells, O.D. 600 When pII expression was induced only during the 50-kDa phase (approximately 0.1), the culture contained a maximum number of cells (10 per L). 11 ~6x10 12 ) nanorod production peaks when the culture reaches a high density. This avoids a decline in nanorod production before the culture reaches a high density. To delay pII production, the gII(gX)-gV-gVII-gIX-gVIII operon expression is driven by the native (constitutive) Ff promoter P AThe vector is placed under an inducible promoter by replacing the promoter with the promoter of the vector (lacUV5, block ii, SEQ ID NO: 90, Figure 49). A A new family of constructs containing the lacUV5 promoter instead of the pII promoter was designed to generate the pPopUp series and pHP1Lac series, respectively (Figures 7 and 8). Analysis of nanorod production revealed that optimal cell density and synchronization of efficient BSF nanorod production with inducible expression of pII resulted in nanorod numbers of 4.6 × 10 14 to 4.8 x 10 15 A ten-fold increase in the ATP concentration is shown (Table 8, Figures 12 and 13, Example 6).

[0132] gII allele - The phage-encoded pII used in this disclosure contains the mutation IR1-B (Enea and Zinder, 1982) that allows efficient replication from the core (+) ori (domain A).

[0133] Coat protein Ff phage (and BSF nanorods) are composed of five distinct coat proteins. Among them, pVIII (50 aa long) is the major coat protein that forms the shaft of the nanorod and is present in multiple copies. The exact number of pVIII copies per nanorod varies depending on the length of the packaged ssDNA (1 pVIII subunit per 2.3 nt (Newman et al., 1977)). The remaining two sets of "minor" coat proteins are present in small, fixed numbers (5 per virion) and form two distinct ends of the virion: pIII and pVI at the proximal end, and pVII and pIX at the distal end. The nanorod itself has a five-fold axial symmetry (Newman et al., 1977).

[0134] As demonstrated in the field of phage display technology, each Ff coat protein represents a platform for displaying functions of interest based on specific applications (O'Neil and Hoess, 1995; Petrenko, 2008; Rakonjac et al., 2011). Protein fusions can be constructed between coat proteins and heterologous protein sequences, resulting in the display of heterologous sequences on the surface of viral particles. Alternatively, specific mutations or additional codons can be introduced into the coat protein coding sequence to serve as handles for site-specific modifications (via a "tag and modify" strategy (Chalker et al., 2011)).

[0135] Insertion of heterologous peptide sequences or modification of coat proteins must be performed in a way that does not interfere with nanorod assembly. Each coat protein has specific sites and / or modifiable segments that are optimal for insertion. Heterologous sequences can be inserted between the signal sequence and the mature portion of pIII and pVIII, at the C-terminus of pIII or pVI, or at the N-terminus of pVII and pIX (Fuh and Sidhu, 2000; Gao et al., 1999; Haaparanta and Huse, 1995; Jespers et al., 1996). Some of these fusions do not interfere with virion assembly when wild-type copies are also present, but they cannot mediate phage assembly alone. In the latter case, coexpression of wild-type and mutated copies (fusions) in the same cell allows nanorod assembly. As described herein, additional copies of wild-type or fusion coat proteins (i.e., mutated counterparts) can be inserted into a single plasmid containing the remaining phage genes or expressed from a second plasmid in the same cell (Barbas III et al., 2001). Fusions that can be incorporated into Ff phage but cannot drive assembly by themselves include insertions at the C-terminus of pIII and pVI (Fuh and Sidhu, 2000; Jespers et al., 1996) and insertions longer than six amino acid residues between the signal sequence of pVIII and its mature portion. The effect of the six-residue insertion is sequence-specific, with some sequences being better tolerated than others (Iannolo et al., 1995). In some embodiments discussed herein, a second copy of pVIII (pVIII fusion to a long peptide or protein) can be expressed from a BSF nanoreplication assembly cassette as described herein (Figure 7, block i). In one non-limiting example, expression of a second copy of the Ff phage protein from a BSF replication assembly cassette filler nucleic acid sequence is demonstrated by the expression of pVII and pIX from such a cassette, as shown in Figure 6B and Figure 41 (SEQ ID NOs:52-55).

[0136] Furthermore, it is envisioned herein that additional expression constructs containing plasmids can be used to supply a secondary copy of the pVIII coat protein if the inserted heterologous sequence prevents nanorod assembly in the absence of a wild-type counterpart. These additional plasmids should contain an origin of replication compatible with the pop-up plasmid, such as chloramphenicol resistance (cat; Cm R ) marker and the ColD origin of replication.

[0137] Further modifications to coat protein-encoding genes to create functionalization handles, known as "tag-and-modify" strategies, allow for targeted chemical or enzymatic modification. For example, engineering mature coat proteins pVIII or pIII or pVII and pIX with three or more extra glycines or two or more alanines at their N-termini (the latter two proteins may require the addition of a heterologous signal sequence) creates motifs that can be used for enzymatic attachment of protein or nonprotein molecules linked to C-terminal LPXTA or LPXTG motifs. In this case, attachment of the molecule of interest is catalyzed by the enzyme sortase A (SrtA) from Streptococcus pyogenes (SrtA Sp) or Staphylococcus aureus (SrtA Sa), respectively (Hess et al., 2012). Interchangeable blocks (Figure 7, block iv) have been generated for NPSs that generate nanorods containing pVIII displaying four glycine residues, or two alanine residues (Figures 30-32 and SEQ NOs: 19-23, 27-28) or five residues (Figure 54, SEQ NOs: 97-98) at the N-terminus.

[0138] Reactive amino acid groups, such as the amine group of the N-terminal residue, lysine, cysteine, tyrosine, aspartic acid, and glutamic acid, can be used for chemical modification (Bernard and Francis, 2014). Alternatively, other motifs that covalently attach non-protein molecules, such as SNAP tags, enzymatically or chemically, can be directly or indirectly inserted into nanorods to allow for the attachment of diverse molecules. We also describe an exchangeable block displaying an unpaired Cys residue in pIII, enabling maleimide-conjugated proteins and modification with small molecules or other chemicals that target -SH groups (Figure 7, Block iv; Figure 36, SEQ ID NOs: 33-34).

[0139] We also demonstrate that inserting an ATG codon into the coding sequence corresponding to exposed residues of pVIII allows for in vivo labeling with the unnatural amino acid azidohomoalanine (structurally similar to the ATG-encoded residue Met) during translation. The azide groups on the surface of the nanorods provide reactive groups for attaching molecules using "click" chemistry (Petrie, 2015). This was achieved by synthesizing an exchangeable block (Figure 7, block iv) containing both the exposed Met residue (Ala9 mutated to Met) and a pVIII variant in which the buried Met residue 28 was mutated to Leu. The latter mutation serves to prevent azide-mediated destabilization of the nanorod structure (Figure 33, SEQ ID NOs: 23 and 24).

[0140] In one non-limiting example, fluorescent labeling of BSF nanorods with the amine-reactive fluorescent dye DyLight 550 is described herein (Figure 20, Example 11). These nanorods are displayed with a binding molecule (the fibronectin-binding domain of Streptococcus pyogenes protein SOF22 (Rakonjac et al., 1995) as a fusion with pIII (SEQ ID NO: 1)). The nanorods are labeled with nucleotides 37 and 38 (Figure 37) and have been used in a lateral flow assay for the detection of the analyte fibronectin (Figure 20C, Example 11). As those skilled in the art will appreciate, using this approach, any amine-reactive fluorophore, other dye, other small molecule, or biological or chemical polymer designed to be amine-reactive is expected to be suitable for attachment to the nanorods. Each pVIII subunit has three acidic amino acid residues, including side-chain carboxyl groups (Glu2, Asp4, and Asp5), exposed on the nanorod surface. Therefore, carboxyl-reactive molecules can also be chemically conjugated to the nanorods. Other reactive groups, such as the aromatic hydroxyl group of Tyr residues, can also be used to attach appropriate reactive groups, as known in the art (Bernard and Francis, 2014). As those skilled in the art will appreciate, using this approach, any carboxyl-reactive fluorophore, other dye, other small molecule, or biological or chemical polymer designed to be amine-reactive is expected to be suitable for attachment to the nanorods.

[0141] Molecules attached to the nanorods described herein are not limited to biotin, but may be any type of organic molecule useful for binding commercially available or in-house fusions of biotin-binding proteins, such as avidin. In this manner, the nanorods described herein may be modified to display a wide array of avidin fusions to antibodies, dyes, or other functional molecules, providing the skilled worker with multiple methods for indirectly visualizing the nanorod. As will be appreciated by those skilled in the art, nanorods displaying the detector molecules described herein can bind to analytes and be visualized indirectly via phage-specific antibodies or directly, such as by chemically attached fluorescent molecules (Figure 20). In some embodiments, the nanorods described herein are labeled with two or more different chemically attached detector molecules, e.g., different fluorescent molecules, allowing such multi-labeled nanorods to be used in differential labeling methods, such as, but not limited to, multiplexed detection.

[0142] A skilled practitioner will appreciate that all known modifications applied in Ff-based phage display and materials science applications can also be applied to nanorod functionalization as described herein. In one non-limiting example, inserting four Gly residues at the N-terminus of our constructed mature pVIII (Figure 33, SEQ ID NO:27, SEQ ID NO:28) slightly reduced nanorod production. On the other hand, inserting an Ala followed by a Gly residue between Ala1 and Gly2 at the N-terminus of our constructed mature pVIII and deleting Pro6 from wild-type mature pVIII (Figure 32, SEQ ID NO:17, SEQ ID NO:18) prevented nanorod production. To overcome this latter issue, we "evolved" the gVIII sequence to increase the efficiency of this functionalized pVIII variant. This was achieved by transferring the coding sequence to the backbone of Ff phage (VCSM13). The resulting engineered phage produced very small plaques and low titers; however, three rounds of phage propagation, in which host cells were infected at a low multiplicity of infection (1 phage to 1,000 E. coli cells), resulted in the emergence of "large plaque" variants. Sequencing of gVIII from the evolved phage identified two gVIII variants, each containing a different compensatory mutation (D5A and L27S, as previously described; Figures 32 and 33, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22). These two alleles were each reintroduced into an inducible p-popup plasmid backbone and shown to generate BSF nanorods. As will be appreciated by those skilled in the art, the inventors believe that the coding sequences of various Ff phage proteins can be evolved to accommodate other modifications that may interfere with the assembly of BSF nanorods.

[0143] In one non-limiting example of enzymatic modification, evolved pVIII (SEQ ID NO: 19, 20) nanorods display an AlaAlaGlyGly motif on each pVIII copy. These were further enzymatically modified with LPETA-(LeuProGluThrAla)-tagged fluorescent dye FITC or the small molecule biotin by enzymatic attachment using Streptococcus pyogenes sortase (SrtA Sp, Figure 21). Analysis by native virus particle electrophoresis shows high-intensity fluorescence corresponding to the nanorod band after LPETA-FITC enzymatic conjugation (Figure 22). Analysis of enzyme-biotinylated nanorods by transmission electron microscopy using avidin-coated gold beads demonstrates sortase-dependent binding along the nanorod length (Figure 23). For immunodetection assays, avidin-alkaline phosphatase may be attached to the nanorods (Figures 24A, 25-27). Enzymatic visualization of such avidin-alkaline phosphatase-labeled nanorods was performed by native agarose gel electrophoresis, blotted onto a membrane, and detected using a chromogenic substrate (Figure 24A).

[0144] In another non-limiting example, LPETG-β-glucosidase (GUS) was enzymatically attached directly to nanorods displaying an N-terminal 5-Gly peptide, and the attachment of GUS to the nanorods was analyzed by agarose gel electrophoresis followed by an in-gel assay using a chromogenic substrate (Figure 24B).

[0145] Labeled nanorods displaying analyte-specific molecules, such as antibodies, can also be used in immunoassays. In one non-limiting example, nanorods displaying pIII fusion proteins that specifically bind to a SARS-CoV-2 pike-specific single-chain antibody (Figure 38, SEQ ID NOs: 39 and 40) or a SARS-CoV-2 nucleoprotein-specific camelid single-domain antibody VHH (Figure 55, SEQ ID NOs: 99 and 100) were fabricated. These pIII fusions were combined with pVIII, displaying an N-terminal Ala-Ala-Gly-Gly (AAGG), and evolved to efficiently assemble nanorods (Figure 32, SEQ ID NO: 18; Figure 33, SEQ ID NO: 20). LPETA-biotin was enzymatically attached to the nanorods using Streptococcus pyogenes sortase A, as described in the Methods section. The engineered nanorods are then used in dot blots, ELISAs, and lateral flow assays (Figures 25-27), as described in the Methods section. Avidin-alkaline phosphatase or avidin-horseradish peroxidase fusions are used as secondary or indirect detection reagents for biotin-modified nanorods, allowing alkaline phosphatase- or horseradish peroxidase-mediated enzyme visualization using chromogenic or chemiluminescent substrates of these two enzymes (Figures 25-27).

[0146] The copy number and location of the displayed functionality will vary depending on the coat protein used as a platform, whether it is a heterologous protein or a "handle" for modification. In one non-limiting example, using pVIII as a display platform results in a high copy number of peptides displayed along the nanorod shaft. The copy number of displayed peptides (or other functionality) depends on the number of pVIII subunits per nanorod, which in turn depends on the length of the ssDNA scaffold. The copy number of functionality (fluorochromes, small molecules, polymers, and / or enzymes) depends on the length of the nanorod. For example, for a 1,000 nm long phage (containing approximately 3,000 pVIII copies per viral particle), approximately 400 copies of fluorophores or biotin are expected to be attached per phage nanorod, whether chemically or enzymatically (approximately one fluorophore per seven pVIII subunits). Multiple different fluorescent dyes can be mixed for labeling purposes, enabling barcoding and other more complex methods and detection.

[0147] Using minor coat proteins as a platform, up to five copies can be displayed per nanorod (reviewed in (Rakonjac et al., 2017) for each of pIII, pVII, and pIX). Furthermore, displaying on both pVII and pIX allows up to 10 copies per nanorod. Using different fusion or attachment molecules to different minor Ff phage coat proteins, different functions can be displayed on a single nanorod. For example, two functions can be displayed on one end of the nanorod (pVII-pIX end) and one function on the other end (pIII end). Such modifications have been demonstrated using various phage display methods using full-length Ff phage.

[0148] As described here, the toxicity of the major coat protein pVIII was overcome by the introduction of amber mutations. The major coat protein pVIII is toxic to E. coli when expressed in the absence of phage assembly. This toxicity is caused by mutations introduced during the cloning process that either remove the gVIII promoter or result in poor growth of transformed E. coli cells expressing pVIII, even when expression is controlled by an inducible promoter. To overcome this problem, a helper plasmid was constructed using a gVIII suppressible (nonsense) mutant. The construction was performed in an E. coli host that did not contain a suppressor mutation, which prevented translation of most of the pVIII protein. Two different amber (TAG) mutants were used: one containing a G to T mutation, converting GAG codon 25, encoding glutamic acid at position 2 of the mature protein, to TAG (SEQ ID NOS: 13-24, Figures 32-33), and the other in which TCT codon 4 for serine in the signal sequence was replaced with TAG (SEQ ID NOS: 25-28, Figure 33). A suppressor D mutation (supD) in serine tRNA was used to suppress these two amber mutations, and E. coli strains containing this mutation were used for nanorod production (Table 1).

[0149] The gVIII suppressor amber mutant described here exhibits reduced cell proliferation compared to E. coli cells expressing wild-type gVIII. An additional benefit is that less pVIII is produced within the vacuole, due to the lower translation efficiency of the suppressor tRNA compared to the cognate tRNA that reads the sense codon, and by reducing the ratio of shaft protein pVIII to end-capping proteins pIII, pVI, pVII, and pIX, the assembly of short nanorods is favored over long nanorods.

[0150] C) Plasmid replication origin and selectable marker Plasmid replication origin In one embodiment, the plasmid replication origin p15A is used for the pPopUp and helper plasmids to enable replication in E. coli. Based on the disclosure herein, one skilled in the art will understand that other suitable plasmid replication origins can be used in the NPS as described herein.

[0151] Selection Marker - markers for selecting transformed E. coli cells, antibiotic selection markers, e.g., kanamycin resistance marker aph(3')-Ia(Kan R Either a nucleotide sequence (nucleotide sequence) or an auxotrophic marker, such as NadC, is required. When nadC is used as the selection marker, an E. coli host strain containing a deletion of the nadC gene (ΔnadC) is used for the construction of pop-up Ns and the production of nanorods using the NPS described herein containing the pPopUp529LacYMN plasmid. Minimal medium containing casamino acids (no NAD) is used for auxotrophic selection using the NadC marker.

[0152] Two-plasmid system The second type of BSF nanoproduction system described here consists of two plasmids. This two-plasmid system is also referred to here as a dual-plasmid system. Similar to the single-plasmid pop-up system described here, these plasmids are transformed into a specific E. coli host strain. This host strain contains a nanorod replication assembly plasmid containing the BSF nanoreplication assembly cassette or its variants (pBSF nanoseries) and a helper plasmid expressing all of the Ff phage proteins required for replication of the nanorod (+)-strand circular ssDNA from the BSF nanoreplication assembly cassette and assembly of short nanorods or their variants (pHP series). The helper plasmid also functions as a display vector, enabling nanorod functionalization. For example, the coding sequence of the helper plasmid can be modified to enable expression of functionalizable Ff phage proteins.

[0153] As described here, the use of two plasmids in NPS allows combining different BSF nanoreplicative assembly cassettes with different functions encoded by helper plasmid variants without the need to create new recombinant DNA constructs.

[0154] Helper plasmids (pHP series) The helper plasmid contains the same components as the pop-up plasmid described above, except that the BSF nanoreplication assembly cassette is absent.

[0155] The Ff phage genes are organized into two operons, gII(gX)-gV-gVII-gIX-gVIII and gIII-gVI-gI-gIV, which encode all the functions required for replication of the BSF nanoreplication assembly cassette and assembly of BSF nanorods. The Ff genes are functionally categorized as gII(gX), which encodes replication; gV (block II of the helper plasmid, Figure 8), which encodes packaging substrate formation; gVII, gIX, gVIII, gIII, and gVI (block III of the helper plasmid, Figure 8), which encode coat proteins; and gI(gXI) and gIV (block IV of the helper plasmid, Figure 8), which encode assembly proteins. In some embodiments, nucleic acid constructs containing these operons further contain multiple elements that serve to increase nanorod production or introduce functional groups in an orthogonal manner at specific nanorod locations and copy numbers, depending on the application for which the system is designed.

[0156] Promoter of gII(gX)-gV-gVII-gIX-gVIII operon A surprising technical advance provided by the present disclosure is the ability to regulate the production of the replication function encoded by gII (gX) and the substrate packaging function encoded by gV to direct replication of the BSF replication assembly cassette at sufficiently high cell densities. The NPS nucleic acid constructs described herein (e.g., pBSFp or pBSFpn plasmids) can be expressed at approximately 10 per liter of culture medium. 7transformed into E. coli with a transformation efficiency of 100 transformed cells (approximately 3 x 10 total per liter) 12 (This is in contrast to a complete culture with cells.) Therefore, the number of generations (cell divisions) from transformation to nanorod harvest is approximately 20. Based on quantitative monitoring and derived mathematical modeling (Smeal et al., 2017a, b), phage production declines to baseline after seven E. coli cell divisions. When applied to a culture of transformed cells (starting number 10 7 / L), while the 7th generation is only 10 per liter 9 cells, which corresponds to 1 mL of a complete overnight culture. Given that each cell produces a finite number of nanorods, this low number of nanorod-producing cells reduces the overall yield of nanorods that can be produced from 1 liter of transformed cells.

[0157] The inventors surprisingly found that after the transformed cell culture reached higher cell densities, the culture was still in the exponential growth phase (approximately 10 cells per liter). 11 Cells, OD< 600 When pII expression was induced only during the 50-kDa phase (approximately 0.1), the culture contained a maximum number of cells (10 per L). 11 ~6×10 12 ) nanorod production peaks when the culture reaches a high density. This prevents nanorod production from declining until the culture reaches a higher density. To achieve delayed pII production, the native (constitutive) Ff promoter P A The gII(gX)-gV-gVII-gIX-gVIII operon expression was placed under an inducible promoter by replacing the pGVVIII-gIX-gVIII operon with an inducible promoter (lacUV5, block i, SEQ ID NO: 90, Figure 49). A A new family of constructs containing the lacUV5 promoter instead of the pII promoter was designed, resulting in the pPopUpLac series and the pHP1Lac series, respectively (Figures 7 and 8). Analysis of nanorod production revealed that optimal cell density and synchronization of efficient BSF nanorod production with inducible expression of pII resulted in nanorod numbers of 4.6 × 1014 to 4.8 x 10 15 (Table 8, Figures 12 and 13, Example 6).

[0158] gII allele - The phage-encoded pII used in this disclosure contains the mutation IR1-B (Enea and Zinder, 1982) that allows efficient replication from the core (+) ori (domain A).

[0159] Coat protein The Ff phage (and BSF nanorods) are composed of five distinct coat proteins. Among them, pVIII (50 aa in length) is the major coat protein that forms the shaft of the nanorod and is present in multiple copies. The exact number of pVIII copies per nanorod depends on the length of the packaged ssDNA (1 pVIII subunit per 2.3 nt (Newman et al., 1977)). The remaining two sets of "minor" coat proteins are present in small, fixed numbers (5 per virion) and form the two distinct ends of the virion (pIII and pVI at the proximal end, and pVII and pIX at the distal end). The nanorod itself has a five-fold axial symmetry (Newman et al., 1977).

[0160] As demonstrated in the field of phage display technology, each Ff coat protein represents a platform for displaying functions of interest based on specific applications (O'Neil and Hoess, 1995; Petrenko, 2008; Rakonjac et al., 2011). Protein fusions can be constructed between coat proteins and heterologous protein sequences, resulting in the display of heterologous sequences on the surface of viral particles. Alternatively, specific mutations or additional codons can be introduced into the coat protein coding sequence to serve as handles for site-specific modifications (via a "tag and modify" strategy; Chalker et al., 2011).

[0161] Insertion of heterologous peptide sequences or modification of coat proteins must be performed in a way that does not interfere with nanorod assembly. Each coat protein has specific sites and / or modifiable segments that are optimal for insertion. Heterologous sequences can be inserted between the signal sequence and the mature portion of pIII and pVIII, at the C-terminus of pIII or pVI, or at the N-terminus of pVII and pIX (Fuh and Sidhu, 2000; Gao et al., 1999; Haaparanta and Huse, 1995; Jespers et al., 1996). Some of these fusions do not interfere with assembly into viral particles when wild-type copies are also present, but they cannot mediate phage assembly by themselves. In the latter case, coexpression of the wild-type copy and the mutated counterpart (fusion) in the same cell enables nanorod assembly. As described here, additional copies of wild-type or fusion coat proteins (i.e., mutated counterparts) can be inserted into a plasmid containing the remaining phage genes or expressed from a second plasmid in the same cell (Barbas III et al., 2001). Fusions that can be incorporated into Ff phage but cannot drive assembly by themselves include insertions at the C-terminus of pIII and pVI (Fuh et al., 2000; Jespers et al., 1996) and insertions longer than six amino acid residues between the signal sequence of pVIII and its mature portion. The effect of the six-residue insertion is sequence-specific, with some sequences being better tolerated than others (Iannolo et al., 1995). In some embodiments discussed herein, a second copy of pVIII (pVIII fusion to a long peptide or protein) can be expressed from a BSF nanoreplicative assembly cassette within the pBSF plasmid, as described herein (Figure 6B; Figure 9, block i). In one non-limiting example, expression of a second copy of the Ff phage protein from a BSF replication assembly cassette filler nucleic acid sequence is demonstrated by the expression of pVII and pIX from such a cassette, as shown in Figure 6B and Figure 41 (SEQ ID NOs:52-55).

[0162] Furthermore, it is envisioned herein that additional expression construct-containing plasmids can be used to supply a secondary copy of the pVIII coat protein if the inserted heterologous sequence prevents nanorod assembly in the absence of a wild-type counterpart. These additional plasmids contain origins of replication (e.g., chloramphenicol resistance (cat; Cm)) compatible with both the helper plasmid (pHP series) and the nanorod replicating plasmid (pBSF nano series) within the two-plasmid system. R >) marker and ColD origin of replication).

[0163] Further modification of coat protein-encoding genes to create functionalization handles is often performed to allow targeted chemical or enzymatic alterations, known as the "tag and modify" strategy. For example, engineering mature coat proteins pVIII or pIII or pVII and pIX with three or more extra glycines or two or more alanines at their N-termini (the latter two proteins may require the addition of a heterologous signal sequence) creates motifs that can be used for enzymatic attachment of protein or nonprotein molecules linked to C-terminal LPXTA or LPXTG motifs. In this case, attachment of the molecule of interest is catalyzed by the enzyme sortase A (SrtA) from Streptococcus pyogenes (SrtA Sp) or Staphylococcus aureus (SrtA Sa), respectively (Hess et al., 2012). Interchangeable blocks (Figure 8, block iii) have been generated for NPSs that generate nanorods containing pVIII displaying four glycine residues, or two alanine residues (Figures 30-32 and SEQ NOS: 19-23, 27-28) or five residues (Figure 54, SEQ NOS: 97-98) at the N-terminus.

[0164] Reactive groups of amino acids, such as the amine group of the N-terminal residue, lysine, cysteine, tyrosine, aspartic acid, glutamic acid, etc., can be used for chemical modification (Bernard and Francis, 2014). Alternatively, other motifs that can be enzymatically or chemically covalently attached to non-protein molecules such as the SNAP tag are inserted directly or indirectly into the nanorods to enable the attachment of various molecules. In addition, this specification also describes an exchangeable block that displays an unpaired Cys residue in pIII, enabling modification with maleimide-conjugated proteins and other chemicals targeting small molecules or -SH groups (Figure 8, block iii, Figure 36, SEQ ID NO: 33 - 34).

[0165] Furthermore, inserting an ATG codon into the coding sequence corresponding to the exposed residue of pVIII enables in vivo labeling with the unnatural amino acid azidohomoalanine (structurally similar to the residue Met encoded by ATG) during translation. The azide group on the surface of the nanorod provides a reactive group for attaching molecules using "click" chemistry (Petrie, 2015). To enable this, an exchangeable block containing a pVIII variant containing an exposed Met residue (Ala9 mutated to Met) and an embedded Met28 residue mutated to Leu (Figure 8, block iii) is also described in this specification (Figure 33, SEQ ID NO: 23, 24). 23, 24). This pVIII variant enables the incorporation of the unnatural amino acid azidohomoalanine (Aha) at the position exposed on the surface of pVIII during translation in vivo (from Ala9 to Met), preventing the aggregation and structural disruption of virus particles that would have been caused by the insertion of Aha at position 28 blocked by the mutation from Met28 to Leu (Petrie, 2015)< / 509. Aha contains an azide group in its side chain, and small molecules can be attached to virus particles using click chemistry targeting the azide group.

[0166] In one non-limiting example, fluorescent labeling of BSF nanorods with the amine-reactive fluorescent dye DyLight550 is described herein (Figure 20, Example 11). These nanorods are labeled with a binding molecule (a fusion of the fibronectin-binding domain of the Streptococcus pyogenes protein SOF22 (Rakonjac et al., 1995) to pIII (SEQ ID NO: 11). These nanorods have been displayed as nanorods (NO:37, 38, Figure 37) and used in lateral flow assays to detect analytes (fibronectin, Figure 20C, Example 11). Using this approach, skilled practitioners will appreciate that amine-reactive fluorophores, other dyes, or other small molecules or biological or chemical polymers designed to be amine-reactive will be suitable for attachment to the nanorods. Each pVIII subunit has three acidic amino acid residues, including side-chain carboxyl groups (Glu2, Asp4, and Asp5), exposed on the surface of the nanorod. Therefore, carboxyl-reactive molecules can also be chemically conjugated to the nanorods. As known in the art, other reactive groups, such as the aromatic hydroxyl group of Tyr residues, can also be used to attach appropriate reactive groups (Bernard and Francis, 2014). Using this approach, skilled practitioners will appreciate that carboxyl-reactive fluorophores, other dyes, or other small molecules or biological or chemical polymers designed to be amine-reactive will be suitable for attachment to the nanorods.

[0167] The molecules attached to nanorods as described herein can be any type of organic molecule, including, but not limited to, biotin, which serves to bind to fusions of commercially available or in-house biotin-binding proteins, such as avidin. Thus, the nanorods described herein can be modified to display a wide variety of avidin fusions to antibodies, dyes, or other functional molecules, providing the skilled worker with multiple methods for indirectly visualizing the nanorods. As will be appreciated by those skilled in the art, nanorods displaying the detector molecules described herein can bind to analytes and be visualized indirectly via phage-specific antibodies or directly, such as by chemically attached fluorescent molecules (Figure 20). In some embodiments, the nanorods described herein are labeled with two or more different chemically attached detector molecules, e.g., different fluorescent molecules, allowing such multi-labeled nanorods to be used in methods of multiplexed detection.

[0168] Skilled practitioners will appreciate that all known modifications applied in Ff-based phage display and materials science applications can also be applied to the nanorod functionalization described herein. In one non-limiting example, inserting four Gly residues into the N-terminus of our constructed mature pVIII (Figure 33, SEQ ID NO:27, SEQ ID NO:28) slightly reduced nanorod production. On the other hand, inserting an Ala followed by a Gly residue into the N-terminus of our constructed mature pVIII and deleting Pro6 from wild-type mature pVIII (Figure 32, SEQ ID NO:17, SEQ ID NO:18) prevented nanorod production. To overcome this latter issue, we "evolved" the gVIII sequence to increase the efficiency of this functionalized pVIII variant. This was achieved by transferring the coding sequence into the backbone of the Ff phage (VCSM13). The resulting engineered phage produced very small plaques and low titers; however, three rounds of phage propagation, in which host cells were infected at a low multiplicity of infection (1 phage to 1,000 E. coli cells), resulted in the emergence of "large plaque" variants. Sequencing of gVIII from the evolved phage identified two gVIII variants, each containing a different compensatory mutation (D5A and L27S, as previously described; Figures 32 and 33, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22). These two alleles were each reintroduced into an inducible p-popup plasmid backbone and shown to generate BSF nanorods. As will be appreciated by those skilled in the art, the inventors believe that the coding sequences of various Ff phage proteins can be evolved to accommodate other modifications that may interfere with the assembly of BSF nanorods.

[0169] In one non-limiting example of enzymatic modification, BSF nanorods were generated containing evolved pVIII (SEQ ID NOs: 19 and 20) that displays an AlaAlaGlyGly motif in each pVIII copy along the nanorod. These two alleles were reintroduced into an inducible pPopUp plasmid backbone and shown to generate BSF nanorods. These were further enzymatically modified with LPETA (LeuProGluThrAla)-tagged fluorescent dye FITC or the small molecule biotin by enzymatic attachment using Streptococcus pyogenes sortase (SrtA Sp; Figure 21). Analysis by native virus particle electrophoresis shows high-intensity fluorescence corresponding to the nanorod band after LPETA-FITC enzymatic conjugation (Figure 22). Analysis of enzymatically biotinylated nanorods by transmission electron microscopy using avidin-coated gold beads demonstrates sortase-dependent attachment along the nanorod length (Figure 23). In immunodetection assays, avidin-alkaline phosphatase may be attached to nanorods (Figures 24A, 25-27). Enzymatic visualization of such avidin-alkaline phosphatase-labeled nanorods was performed by native agarose gel electrophoresis, blotted onto a membrane, and detected using a chromogenic substrate (Figure 24A).

[0170] In another non-limiting example, LPETG-β-glucosidase (GUS) was enzymatically attached directly to nanorods displaying an N-terminal 5-Gly peptide, and the attachment of GUS to the nanorods was analyzed by agarose gel electrophoresis followed by an in-gel assay using a chromogenic substrate (Figure 24B).

[0171] Labeled nanorods displaying analyte-specific molecules, such as antibodies, can also be used in immunoassays. In one non-limiting example, nanorods displaying pIII fusion proteins that specifically bind to a SARS-CoV-2 spike-specific single-chain antibody (Figure 38, SEQ ID NOs: 39 and 40) or a SARS-CoV-2 nucleoprotein-specific camelid single-domain antibody VHH (Figure 55, SEQ ID NOs: 99 and 100) were generated. These pIII fusions were combined with pVIII, displaying an N-terminal Ala-Ala-Gly-Gly (AAGG), and evolved to efficiently assemble nanorods (Figure 32, SEQ ID NO: 18; Figure 33, SEQ ID NO: 20). LPETA-biotin was enzymatically attached to the nanorods using Streptococcus pyogenes sortase A, as described in the Methods section. Therefore, the modified nanorods are used in dot blots, ELISAs, and lateral flow assays, as described in the Methods section (Figures 25-27). Avidin-alkaline phosphatase or avidin-horseradish peroxidase fusions are used as secondary or indirect detection reagents for biotin-modified nanorods, allowing alkaline phosphatase- or horseradish peroxidase-mediated enzyme visualization using chromogenic or chemiluminescent substrates of these two enzymes (Figures 25-27).

[0172] The copy number and location of the displayed functionality, whether a heterologous protein or a "handle" for modification, will vary depending on the coat protein used as a platform. In one non-limiting example, using pVIII as a display platform allows for high-copy display of peptides along the shaft of nanorods. The copy number of displayed peptides (or other functionality) depends on the number of pVIII subunits per nanorod, which in turn depends on the length of the ssDNA scaffold. The copy number of functionality (fluorochrome, small molecule, or enzyme) depends on the length of the nanorod. For example, for a 1,000 nm long phage, approximately 400 copies of fluorophores or biotin are expected to be chemically or enzymatically attached per phage particle (Hess et al., 2012; Li et al., 2010). For labeling purposes, multiple different fluorescent dyes can be mixed to enable barcoding and other more complex methods and detection.

[0173] Using minor coat proteins as a platform, up to five copies can be displayed per nanorod (reviewed in Rakonjac et al., 2017 for each of pIII, pVII, and pIX). Furthermore, display on both pVII and pIX allows up to 10 copies per nanorod. By using different fusions or attachment molecules to different minor Ff phage coat proteins, it is possible to display various functions on a single nanorod, such as displaying two functions on one end (pVII-pIX end) and one function on the other end (pIII end). Such modifications have been demonstrated for full-length Ff phage, as is known in phage display technology.

[0174] Overcoming the toxicity of -pVIII and amber mutations Importantly, the major coat protein pVIII is toxic when expressed in E. coli without the phage assembly. This toxicity causes poor growth of transformed E. coli cells expressing pVIII, even when the gVIII promoter is removed by mutation during the cloning process or when expression is controlled by an inducible promoter. To overcome this problem, helper plasmids were constructed using gVIII suppressible (nonsense) mutants. The construction was carried out in an E. coli host without suppressor mutations, which prevented most of the translation of the pVIII protein. Two different amber (TAG) mutants were used. One contains a G-to-T mutation that converts codon 25 of GAG, which encodes glutamic acid at position 2 of the mature protein, to TAG (SEQ NO:13 - 24, Figures 32 - 33), and the other has the TCT codon 4 of serine within the signal sequence replaced by TAG (SEQ NO:25 - 28, Figure 33). The suppressor D mutation (supD) of serine tRNA was used to suppress these two amber mutations, and a strain of E. coli containing this mutation was used to generate nanorods (Table 1).

[0175] The gVIII suppressible amber mutants described here have the additional advantage that the pVIII produced intracellularly is reduced compared to E. coli cells expressing wild-type gVIII. This reduction is due to the lower translation efficiency of suppressor tRNAs compared to cognate tRNAs that read sense codons, which favors the assembly of shorter nanorods over longer ones by reducing the ratio of shaft protein pVIII to end-cap proteins pIII, pVI, pVII, pIX.

[0176] BSF nano-replication assembly plasmid (pBSF nano series) The components of the BSF nano-replication assembly plasmid used in the two-plasmid system are the BSF nano-replication cassette, the plasmid replication origin, and the selection marker.

[0177] BSF nanoreplication assembly cassette variants are equivalent to those described for pop-up plasmids (e.g., BSFp and BSFpn). A "filler" nucleic acid sequence of a predetermined length can be inserted between (+)Ori1 and (+)Ori2 to construct nanorods of a specific length, as described herein (Figures 5, 6, 9, 40, 42, 44, 46, 48, 57, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:56; SEQ ID NO:60, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:75; SEQ ID NO:82, SEQ ID NO:85; SEQ ID NO:86, SEQ ID NO:104). In some embodiments, no filler nucleic acid sequence is inserted.

[0178] In one non-limiting example, a BSF nanoreplicative assembly cassette in the pBSF plasmid series is a combination of the following units:

[0179] - Initiator (+)ori1, a functional positive strand replication origin (+)ori that allows binding of replication protein pII (a DNA strand transferase) and cleavage of the (+)strand to form a primer (Figures 4-6, 39-48, 56-57, SEQ ID NO:45, SEQ ID NO:65, SEQ ID NO:81; SEQ ID NO:103) -The packaging signal (PS) required to target the (+)-strand circular ssDNA replicated from the BSF nanoreplication assembly cassette to the trans-envelope assembly machinery for nanorod assembly (Figures 4-6, 40, 42, 44, 46, 48, 57, SEQ ID NO:48, SEQ ID NO:68, SEQ ID NO:76, SEQ ID NO:83, SEQ ID NO:84). -(-) Ori, allowing replication of the negative strand using the short BSF nano (+) strand ssDNA as a template, increasing the copy number of the (+) strand circular ssDNA generated from the BSF nanoreplication assembly cassette (Figures 4-6, 40, 42, 44, 46, 57, SEQ ID NO: 50).

[0180] -terminator ((+)Ori2), which cleaves the template (+) strand where replication initiated at (+)Ori1 and ligates both ends of the (+) strand to generate a truncated (+)Ori mutant (△29) that can generate a (+) strand circular ssDNA that serves as the backbone for nanorod assembly described herein (Figures 4-6, 40, 42, 44, 46, 48, 57, SEQ ID NO:51, SEQ ID NO:69, SEQ ID NO:87).

[0181] Characterization and variants of the -BSF nanoreplicative assembly cassette The initiator, (+)Ori, is either the minimal or core domain of (+)Ori (A or I) alone (Figures 4-6, 46, 48; SEQ ID NO:74, SEQ ID NO:81), or the complete (+)Ori (both A and B domains) (Figures 4-6, 40, 42, 44, 57; SEQ ID NO:45, SEQ ID NO:65, SEQ ID NO:103), the latter being more efficient for initiation than the former due to the presence of the complete pII-binding sequence.

[0182] The length of the nanorods generated is determined by the size of the scaffold nucleic acid sequence included in the BSF nanoreplication assembly cassette described herein. The scaffold nucleic acid sequence is located between the first pII nick site of (+)Ori1 and the second pII nick site (gttcttt(AATA) (SEQ ID NO: 88) of (+)Ori2, which are included in the BSF nanoreplication assembly cassette (Figures 4-6, 39-48, 56-57, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 101). BSFp replication assembly cassette For example, a BSF nanoreplication assembly cassette (here named BSFp) consisting of the initiator (+)Ori1 (containing only the (+)Ori core (or domain A)), the packaging signal, and the terminator (+)Ori2 corresponding to (+)OriΔ29 generates 152- or 221-nt circular (+)ssDNA and the assembly of nanorods 40 or 50 nm in length, respectively (Figures 1, 4-6, 47-48, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:77, SEQ ID NO:78). The 40-nm nanorods are the shortest Ff-derived nanorods generated to date.

[0183] BSFpn replication assembly cassette In another example, a replication assembly cassette, designated BSFpn, contains a combination of an initiator ((+)Ori1), a packaging signal (-)Ori, and a terminator ((+)Ori△29) corresponding to the complete (+)Ori (domain AB). In the presence of pII, this replication assembly cassette replicates (+)-strand ssDNA of 395, 529, 707, 711, 728, and 748 nt and nanorods of 70, 80, 100, or 110 nm in length (Figures 1, 4-6, 39-44, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:61, SEQ ID NO:63). Inserting a DNA sequence between (+)Ori1 and PS allows the generation of longer BSF nanorods.

[0184] Another variation of the BSFpn replication assembly cassette is one in which the (+)Ori contains only the core (+)Ori (Domain A) as the initiator, but also contains the (-)Ori (Figures 45 and 46, SEQ ID NO:70, SEQ ID NO:72; SEQ ID NO:74). The ssDNA generated from such BSFpn cassettes is 313 or 289 nt, resulting in nanorods with calculated lengths of 57 or 54 nm (approximately 50-60 nm), respectively.

[0185] Scalability of BSF nanorods In both the single- and two-plasmid NPSs described herein, a scaffold nucleic acid sequence is included between (+)Ori1 (initiator) and (+)Ori2 (terminator, Figures 1, 4-6, 39-48) at the pII cleavage site ((GTTCTTTT(AATA) (SEQ ID NO: 88, Figure 49)) within the BSF nanoreplication assembly cassette. One of skill in the art will recognize, based on the disclosure herein and as known in the art, that a scaffold nucleic acid sequence of an appropriate size to generate nanorods and / or multiple nanorods of a desired size can be readily selected for use in an NPS, as described herein.

[0186] As previously mentioned, the length of the (+)-strand circular ssDNA backbone (scaffold) generated by rolling circle replication of the BSF nanoreplication assembly cassette is determined by the number of nucleotides between the pII cleavage sites of (+)Ori1 (initiator) and (+)Ori2 (terminator). To reduce the size of the nanorods, the length of the scaffold nucleic acid sequence can be shortened. To achieve this, the (-)Ori is removed as was done in the BSFp replication assembly cassette, the filler sequence is completely removed, and the size of (+)Ori1 and (+)Ori2 in the BSFpn replication assembly cassette is reduced (Table 9, e.g., Figures 43 and 44, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:66 vs SEQ ID NO:67; Figures 45 and 46, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73; SEQ ID NO:74 vs SEQ ID NO:65; Figures 47 and 48, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:74). Conversely, the length of nanorods can be extended by inserting "filler" nucleic acid sequences between the initiator ((+)Ori1) and PS, between the PS and (-)Ori of BSFpn, or between the PS and (+)Ori2 of the BSFp replication assembly cassette (Figure 5B and C, Figure 6, Figures 56-57, SEQ ID NO: 104). Consequently, by designing filler nucleic acid sequences of appropriate length, nanorods can be extended to the desired length. Based on structural analysis of the Ff phage shaft, it can be accurately calculated that the length of nanorods increases by 0.133 nm for each nucleotide added to the ssDNA genome (Newman et al., 1977).

[0187] Protein-coding genes within the replicate assembly cassette Also herein, the filler nucleic acid sequence can encode a second copy of gVIII, used as a platform for the expression of pVIII fusions to longer peptides or proteins (Figure 6B, Figure 9, block i). Alternatively, the second copy of gVIII can be encoded on a compatible plasmid and provide the Ff phage protein, which is incorporated into the generated nanorods, as is commonly done in phage display technology. Examples of expression from BSF replicative assembly cassettes are shown by the expression of pVII and pIX from said cassettes (Figure 6B, Figure 41, SEQ ID NOs: 52-55). In addition to the Ff protein expressed in E. coli, the filler sequence can be used to accommodate eukaryotic gene expression cassettes.

[0188] Plasmid replication origin and selectable marker For nanorod replication assembly plasmids, any theta replication plasmid origin can be used as long as it is compatible with the plasmid origin of the helper plasmid, such as MB1 or ColEI for the pBSF nanoreplication assembly plasmid, or pA15 for the pHP helper plasmid (Figure 9, block iii).

[0189] The selectable marker for maintenance after transformation of the nanoreplicate assembly plasmid into E. coli (Figure 9, block ii) can be an antibiotic selectable marker, as long as it is different from the marker on the helper plasmid (e.g., the ampicillin resistance marker β-lactamase-encoding bla gene). Alternatively, an auxotrophic marker (e.g., nadC) can be used. 6 This also avoids the generation of nanorods containing antibiotic resistance, which is detected at a low frequency. These rare antibiotic resistance-encoding nanorods, which comprise the entire nanorod replication assembly plasmid described herein, are the result of aborted termination at (+)Ori2 or recombination between (+)Ori1 and (+)Ori2, resulting in the presence of a single positive replication origin. In certain preferred embodiments of the invention provided herein, the selectable marker on the nanorod replication plasmid is an auxotrophic marker, as described herein.

[0190] Additional plasmids Furthermore, it is contemplated herein that additional plasmids can be used to supply secondary copies of coat proteins if the inserted heterologous sequence prevents nanorod assembly in the absence of the wild-type counterpart. These additional plasmids contain origins of replication (e.g., chloramphenicol resistance markers (cat, Cm)) that are compatible with both the helper plasmid (pHP series) and the nanorod replicating plasmid (pBSF nano series) within the two-plasmid system. R ) and the ColD origin of replication).

[0191] In a first aspect, the present invention relates to a nanorod generating system (NPS) comprising a single nucleic acid expression construct, the construct comprising: a BSF nanoreplication assembly cassette; at least one auxotrophic marker; at least one inducible promoter operably linked to a nucleic acid sequence encoding at least one Ff phage protein; and and at least one plasmid origin of replication that is not present in the BSF nanoreplication assembly cassette.

[0192] In one embodiment, the nucleic acid expression construct is a vector or is contained within a vector. In one embodiment, the nucleic acid expression construct is a vector.

[0193] In one embodiment, the vector is selected from the group consisting of a plasmid, a bacterial artificial chromosome (BAC), a P1-derived artificial chromosome (PAC), a yeast artificial chromosome (YAC), a bacteriophage, a phagemid, and a cosmid. In one embodiment, the vector is a plasmid.

[0194] In one embodiment, the nucleic acid expression construct is or is contained in a plasmid. In one embodiment, the nucleic acid expression construct is a plasmid.

[0195] In one embodiment, the BSF nanoreplication assembly cassette comprises at least two (+) oligonucleotides. In one embodiment, the BSF nanoreplication assembly cassette comprises at least one (-) oligonucleotide. In terms of morphology, the BSF nanoreplication assembly cassette consists of two (+) Ori and one (-) Ori.

[0196] In one embodiment, one (+) ori is a DNA replication initiator. A (+) ori that is a DNA replication initiator is referred to herein as (+) ori 1. In one embodiment, one (+) ori is a DNA replication terminator. A (+) ori that is a DNA replication terminator is referred to herein as (+) ori 2.

[0197] In one embodiment, one (+) Ori is a DNA replication initiator ("(+) Ori 1") and one (+) Ori is a DNA replication terminator ("(+) Ori 2"). In one embodiment, the BSF nanoreplication assembly cassette comprises (+) Ori 1, (+) Ori 2, and one (-) Ori.

[0198] In one embodiment, the BSF nanoreplication assembly cassette comprises a packaging signal (PS). In one embodiment, the PS is between (+)Ori1 and (+)Ori2. In one embodiment, the PS is between (+)Ori1 and (-)Ori. In one embodiment, (+)Ori1 and (+)Ori2 comprise a pII cleavage site.

[0199] In one embodiment, the BSF nanoreplication assembly cassette comprises a scaffold nucleic acid sequence.

[0200] In one embodiment, the BSF nanoreplication assembly cassette comprises a scaffold nucleic acid sequence and flanking sequences required for (+) strand replication.

[0201] In one embodiment, the flanking sequences are located upstream of the pII cleavage site in ori (1) and downstream of the pII cleavage site in ori (2). In one embodiment, the flanking nucleic acid sequences bind to pII and / or bind to modified pII.

[0202] In one embodiment, the scaffold nucleic acid sequence is positioned between (+)Ori1 and (+)Ori2. In one embodiment, the scaffold nucleic acid sequence is positioned between the pII cleavage sites of (+)Ori1 and (+)Ori2.

[0203] In one embodiment, the scaffold nucleic acid sequence is placed between the (+)Ori1 (initiator) and (+)Ori2 (terminator) sequences (GTTCTTAATA, SEQ ID NO:88, Figure 49).

[0204] In one embodiment, the scaffold nucleic acid sequence is arranged in a BSF nanoreplication assembly cassette, as shown in FIGS.

[0205] In one embodiment, replication of the scaffold nucleic acid sequence in the presence of pII generates circular ssDNA.

[0206] In one embodiment, the scaffold nucleic acid sequence does not include a filler nucleic acid sequence. In one embodiment, the scaffold nucleic acid sequence includes at least one filler nucleic acid sequence. In one embodiment, the scaffold nucleic acid sequence includes two filler nucleic acid sequences.

[0207] In one embodiment, the scaffold nucleic acid sequence comprises a filler nucleic acid sequence that comprises an additional nucleic acid sequence arranged to extend the length of the (+) strand ssDNA produced by replication of the scaffold nucleic acid sequence.

[0208] In one embodiment, the filler nucleic acid sequence is arranged as shown in "Filler" in FIGS.

[0209] In one embodiment, the scaffold nucleic acid sequence comprises a filler nucleic acid sequence disposed between (+)Ori1 and PS. In one embodiment, the scaffold nucleic acid sequence comprises a filler nucleic acid sequence disposed between PS and (+)Ori2. In one embodiment, the scaffold nucleic acid sequence comprises filler nucleic acid sequences between (+)Ori1 and PS and between PS and (+)Ori2.

[0210] In one embodiment, the scaffold nucleic acid sequence comprises a filler nucleic acid sequence comprising 0 to about 6000 nt, 0 to about 5000, 0 to about 4000, 0 to about 3000, 0 to about 2000, 0 to about 1000, 0 to about 750, 0 to about 500, 0 to about 400, 0 to about 300, 0 to about 200, 0 to about 100, 0 to about 50, 0 to about 40, 0 to about 30, 0 to about 25, 0 to about 20, 0 to about 15, 0 to about 10, 0 to about 5, or 0 nt. In one embodiment, the scaffold nucleic acid sequence comprises a filler nucleic acid sequence comprising 0 to 6000 nt, 0 to 5000, 0 to 4000, 0 to 3000, 0 to 2000, 0 to 1000, 0 to 750, 0 to 500, 0 to 400, 0 to 300, 0 to 200, 0 to 100, 0 to 50, 0 to 40, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, 0 to 5, or 0 nt.

[0211] In one embodiment, the scaffold nucleic acid sequence comprises a filler nucleic acid sequence comprising 0, 5, 23, 24, 31, 145, 315, 319, 336, 356, 700, 1400, or 2100 nucleotides. In one embodiment, the filler nucleic acid sequence comprises, consists essentially of, or consists of a filler nucleic acid sequence identified in Table 9. The skilled worker will appreciate that the size of the filler can be varied to accommodate the production of nanorods of various sizes depending on the length (i.e., number of nucleotides) of other functional sequence elements, including (+)Ori1, (-)Ori, and (+)Ori2, of the scaffold nucleic acid sequence.

[0212] In one embodiment, the single nucleic acid construct comprises SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:77, SEQ ID NO:79 (Figures 39-48) or SEQ ID NO:101 (Figure 56).

[0213] In one embodiment, the scaffold nucleic acid sequence comprises a filler nucleic acid sequence encoding at least one, preferably at least two, Ff phage coat and / or modified coat proteins. In one embodiment, at least one coat and / or modified coat protein is pVII or pIX. In one embodiment, at least two coat and / or modified coat proteins are pVII and pIX.

[0214] In one embodiment, the at least two coat and / or modified coat proteins are operably linked to a promoter. In one embodiment, the promoter is a constitutive or inducible promoter. In one embodiment, the promoter is a constitutive promoter. In one embodiment, the promoter is an inducible promoter. In one embodiment, the constitutive promoter is a phage promoter, preferably pA. In one embodiment, the inducible promoter is selected from the group consisting of lac, tac, araC, or trp promoters, preferably a lac promoter. In one embodiment, the lac promoter is a lac promoter controlled by an inducing agent (IPTG). In one embodiment, the lac promoter mutant is susceptible to repression by glucose (catabolite repression). In one embodiment, the lac promoter is a lac promoter (Figure 42, SEQ ID NO:58).

[0215] In one embodiment, the lac promoter is a lac promoter mutant that is regulated only by an inducing agent (IPTG). In one embodiment, the lac promoter mutant is not subject to repression by glucose (catabolite repression).

[0216] In one embodiment, the lac promoter is the lacUV5 promoter (Figure 49, SEQ ID NO:90). In one embodiment, multiple replicated (+) strand circular ssDNA molecules are generated by enzymatic replication of the scaffold nucleic acid sequence. In one embodiment, the enzymatic replication is rolling circle replication.

[0217] In one embodiment, the replicated (+) strand ssDNA binds to at least one Ff phage coat protein or Ff phage modified coat protein, or both. In one embodiment, the replicated (+) strand ssDNA binds to multiple different Ff phage coat proteins and / or Ff phage modified coat proteins.

[0218] In one embodiment, the replicated (+) strand ssDNA is bound by at least one Ff phage coat protein, at least one modified Ff phage coat protein, and / or a plurality of different Ff phage coat proteins and / or modified coat proteins within a plurality of nanorods.

[0219] In one embodiment, the replicated (+) strand ssDNA sequence comprises 152-221 nucleotides (Figures 47-48, SEQ ID NO:80, SEQ ID NO:78). In one embodiment, the replicated (+) strand ssDNA comprises, consists of, or consists essentially of 152 nt.

[0220] In one embodiment, the replicated (+) strand ssDNA comprises 289, 313, 395, 529, 707, 711, 728, 748 nt or 1400 nt (Figure 45, SEQ ID NO:73, SEQ ID NO:71, Figure 43, SEQ ID NO:63, SEQ ID NO:61, Figure 41, SEQ ID NO:55, SEQ ID NO:53, Figure 39, SEQ ID NO:44, SEQ ID NO:42, Figure 56, SEQ ID NO:102, Table 9).

[0221] In one embodiment, the at least one auxotrophic marker is selected from the group consisting of metE, glyA, infA, thyA, argE, delta-thi-1, thi1, leuB, proAB, ara, and nadC. In one embodiment, the at least one auxotrophic marker comprises nadC (Figure 50, SEQ ID NO:91, SEQ ID NO:93). In one embodiment, the at least one inducible promoter is selected from the group consisting of lac, tac, araC, or trp promoters. In one embodiment, the at least one inducible promoter is a lac promoter. In one embodiment, the lac promoter is a lac promoter mutant that is controlled only by an inducing agent (IPTG). In one embodiment, the lac promoter mutant is not susceptible to repression by glucose (catabolite repression). In one embodiment, the lac promoter comprises the lacUV5 promoter (Figure 49, SEQ ID NO:90).

[0222] In one embodiment, at least one inducible promoter is operably linked to a nucleic acid sequence encoding at least one Ff phage replication protein or at least one Ff phage coat protein, or both.

[0223] In one embodiment, at least one inducible promoter is operably linked to a nucleic acid sequence encoding at least one Ff phage protein selected from the group consisting of pII, pV, pVII, pVIII, and pIX.

[0224] In one embodiment, at least one inducible promoter is operably linked to a nucleic acid sequence encoding Ff phage proteins pII, pV, pVII, pVIII, and pIX.

[0225] In one embodiment, at least one Ff phage replication protein is pII.

[0226] In one embodiment, the amino acid sequence of pII comprises, consists of, or consists essentially of SEQ ID NO:1 (Figure 29). In one embodiment, the nucleic acid sequence encoding pII has at least 70%, 80%, 90%, 95%, or 99% nucleic acid sequence identity to SEQ ID NO:2 (Figure 29). In one embodiment, the nucleic acid sequence encoding pII comprises, consists of, or consists essentially of SEQ ID NO:2 (Figure 29).

[0227] In one embodiment, the at least one Ff phage coat protein is pVIII.

[0228] In one embodiment, the amino acid sequence of pVIII comprises, consists of, or consists essentially of SEQ ID NO:11 (Figure 32). In one embodiment, the nucleic acid sequence encoding pVIII has at least 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:12 (Figure 32). In one embodiment, the nucleic acid sequence encoding pVIII comprises, consists of, or consists essentially of SEQ ID NO:12 (Figure 32).

[0229] In one embodiment, the inducible promoter is operably linked to a nucleic acid sequence encoding at least one modified Ff phage replication protein or at least one modified Ff phage coat protein, or both.

[0230] In one embodiment, at least one modified Ff phage replication or coat protein comprises at least one amino acid addition, deletion or substitution compared to the corresponding wild-type Ff phage coat protein.

[0231] In one embodiment, the inducible promoter is operably linked to a nucleic acid sequence encoding at least one modified Ff phage replication protein. In one embodiment, the modified Ff phage-encoded replication protein is a modified pII protein.

[0232] In one embodiment, the amino acid sequence of the modified pII protein comprises, consists of, or consists essentially of SEQ ID NO:3, which comprises a Thr182IIe amino acid change compared to wild-type pII (Figure 30).

[0233] In one embodiment, the nucleic acid sequence encoding the modified pII protein comprises, consists of, or consists essentially of SEQ ID NO:4, which includes a C545T change. The skilled worker will understand that the C545T change is recognized by counting from the ATG start codon of the nucleic acid sequence encoding the modified pII protein.

[0234] In one embodiment, the inducible promoter is operably linked to a nucleic acid sequence encoding at least one modified Ff phage coat protein. In one embodiment, the at least one modified Ff phage coat protein is modified pVIII.

[0235] In one embodiment, the modified pVIII comprises at least one amber mutation. In one embodiment, the amino acid sequence of the modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 13. In one embodiment, the amino acid sequence of the modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 19.

[0236] In one embodiment, the amino acid sequence of pV comprises, consists of, or consists essentially of SEQ ID NO:5 (Figure 31). In one embodiment, the nucleic acid sequence encoding pV has at least 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:6 (Figure 31). In one embodiment, the nucleic acid sequence encoding pV comprises, consists of, or consists essentially of SEQ ID NO:6 (Figure 31).

[0237] In one embodiment, the amino acid sequence of pVII comprises, consists of, or consists essentially of SEQ ID NO:7 (Figure 31). In one embodiment, the nucleic acid sequence encoding pVII has at least 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:8 (Figure 31). In one embodiment, the nucleic acid sequence encoding pVII comprises, consists of, or consists essentially of SEQ ID NO:8 (Figure 31).

[0238] In one embodiment, the amino acid sequence of pIX comprises, consists of, or consists essentially of SEQ ID NO:9 (Figure 31). In one embodiment, the nucleic acid sequence encoding pIX has at least 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:10 (Figure 31). In one embodiment, the nucleic acid sequence encoding pIX comprises, consists of, or consists essentially of SEQ ID NO:10 (Figure 31).

[0239] In one embodiment, a single nucleic acid expression construct comprises nucleic acid sequences encoding at least one additional Ff phage protein, preferably at least two additional Ff phage proteins.

[0240] In one embodiment, the nucleic acid sequence encoding at least one additional Ff phage protein is operably linked to a promoter. In one embodiment, the promoter is an inducible promoter or a constitutive promoter, preferably the promoter is a constitutive promoter, preferably pZ.

[0241] In one embodiment, the additional Ff phage protein is selected from the group consisting of pill and pVI, hi one embodiment, the additional Ff phage protein is pill or pVI or both.

[0242] In one embodiment, the amino acid sequence of pIII comprises, consists of, or consists essentially of SEQ ID NO:29 (Figure 34). In one embodiment, the nucleic acid sequence encoding pIII has at least 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:30 (Figure 34). In one embodiment, the nucleic acid sequence encoding pIII comprises, consists of, or consists essentially of SEQ ID NO:30 (Figure 34).

[0243] In one embodiment, the amino acid sequence of the modified pill comprises, consists of, or consists essentially of SEQ ID NO:31 (Figure 35). In one embodiment, the nucleic acid sequence encoding pill has at least 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:32 (Figure 35). In one embodiment, the nucleic acid sequence encoding the modified pill comprises, consists of, or consists essentially of SEQ ID NO:32 (Figure 35).

[0244] In one embodiment, the amino acid sequence of the modified pill comprises, consists of, or consists essentially of SEQ ID NO:33 (Figure 36). In one embodiment, the nucleic acid sequence encoding the modified pill has at least 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:34 (Figure 36). In one embodiment, the nucleic acid sequence encoding the modified pill comprises, consists of, or consists essentially of SEQ ID NO:34 (Figure 36).

[0245] In one embodiment, the amino acid sequence of pVI comprises, consists of, or consists essentially of SEQ ID NO:35 (Figure 36). In one embodiment, the nucleic acid sequence encoding pVI has at least 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:36 (Figure 36). In one embodiment, the nucleic acid sequence encoding pVI comprises, consists of, or consists essentially of SEQ ID NO:36 (Figure 36).

[0246] In one embodiment, the nucleic acid expression construct comprises a nucleic acid sequence encoding a fusion protein comprising at least one Ff phage protein or modified Ff phage protein or functional portion thereof fused to a binding protein or binding portion thereof, in one embodiment, the Ff phage protein or modified Ff phage protein or functional portion thereof is an Ff phage coat or modified Ff phage coat protein or functional portion thereof.

[0247] In one embodiment, the nucleic acid sequence encoding the fusion protein comprises a first nucleic acid coding sequence encoding at least one Ff phage protein or at least one modified Ff phage protein.

[0248] In one embodiment, the nucleic acid sequence encoding the fusion protein comprises a second nucleic acid coding sequence, and expression of the first and second nucleic acid sequences produces the fusion protein.

[0249] In one embodiment, the second nucleic acid coding sequence encodes a protein or functional portion thereof to be displayed on the surface of the nanorod, hi one embodiment, the second nucleic acid sequence encodes an antibody or antigen-binding portion thereof, or a binding protein or binding portion thereof.

[0250] In one embodiment, the antibody or antigen-binding portion thereof is selected from the group consisting of a SARSCoV-2 spike-specific single chain antibody (preferably C121), a SARSCoV-2 nucleocapsid-specific antigen-binding fragment of a heavy chain-only antibody (VHH) (preferably N3 (VHHN3)), and a botulinum neurotoxin-specific VHH.

[0251] In one embodiment, the binding protein or binding portion thereof is selected from the group consisting of the FnB fibronectin binding domain of Streptococcus pyogenes M type 22 protein Sof, the botulinum toxin binding domain of synaptic vesicle glycoprotein 2C (SV2C), and a SARS-CoV-2 spike (S) or matrix (M) derived peptide that interacts with the SARS-CoV-2 nucleocapsid protein (N).

[0252] In one embodiment, the first nucleic acid sequence comprises, consists essentially of, or consists of modified gill (SEQ ID NO:32, Figure 35).

[0253] In one embodiment, the nucleic acid sequence encoding the fusion protein comprises, consists essentially of, or consists of a nucleic acid sequence encoding the single-chain variable domain of antibody C121 (scFvC121) fused to a nucleic acid sequence encoding full-length pIII (SEQ ID NO:40, Figure 38). In one embodiment, the fusion protein comprises SEQ ID NO:40.

[0254] In one embodiment, the nucleic acid sequence encoding the fusion protein comprises, consists essentially of, or consists of a nucleic acid sequence encoding an antigen-binding fragment of the heavy chain-only antibody N3 (VHHN3) fused with a nucleic acid sequence encoding full-length pIII (SEQ ID NO:1) (SEQ ID NO:100, Figure 55). In one embodiment, the fusion protein comprises SEQ ID NO:99.

[0255] In one embodiment, the nucleic acid sequence encoding the fusion protein comprises, consists essentially of, or consists of the nucleic acid coding sequence of the FnB fibronectin binding domain of Streptococcus pyogenes M type 22 protein Sof fused to the full-length gill coding sequence (SEQ ID NO:1) (SEQ ID NO:38, Figure 37). In an embodiment, the fusion protein comprises SEQ ID NO:37 (Figure 37). Those skilled in the art will appreciate that the amino acid sequence of any of pIII, pVI, pVII, pVIII, or pIX can be modified for peptide display purposes as described herein and as known in the art, and all such modifications are contemplated herein and, when combined with the disclosure herein, are considered to be within the skill of the art.

[0256] In one embodiment, the inducible promoter is operably linked to a first operon comprising, consisting of, or consisting essentially of the Ff phage genes gII (gX), gV, gVII, gIX, and gVIII.

[0257] In one embodiment, the Ff phage genes gII (gX), gV, gVII, gIX and gVIII have at least 70%, 80%, 90%, 95% or 99% nucleic acid sequence identity to one of SEQ ID NO:4 (Figure 30), SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 (Figure 31), and SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 (Figure 32), SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26 or SEQ ID NO:28 (Figure 33), SEQ ID NO:98 (Figure 54), respectively.

[0258] In one embodiment, the Ff phage genes gII (gX), gV, gVII, gIX and gVIII comprise, consist of, or consist essentially of one of SEQ ID NO:4 (Figure 30), SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 (Figure 31) and SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 (Figure 32), SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26 or SEQ ID NO:28 (Figure 33), SEQ ID NO:98 (Figure 54), respectively.

[0259] In one embodiment, the Ff phage genes gIII and gVIII are modified to encode modified Ff phage coat proteins pIII and pVIII, respectively.

[0260] In one embodiment, the modified pVIII comprises at least one amber mutation.

[0261] In one embodiment, the amino acid sequence of modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:13 (Figure 32). In one embodiment, the amino acid sequence of modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:15 (Figure 32). In one embodiment, the amino acid sequence of modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:17 (Figure 32). In one embodiment, the amino acid sequence of modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:19 (Figure 32). In one embodiment, the amino acid sequence of modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:21 (Figure 33). In one embodiment, the amino acid sequence of modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:23 (Figure 33). In one embodiment, the amino acid sequence of the modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:25 (Figure 33). In one embodiment, the amino acid sequence of the modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:27 (Figure 33). In one embodiment, the amino acid sequence of the modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:97 (Figure 54).

[0262] In one embodiment, at least one plasmid origin of replication (p-ori) is a theta origin of plasmid replication. In one embodiment, the p-ori is selected from the group consisting of ColE1, pMB1, pSC101, R6K, ColD, and 15A. In one untranslated (0%) embodiment, the p-ori is 15A.

[0263] In one embodiment, the nucleic acid construct comprises a second operon comprising, consisting of, or consisting essentially of the Ff phage genes gIII, gVI, gI (gXI), and gIV. In one embodiment, the second operon is operably linked to a constitutive or inducible promoter, preferably a constitutive promoter, preferably an inducible promoter. In one embodiment, the inducible promoter is as described herein for the NPS aspect of the invention.

[0264] In one embodiment, the Ff phage gene gIII has at least 70%, 80%, 90%, 95%, or 99% nucleic acid sequence identity to SEQ ID NO:30 (Figure 34), SEQ ID NO:32 (Figure 35), or SEQ ID NO:34 (Figure 36). In one embodiment, the Ff phage gene gIII comprises, consists of, or consists essentially of SEQ ID NO:30 (Figure 34), SEQ ID NO:32 (Figure 35), or SEQ ID NO:34 (Figure 36).

[0265] In one embodiment, the Ff phage gene gVI has at least 70%, 80%, 90%, 95%, or 99% nucleic acid sequence identity to SEQ ID NO: 36 (Figure 36). In one embodiment, the Ff phage gene gVI comprises, consists of, or consists essentially of SEQ ID NO: 36 (Figure 36).

[0266] In a second aspect, the present invention provides a method for producing a pharmaceutical composition comprising: i) a nucleic acid replicative assembly construct, BSF nanoreplication assembly cassette, at least one auxotrophic marker, and a nucleic acid replication assembly construct comprising at least one plasmid origin of replication that is not present in the BSF nanoreplication assembly cassette; ii) a helper nucleic acid expression construct, at least one selectable marker, and The present invention relates to a nanorod generating system (NPS) comprising a helper nucleic acid expression construct comprising at least one inducible promoter operably linked to a nucleic acid sequence encoding at least one Ff phage protein.

[0267] In one embodiment, the nucleic acid replication construct of i) is a vector or is included in a vector. In one embodiment, the nucleic acid replication construct of i) is a vector. In one embodiment, the vector is selected from the group consisting of a plasmid, a bacterial artificial chromosome (BAC), a P1-derived artificial chromosome (PAC), a yeast artificial chromosome (YAC), a bacteriophage, a phagemid, and a cosmid. In one embodiment, the vector is a plasmid.

[0268] In one embodiment, the nucleic acid expression construct in i) is a plasmid or is contained in a plasmid. In one embodiment, the nucleic acid replication construct in i) is a plasmid. In this embodiment, the plasmid is referred to as a BSF nanoreplication assembly plasmid.

[0269] In one embodiment, the helper nucleic acid expression construct of ii) is a vector or is contained in a vector. In one embodiment, the helper nucleic acid expression construct of ii) is a vector. In one embodiment, the vector is selected from the group consisting of a plasmid, a bacterial artificial chromosome (BAC), a P1-derived artificial chromosome (PAC), a yeast artificial chromosome (YAC), and a cosmid. In one embodiment, the vector is a plasmid.

[0270] In one embodiment, the helper nucleic acid expression construct of ii) is or is contained in a plasmid. In one embodiment, the helper nucleic acid expression construct of ii) is a plasmid. In this embodiment, the plasmid is referred to as a helper plasmid.

[0271] In one embodiment, the BSF nanoreplication assembly cassette comprises at least two (+) oligonucleotides. In one embodiment, the BSF nanoreplication assembly cassette comprises at least one (-) oligonucleotide. In one embodiment, the BSF nanoreplication assembly cassette is comprised of two (+) oligonucleotides and one (-) oligonucleotide.

[0272] In one embodiment, one (+) ori is a DNA replication initiator. A (+) ori that is a DNA replication initiator is referred to herein as (+) ori 1. In one embodiment, one (+) ori is a DNA replication terminator. A (+) ori that is a DNA replication terminator is referred to herein as (+) ori 2.

[0273] In one embodiment, one (+) Ori is a DNA replication initiator ("(+) Ori 1") and one (+) Ori is a DNA replication terminator ("(+) Ori 2"). In one embodiment, the BSF nanoreplication assembly cassette comprises (+) Ori 1, (+) Ori 2, and one (-) Ori.

[0274] In one embodiment, the BSF nanoreplication assembly cassette comprises a packaging signal (PS). In one embodiment, the PS is between (+)Ori1 and (+)Ori2. In one embodiment, the PS is between (+)Ori1 and (-)Ori. In one embodiment, (+)Ori1 and (+)Ori2 comprise a pII cleavage site.

[0275] In one embodiment, the BSF nanoreplication assembly cassette comprises a scaffold nucleic acid sequence. In one embodiment, the BSF nanoreplication assembly cassette comprises a scaffold nucleic acid sequence and flanking sequences required for (+) strand replication.

[0276] In one embodiment, the scaffold nucleic acid sequence is positioned between (+)Ori1 and (+)Ori2. In one embodiment, the scaffold nucleic acid sequence is located between the pII cleavage sites of (+)Ori1 and (+)Ori2.

[0277] In one embodiment, the scaffold nucleic acid sequence is located between the (+)Ori1 (initiator) and (+)Ori2 (terminator) sequences [(GTTCTTAATA) (SEQ ID NO:88, Figure 49)]. In one embodiment, the scaffold nucleic acid sequence is located within a BSF nanoreplication assembly cassette, as shown in Figures 5 and 6.

[0278] In one embodiment, replication of the scaffold nucleic acid sequence in the presence of pII generates circular ssDNA.

[0279] In one embodiment, the scaffold nucleic acid sequence does not include a filler nucleic acid sequence. In one embodiment, the scaffold nucleic acid sequence includes at least one filler nucleic acid sequence. In one embodiment, the scaffold nucleic acid sequence includes two filler nucleic acid sequences.

[0280] In one embodiment, the scaffold nucleic acid sequence comprises a filler nucleic acid sequence, which comprises an additional nucleic acid sequence arranged to extend the length of the (+) strand ssDNA produced by replication of the scaffold nucleic acid sequence. In one embodiment, the filler nucleic acid sequence is arranged as shown in "Filler" in Figures 5 and 6.

[0281] In one embodiment, the scaffold nucleic acid sequence comprises a filler nucleic acid sequence positioned between (+)Ori1 and PS. In one embodiment, the scaffold nucleic acid sequence comprises a filler nucleic acid sequence located between PS and (+)Ori2. In one embodiment, the scaffold nucleic acid sequence comprises a filler nucleic acid sequence located between (+)Ori1 and PS and between PS and (+)Ori2.

[0282] In one embodiment, the scaffold nucleic acid sequence comprises a filler nucleic acid sequence comprising 0 to about 6000 nt, 0 to about 5000, 0 to about 4000, 0 to about 3000, 0 to about 2000, 0 to about 1000, 0 to about 750, 0 to about 500, 0 to about 400, 0 to about 300, 0 to about 200, 0 to about 100, 0 to about 50, 0 to about 40, 0 to about 30, 0 to about 25, 0 to about 20, 0 to about 15, 0 to about 10, 0 to about 5, or 0 nt. In one embodiment, the scaffold nucleic acid sequence comprises a filler nucleic acid sequence comprising 0 to 6000 nt, 0 to 5000, 0 to 4000, 0 to 3000, 0 to 2000, 0 to 1000, 0 to 750, 0 to 500, 0 to 400, 0 to 300, 0 to 200, 0 to 100, 0 to 50, 0 to 40, 0 to 30, 0 to 25, 0 to 20, 0 to 15, 0 to 10, 0 to 5, or 0 nt.

[0283] In one embodiment, the scaffold nucleic acid sequence comprises a filler nucleic acid sequence comprising 0, 5, 23, 24, 31, 145, 315, 319, 336, 356, 700, 1400, or 2100 nucleotides. In one embodiment, the filler nucleic acid sequence comprises, consists essentially of, or consists of a filler nucleic acid sequence identified in Table 9. A skilled artisan may vary the size of the filler depending on the length (i.e., number of nucleotides) of other functional sequence elements of the scaffold nucleic acid sequence, including (+)Ori1, (-)Ori, and (+)Ori2, to allow for the generation of nanorods of various sizes.

[0284] In one embodiment, the single nucleic acid construct comprises SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:77, SEQ ID NO:79 or SEQ ID NO:101 (Figures 39-48, 56-57).

[0285] In one embodiment, the scaffold nucleic acid sequence comprises a filler nucleic acid sequence encoding at least one, preferably at least two, Ff phage coat and / or modified coat proteins. In one embodiment, at least one coat and / or modified coat protein is pVII or pIX. In one embodiment, at least two coat and / or modified coat proteins are pVII and pIX.

[0286] In one embodiment, at least two Ff phage coat and / or modified coat proteins are operably linked to a promoter. In one embodiment, the promoter is a constitutive or inducible promoter. In one embodiment, the promoter is a constitutive promoter. In one embodiment, the promoter is an inducible promoter. In one embodiment, the constitutive promoter is a phage promoter, preferably pA. In one embodiment, the inducible promoter is selected from the group consisting of lac, tac, araC, or trp promoter.

[0287] In one embodiment, the promoter is a lac promoter. In one embodiment, the lac promoter is regulated by an inducer (IPTG). In one embodiment, the lac promoter is subject to repression by glucose (catabolite repression). In one embodiment, the lac promoter is a lac promoter (Figure 42, SEQ ID NO:58).

[0288] In one embodiment, the promoter is a lac promoter. In one embodiment, the lac promoter is a lac promoter mutant that is regulated only by an inducing agent (IPTG). In one embodiment, the lac promoter mutant is not susceptible to repression by glucose (catabolite repression). In one embodiment, the lac promoter is a lacUV5 promoter (Figure 49, SEQ ID NO:90).

[0289] In one embodiment, the BSF nanoreplication assembly cassette comprises a scaffold nucleic acid sequence comprising flanking nucleic acid sequences within (+)Ori 1 and (+)Ori 2. In one embodiment, the flanking nucleic acid sequences bind to pII and / or bind to modified pII.

[0290] In one embodiment, multiple replicated (+) strand circular ssDNA molecules are generated by enzymatic replication of the scaffold nucleic acid sequence. In one embodiment, the enzymatic replication is rolling circle replication.

[0291] In one embodiment, the replicated (+) strand ssDNA binds to at least one Ff phage coat protein or Ff phage modified coat protein, or both. In one embodiment, the replicated (+) strand ssDNA binds to multiple different Ff phage coat and / or modified coat proteins.

[0292] In one embodiment, the replicated (+) strand ssDNA is bound by at least one Ff phage coat protein, at least one modified Ff phage coat protein, and / or a plurality of different Ff phage coat and / or modified coat proteins within a plurality of nanorods.

[0293] In one embodiment, the replicated (+) strand ssDNA is 152-221 nucleotides (nt) (Figure 47, SEQ ID NO:80, SEQ ID NO:78). In one embodiment, the replicated (+) strand ssDNA comprises, consists of, or consists essentially of 152 nt.

[0294] In one embodiment, the replicated +ssDNA comprises 289, 313, 395, 529, 707, 711, 728, 748 or 1400 nt (Figure 45, SEQ ID NO:73, SEQ ID NO:71, Figure 43, SEQ ID NO:63, SEQ ID NO:61, Figure 41, SEQ ID NO:55, SEQ ID NO:53, Figure 39, SEQ ID NO:44, SEQ ID NO:42, Figure 56, SEQ ID NO:102, Table 9).

[0295] In one embodiment, the auxotrophic marker is selected from the group consisting of metE, glyA, infA, thyA, argE, delta-thi-1, thi1, leuB, proAB, ara, and nadC. In one embodiment, the auxotrophic marker is nadC (Figure 50, SEQ ID NO:91, SEQ ID NO:92).

[0296] In one embodiment, the plasmid replication origin (p-ori) in i) is a theta origin of plasmid replication. In one embodiment, the p-ori is selected from the group consisting of ColE1, pMB1, pSC101, R6K, ColD, and pA15. In one embodiment, the p-ori is pMB1.

[0297] In one embodiment, the helper plasmid in ii) comprises a plasmid replication origin. In one embodiment, the plasmid replication origin (p-ori) in ii) is a theta origin of plasmid replication. In one embodiment, the p-ori is selected from the group consisting of ColE1, pMB1, pSC101, R6K, ColD, and pA15.

[0298] In one embodiment, the at least one selectable marker in ii) is an antibiotic resistance or auxotrophic marker. In one embodiment, the at least one selectable marker is an antibiotic resistance marker. In one embodiment, the at least one selectable marker is an auxotrophic marker.

[0299] In one embodiment, the at least one inducible promoter in ii) is selected from the group consisting of lac, tac, araC, or trp promoters. In one embodiment, the at least one inducible promoter is a lac promoter. In one embodiment, the lac promoter is a lac promoter mutant that is controlled only by an inducing agent (IPTG). In one embodiment, the lac promoter mutant is not susceptible to repression by glucose (catabolite repression). In one embodiment, the lac promoter is a lacUV5 promoter (Figure 49, SEQ ID NO:90).

[0300] In one embodiment, the at least one inducible promoter of ii) is operably linked to a nucleic acid sequence encoding at least one Ff phage replication protein or at least one Ff phage coat protein, or both.

[0301] In one embodiment, the at least one inducible promoter of ii) is operably linked to a nucleic acid sequence encoding at least two Ff phage replication proteins or at least two Ff phage coat proteins, or both.

[0302] In one embodiment, the at least one inducible promoter is operably linked to a nucleic acid sequence encoding at least two Ff phage coat proteins, hi one embodiment, the at least two Ff phage coat proteins are minor coat proteins.

[0303] In one embodiment, the at least two minor coat proteins are pVII and pIX.

[0304] In one embodiment, at least one inducible promoter is operably linked to a nucleic acid sequence encoding at least one, and preferably at least two, Ff phage replication proteins.

[0305] In one embodiment, the inducible promoter is operably linked to a nucleic acid sequence encoding at least one Ff phage protein selected from the group consisting of pII, pV, pVII, pVIII, and pIX.

[0306] In one embodiment, the inducible promoter is operably linked to a nucleic acid sequence encoding Ff phage proteins pII, pV, pVII, pVIII, and pIX.

[0307] In one embodiment, at least one Ff phage replication protein is pII.

[0308] In one embodiment, the amino acid sequence of pII is SEQ ID NO:1 (Figure 29). In one embodiment, the nucleic acid sequence encoding pII has at least 70%, 80%, 90%, 95%, or 99% nucleic acid sequence identity to SEQ ID NO:2 (Figure 29). In one embodiment, the nucleic acid sequence encoding pII comprises, consists of, or consists essentially of SEQ ID NO:2 (Figure 29).

[0309] In one embodiment, the at least one Ff phage coat protein is pVIII.

[0310] In one embodiment, the amino acid sequence of pVIII comprises, consists of, or consists essentially of SEQ ID NO:211 (Figure 32). In one embodiment, the nucleic acid sequence encoding pVIII has at least 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:12 (Figure 32). In one embodiment, the nucleic acid sequence encoding pVIII comprises, consists of, or consists essentially of SEQ ID NO:12 (Figure 32).

[0311] In one embodiment, the inducible promoter is operably linked to a nucleic acid sequence encoding at least one modified Ff phage replication protein or at least one modified Ff phage coat protein, or both. In one embodiment, the at least one modified Ff phage replication or coat protein comprises at least one amino acid addition, deletion, or substitution compared to the corresponding wild-type Ff phage coat protein.

[0312] In one embodiment, the inducible promoter is operably linked to a nucleic acid sequence encoding at least one modified Ff phage replication protein. In one embodiment, the modified Ff phage-encoded replication protein is a modified pII protein.

[0313] In one embodiment, the amino acid sequence of the modified pII protein comprises, consists of, or consists essentially of SEQ ID NO:3, which comprises a Thr182IIe amino acid change compared to wild-type pII (Figure 30).

[0314] In one embodiment, the nucleic acid sequence encoding the modified pII protein comprises, consists of, or consists essentially of SEQ ID NO:4, which includes a C545T change. The skilled worker will understand that the C545T change is recognized by counting from the ATG start codon of the nucleic acid sequence encoding the modified pII protein.

[0315] In one embodiment, the inducible promoter is operably linked to a nucleic acid sequence encoding at least one modified Ff phage coat protein. In one embodiment, the at least one modified Ff phage coat protein is modified pVIII. In one embodiment, the modified pVIII comprises at least one amber mutation.

[0316] In one embodiment, the amino acid sequence of the modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO: 13. In one embodiment, the amino acid sequence of the modified pVIII comprises, consists of, or consists essentially of SEQ ID NO: 19.

[0317] In one embodiment, the amino acid sequence of pV comprises, consists of, or consists essentially of SEQ ID NO:5 (Figure 31). In one embodiment, the nucleic acid sequence encoding pV has at least 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:6 (Figure 31). In one embodiment, the nucleic acid sequence encoding pV comprises, consists of, or consists essentially of SEQ ID NO:6 (Figure 31).

[0318] In one embodiment, the amino acid sequence of pVII comprises, consists of, or consists essentially of SEQ ID NO:7 (Figure 31). In one embodiment, the nucleic acid sequence encoding pVII has at least 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:8 (Figure 31). In one embodiment, the nucleic acid sequence encoding pVII comprises, consists of, or consists essentially of SEQ ID NO:8 (Figure 31).

[0319] In one embodiment, the amino acid sequence of pIX comprises, consists of, or consists essentially of SEQ ID NO:9 (Figure 31). In one embodiment, the nucleic acid sequence encoding pIX has at least 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:10 (Figure 31). In one embodiment, the nucleic acid sequence encoding pIX comprises, consists of, or consists essentially of SEQ ID NO:10 (Figure 31).

[0320] In one embodiment, the helper plasmid ii) comprises a nucleic acid sequence encoding at least one additional Ff phage protein, preferably at least two additional Ff phage proteins. In one embodiment, the nucleic acid sequence encoding the at least one additional Ff phage protein is operably linked to a promoter. In one embodiment, the promoter is an inducible promoter or a constitutive promoter, preferably a constitutive promoter, preferably pZ.

[0321] In one embodiment, the additional Ff phage protein is selected from the group consisting of pill and pVI, hi one embodiment, the additional Ff phage protein is pill or pVI or both.

[0322] In one embodiment, the amino acid sequence of pill comprises, consists of, or consists essentially of SEQ ID NO:29 (Figure 34). In one embodiment, the nucleic acid sequence encoding pill has at least 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:30 (Figure 34). In one embodiment, the nucleic acid sequence encoding the modified pill comprises, consists of, or consists essentially of SEQ ID NO:30 (Figure 34).

[0323] In one embodiment, at least one modified Ff phage coat protein is a modified pill protein. In one embodiment, the modified pill comprises, consists essentially of, or consists of SEQ ID NO:31 (Figure 35). In one embodiment, the nucleic acid sequence encoding the modified pill has at least 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:32 (Figure 35). In one embodiment, the nucleic acid sequence encoding the modified pill comprises, consists of, or consists essentially of SEQ ID NO:32 (Figure 35).

[0324] In one embodiment, the amino acid sequence of the modified pill comprises, consists of, or consists essentially of SEQ ID NO:33 (Figure 36). In one embodiment, the nucleic acid sequence encoding the modified pill has at least 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:34 (Figure 36). In one embodiment, the nucleic acid sequence encoding the modified pill comprises, consists of, or consists essentially of SEQ ID NO:34 (Figure 36).

[0325] In one embodiment, the amino acid sequence of pVI comprises, consists of, or consists essentially of SEQ ID NO:35 (Figure 36). In one embodiment, the nucleic acid sequence encoding pVI has at least 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:36 (Figure 36). In one embodiment, the nucleic acid sequence encoding pVI comprises, consists of, or consists essentially of SEQ ID NO:36 (Figure 36).

[0326] In one embodiment, the helper plasmid ii) comprises a nucleic acid sequence encoding a fusion protein comprising at least one Ff phage protein or modified Ff phage protein or a functional portion thereof fused to a binding protein or a binding portion thereof.

[0327] In one embodiment, the Ff phage protein or modified Ff phage protein or functional portion thereof is an Ff phage coat or modified Ff phage coat protein or functional portion thereof. In one embodiment, the nucleic acid sequence encoding the fusion protein comprises a first nucleic acid coding sequence encoding at least one Ff phage protein or at least one modified Ff phage protein.

[0328] In one embodiment, the nucleic acid sequence encoding the fusion protein comprises a second nucleic acid coding sequence, and expression of the first and second nucleic acid sequences produces the fusion protein.

[0329] In one embodiment, the second nucleic acid coding sequence encodes a binding protein or binding portion thereof that is displayed on the surface of the nanorod. In one embodiment, the binding protein is an antibody or antigen-binding portion thereof, or a binding protein or binding portion thereof.

[0330] In one embodiment, the antibody or antigen-binding portion thereof is selected from the group consisting of a SARSCoV-2-spike-specific single chain antibody, preferably C121 (scFvC121), a SARSCoV-2 nucleocapsid-specific antigen-binding fragment of a heavy chain-only antibody (VHH), preferably N3 (VHHN3), and a botulinum neurotoxin-specific VHH.

[0331] In one embodiment, the binding protein or binding portion thereof is selected from the group consisting of the FnB fibronectin binding domain of Streptococcus pyogenes M type 22 protein Sof, the botulinum toxin binding domain of synaptic vesicle glycoprotein 2C (SV2C), and a SARS-CoV-2 spike (S) or matrix (M) derived peptide that interacts with the SARS-CoV-2 nucleocapsid protein (N).

[0332] In one embodiment, the first nucleic acid sequence comprises, consists essentially of, or consists of modified gill (SEQ ID NO:32, Figure 35).

[0333] In one embodiment, the nucleic acid sequence encoding the fusion protein comprises, consists essentially of, or consists of a nucleic acid sequence encoding the single chain variable domain of antibody C121 (scFvC121) fused to a nucleic acid sequence encoding full-length pIII (SEQ ID NO:40, Figure 38). In one embodiment, the fusion protein is SEQ ID NO:40.

[0334] In one embodiment, the nucleic acid sequence encoding the fusion protein comprises, consists essentially of, or consists of a nucleic acid sequence encoding an antigen-binding fragment of the heavy chain-only antibody N3 (VHHN3) fused with a nucleic acid sequence encoding full-length pIII (SEQ ID NO:100, Figure 55). In one embodiment, the fusion protein is SEQ ID NO:99.

[0335] In one embodiment, the nucleic acid sequence encoding the fusion protein comprises, consists essentially of, or consists of the nucleic acid coding sequence of the FnB fibronectin binding domain of Streptococcus pyogenes M type 22 protein Sof fused to the full-length gill coding sequence (SEQ ID NO:38, Figure 37). In one embodiment, the fusion protein is SEQ ID NO:37 (Figure 37).

[0336] Those skilled in the art will understand that the amino acid sequence of any of pIII, pVI, pVII, pVIII, or pIX can be modified as described herein and known in the art, such as for purposes of peptide display. All such modifications are contemplated herein and are considered to be within the skill of the art in combination with the disclosure herein.

[0337] In one embodiment, the inducible promoter of ii) is operably linked to a first operon comprising, consisting of, or consisting essentially of the Ff phage genes gII (gX), gV, gVII, and gVIII.

[0338] In one embodiment, the Ff phage genes gII (gX), gV, gVII, and gVIII have at least 70%, 80%, 90%, 95%, or 99% nucleic acid sequence identity to SEQ ID NO:4 (Figure 30), SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 (Figure 31), SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 (Figure 32), SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, and SEQ ID NO:28 (Figure 33), SEQ ID NO:98 (Figure 54).

[0339] In one embodiment, the Ff phage genes gII (gX), gV, gVII, gIX and gVIII comprise, consist of, or consist essentially of one of SEQ ID NO:4, (Figure 30), SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10 (Figure 31), SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18 (Figure 32), SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26 or SEQ ID NO:28 (Figure 33), SEQ ID NO:98 (Figure 54), respectively.

[0340] In one embodiment, the Ff phage genes gIII and gVIII encode modified Ff phage coat proteins pIII and pVIII, respectively.

[0341] In one embodiment, the modified pVIII comprises at least one amber mutation.

[0342] In one embodiment, the amino acid sequence of modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:13 (Figure 32). In one embodiment, the amino acid sequence of modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:15 (Figure 32). In one embodiment, the amino acid sequence of modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:17 (Figure 32). In one embodiment, the amino acid sequence of modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:19 (Figure 32). In one embodiment, the amino acid sequence of modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:21 (Figure 33). In one embodiment, the amino acid sequence of modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:23 (Figure 33). In one embodiment, the amino acid sequence of the modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:25 (Figure 33). In one embodiment, the amino acid sequence of the modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:27 (Figure 33). In one embodiment, the amino acid sequence of the modified pVIII comprises, consists of, or consists essentially of the amino acid sequence of SEQ ID NO:97 (Figure 54).

[0343] In one embodiment, the helper plasmid of ii) comprises a second operon comprising, consisting of, or consisting essentially of the Ff phage genes gIII, gVI, gI (gXI), and gIV. In one embodiment, the second operon is operably linked to a constitutive or inducible promoter, preferably a constitutive promoter, preferably an inducible promoter. In one embodiment, the inducible promoter is as described herein for the NPS aspect of the invention.

[0344] In one embodiment, the Ff phage gene gIII has at least 70%, 80%, 90%, 95%, or 99% nucleic acid sequence identity to SEQ ID NO:30 (Figure 34), SEQ ID NO:32 (Figure 35), or SEQ ID NO:34 (Figure 36). In one embodiment, the Ff phage gene gIII comprises, consists of, or consists essentially of SEQ ID NO:30 (Figure 34), SEQ ID NO:32 (Figure 35), or SEQ ID NO:34 (Figure 36).

[0345] In one embodiment, the Ff phage gene gVI has at least 70%, 80%, 90%, 95%, or 99% nucleic acid sequence identity to SEQ ID NO: 36 (Figure 36). In one embodiment, the Ff phage gene gVI comprises, consists of, or consists essentially of SEQ ID NO: 36 (Figure 36).

[0346] In another aspect, the invention relates to a composition comprising a plurality or population of nanorods described herein, or nanorods produced from an NPS described herein, or nanorods produced by the method of producing nanorods described herein.

[0347] In one embodiment, the composition has a viscosity of at least 1.0×10 14 , preferably at least 1.0 x 10 15 In one embodiment, the composition comprises about 1.0×10 nanorods / L. 14 , preferably about 1.0 x 10 15 , preferably about 1.0 x 10 16 In one embodiment, the composition comprises 1.0×10 nanorods / L. 14 , preferably 1.0 x 10 15 , preferably 1.0 x 10 16 Contains nanorods / L.

[0348] Those skilled in the art will understand that with respect to the lengths of the nanorods described in the following embodiments and other embodiments throughout this specification, the stated length values ​​refer to the stated length value + / - 5 nm.

[0349] In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are between about 40 nm and about 1000 nm in length, preferably between about 40 nm and about 400 nm, and preferably between about 100 nm and 300 nm in length. In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are at least 40 nm, preferably at least 50 nm (FIG. 17), 60 nm, 70 nm, 80 nm (FIG. 19), 100 nm, 110 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 2000 nm, 3000 nm, 4000 nm, 5000 nm, or at least 6000 nm in length. In one embodiment, at least 70% of the nanorods have a length of about 40 nm, preferably about 50 nm (Figure 17), 60 nm, 70 nm, 80 nm (Figure 19), 100 nm, 110 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 2000 nm, 3000 nm, 4000 nm, 5000 nm, or 6000 nm. In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are 40 nm, 50 nm (FIG. 17), 60 nm, 70 nm, 80 nm (FIG. 19), 100 nm, 110 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 2000 nm, 3000 nm, 4000 nm, 5000 nm, or 6000 nm in length. In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are 40 nm in length.

[0350] In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods have a length of at least 40 nm, 50 nm (FIG. 17), 60 nm, 70 nm, or 80 nm (FIG. 19). In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods have a length of about 40 nm, 50 nm (FIG. 17), 60 nm, 70 nm, or 80 nm (FIG. 19). In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods have a length of 40 nm, 50 nm (FIG. 17), 60 nm, 70 nm, or 80 nm (FIG. 19). In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods have a length of at least 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or at least 1000 nm. In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or about 1000 nm in length. In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or 1000 nm in length.

[0351] In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are about 80 nm in length (FIG. 19). In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are 80 nm in length. In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are about 100 nm, 110 nm, 200 nm, or 300 nm in length. In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are 100 nm, 110 nm, 200 nm, or 300 nm in length.

[0352] In one embodiment, the nanorods comprise (+) strand ssDNA containing an Ff phage origin of replication. In one embodiment, the nanorods comprise (+) strand ssDNA without a selectable marker. In one embodiment, the nanorods comprise (+) strand ssDNA without an antibiotic resistance marker.

[0353] In one embodiment, the nanorod comprises a (+) strand ssDNA encoding at least one, and preferably at least two, Ff phage coat proteins described herein.

[0354] In some embodiments, the nanorods comprise at least one modified Ff phage protein described herein. In one embodiment, the nanorods comprise at least one fusion protein described herein.

[0355] Specifically contemplated as embodiments of this aspect of the invention relating to compositions comprising a plurality or population of nanorods are any and / or all of the embodiments described in other aspects of the invention relating to the nanorod generating systems (NPS), nanorods, nanorod conjugates, and methods of generating nanorods described herein, including, but not limited to, embodiments relating to nucleic acid expression constructs, scaffold and filler nucleic acid sequences, vectors, plasmids, nanorod replication assembly plasmids, helper plasmids, BSF nanoreplication assembly cassettes, origins of replication, Ff phage proteins, modified Ff phage proteins, Ff phage genes, modified Ff phage genes, promoters, selectable markers, scaffold and filler nucleic acid sequences, replicated +strand ccssDNA, binders, detection moieties, and fusion proteins.

[0356] In another aspect, the present invention relates to a nanorod generation system (NPS) comprising a nucleic acid expression construct comprising a filamentous phage (+)Ori1, a replication assembly cassette comprising a packaging signal (PS) and (+)Ori2, at least one plasmid origin of replication not located within the replication assembly cassette that enables replication of the construct in bacteria, and at least one inducible promoter operably linked to a nucleic acid sequence encoding at least one Ff phage replication protein, wherein the expression construct expresses the Ff phage replication protein to generate excised and replicated DNA sequences from the replication assembly cassette, forming circular single-stranded DNA encapsulated within nanorods.

[0357] In some embodiments, the nucleic acid construct comprises a BSF nanoreplication assembly construct or a variant thereof described herein. The replication assembly construct expresses Ff phage proteins and can generate excised and replicated DNA sequences from the replication assembly cassette, forming circular single-stranded DNA encapsulated within nanorods. Excision occurs by cleavage within (+)Ori1 and (+)Ori2. Thus, the excised and replicated sequences from the replication assembly cassette (referred to herein as the scaffold; see Figure 6) contain the intervening sequence between the cleaved (+)Ori1 and (+)Ori2 sequences, flanked by the remaining portions of (+)Ori1 and (+)Ori2. In some embodiments, the NPS can also contain a (-)Ori between the packaging signal and (+)Ori2 to enhance the efficiency of nanorod generation. In one embodiment, the expression construct is a plasmid. In some embodiments, the expression construct encodes at least one Ff phage replication protein that mediates cleavage of (+)Ori1 and (+)Ori2. In some embodiments, the expression construct encodes one to all of the Ff phage proteins pI-pXI. In one embodiment, the Ff phage replication protein is pII. In one embodiment, the NPS lacks a second nucleic acid construct encoding another filamentous phage protein.

[0358] In some embodiments, any Ff phage proteins pI through pXI not encoded by the nucleic acid expression construct may be encoded by a second expression construct, sometimes referred to as a helper construct. If the nucleic acid expression construct containing the replication assembly cassette encodes all of the Ff phage proteins pI-pXI, a helper construct is not required. In some embodiments, any of pIII, pVI, pVII, pVIII, and pIX can be fused to a heterologous polypeptide, regardless of whether they are encoded by the expression construct containing the replication assembly cassette or by another helper construct. In a preferred embodiment, the nucleic acid expression construct containing the replication assembly cassette includes a nucleic acid sequence encoding the Ff phage replication protein pII, which is operably linked to an inducible promoter. Induction of the promoter and the resulting expression of pII initiates excision, replication, and packaging of the scaffold DNA from the replication assembly cassette.

[0359] In some embodiments, the expression construct containing the replication assembly cassette includes a sequence encoding the Ff phage protein pVIII containing an amber mutation to reduce the toxicity of pVIII to bacterial cells. In some embodiments, the expression construct also includes a nucleic acid sequence encoding a marker to facilitate selection of cells that have taken up the construct. In some embodiments, the marker is an auxotrophic marker. In some embodiments, the marker is not an auxotrophic marker. In some embodiments, the replication assembly cassette includes a filler nucleic acid sequence between (+)Ori1 and PS, or between PS and (-)Ori (if present), or between PS and (+)Ori2 (if (-)Ori is absent). In some embodiments, the replication assembly cassette does not include a filler nucleic acid sequence. In some embodiments, the filler nucleic acid sequence encodes at least one filamentous phage protein. In some embodiments, the filler nucleic acid sequence encodes pVII and pIX, which may lead to increased production of nanorods. In some embodiments, the filler nucleic acid sequence encodes pVII, pVIII, and / or pIX. In some embodiments, the filler nucleic acid sequence encodes a heterologous protein and / or peptide fused to pVII, pVIII, or pIX. In some embodiments, these fusions facilitate the display of long peptides. In addition to the Ff phage protein expressed in E. coli, the filler nucleic acid sequence can be used to house another eukaryotic gene expression cassette to enable expression in eukaryotic cells. In some embodiments, the filler nucleic acid sequence further encodes a prokaryotic or eukaryotic protein of interest.

[0360] Specifically contemplated as embodiments of this aspect of the invention relating to NPSs are any and / or all of the embodiments relating to the nanorod generating systems (NPSs), nanorods, nanorod conjugates, and methods of generating nanorods described in other aspects of the invention, including, but not limited to, embodiments relating to nucleic acid expression constructs, scaffold and filler nucleic acid sequences, vectors, plasmids, nanorod replication assembly plasmids, helper plasmids, BSF nanoreplication assembly cassettes, origins of replication, Ff phage proteins, modified Ff phage proteins, Ff phage genes, modified Ff phage genes, promoters, selectable markers, scaffold and filler nucleic acid sequences, replicated +strand ccssDNA, binders, detection moieties, and fusion proteins.

[0361] In another aspect, the present invention relates to a nanorod generation system (NPS) comprising: i) a nucleic acid expression construct comprising a replication assembly cassette containing a filamentous phage (+)Ori1, a packaging signal (PS), and a (+)Ori2, and at least one plasmid origin of replication not located within the replication assembly cassette (which enables the construct to replicate in bacteria); and ii) a helper nucleic acid expression construct (referred to as a "helper construct") comprising at least one selectable marker and at least one inducible promoter operably linked to a nucleic acid sequence encoding at least one Ff phage protein. Here, the helper nucleic acid construct expresses the Ff phage replication protein, generating a DNA sequence excised and replicated from the replication assembly cassette to form a circular single-stranded DNA packaged within a nanorod. In some embodiments, the helper nucleic acid construct expresses the Ff phage replication protein, generating a scaffold DNA sequence excised and replicated from the replication assembly cassette to form a circular single-stranded DNA packaged within a nanorod. This NPS operates similarly to the NPS described in the previous paragraph, except that the replication assembly construct does not necessarily encode Ff phage proteins. Rather, this system includes a helper construct encoding the Ff phage proteins necessary to form nanorods that encapsulate the scaffold DNA. In some embodiments, a single helper construct encodes any and / or all of the Ff phage proteins pI-pXI, although multiple helper constructs can be used that are expressed together to provide all of the Ff phage proteins pI-pXI necessary to form nanorods that encapsulate the scaffold DNA. In some embodiments, the replication assembly cassette further comprises a (-)Ori between the packaging signal and the (+)Ori2. In one embodiment, the helper construct includes a nucleic acid sequence encoding the Ff phage replication protein pII operably linked to an inducible promoter, such that upon induction, pII is expressed and initiates excision and replication of DNA from the replication assembly cassette. In some embodiments, the replication assembly cassette encodes a selectable marker to facilitate selection of cells containing the construct. In one embodiment, the selectable marker is an auxotrophic marker.In one embodiment, the selectable marker is not an auxotrophic marker.

[0362] Specifically contemplated as embodiments of this aspect of the invention relating to NPSs are any and / or all of the embodiments relating to the nanorod generating systems (NPSs), nanorods, nanorod conjugates, and methods of generating nanorods described in other aspects of the invention, including, but not limited to, embodiments relating to nucleic acid expression constructs, scaffold and filler nucleic acid sequences, vectors, plasmids, nanorod replication assembly plasmids, helper plasmids, BSF nanoreplication assembly cassettes, origins of replication, Ff phage proteins, modified Ff phage proteins, Ff phage genes, modified Ff phage genes, promoters, selectable markers, scaffold and filler nucleic acid sequences, replicated +strand ccssDNA, binders, detection moieties, and fusion proteins.

[0363] In another aspect, the invention relates to an isolated host cell comprising an NPS described herein.

[0364] In another aspect, the present invention relates to a method for producing nanorods, comprising culturing isolated host cells containing the NPS described herein and supplying the host cells with an inducer for an inducible promoter during an optimal growth phase, thereby expressing an Ff phage replication protein within the cells and generating an excised and replicated DNA sequence that forms a circular single-stranded DNA encapsulated within the nanorod. In one embodiment, the optimal growth phase is determined by the optical density (OD600) of the host cells. In one embodiment, the Ff phage replication protein is pII.

[0365] Specifically contemplated as embodiments of this aspect of the invention relating to methods of generating nanorods are any and / or all of the nanorod generating systems (NPSs), nanorods, nanorod conjugates described in other aspects of the invention, and embodiments of methods of generating nanorods described herein, including, but not limited to, embodiments relating to nucleic acid expression constructs, scaffold and filler nucleic acid sequences, vectors, plasmids, nanorod replication assembly plasmids, helper plasmids, BSF nanoreplication assembly cassettes, origins of replication, Ff phage proteins, modified Ff phage proteins, Ff phage genes, modified Ff phage genes, promoters, selectable markers, scaffold and filler nucleic acid sequences, replicated +strand ccssDNA, binders, detection moieties, and fusion proteins.

[0366] In another aspect, the present invention relates to nanorods approximately 60-800 nm long encapsulating circular single-stranded DNA, called scaffolds, excised by pII cleavage of a replication assembly cassette comprising a filamentous phage (+) ORI1, a packaging signal (PS), and a (+) ORI2, and a filler nucleic acid sequence encoding at least one filler nucleic acid sequence. In some embodiments, the filler nucleic acid sequence is located between the (+) ORI1 and the PS (filler I; Table 9, SEQ ID NOs:46, 47 (Figure 40), SEQ ID NOs:56-59 (Figure 42), SEQ ID NOs:66-67 (Figure 44), SEQ ID NO:75 (Figure 46), SEQ ID NO:82 (Figure 48), SEQ ID NO:104 (Figure 57)). In some embodiments, the filler nucleic acid is located between the PS and the (+) ORI2. In some embodiments, the replication assembly cassette comprises a (-) ORI between the PS and the (+) ORI2. In some embodiments, a filler nucleic acid is located between the PS and the (-) oligonucleotide (filler II; Table 9, SEQ ID NO: 49, Figures 40, 42, 44, 57, SEQ ID NO: 85, 86, Figure 48).

[0367] These nanorods differ from previously described nanorods in that filler DNA is used to encode at least one Ff protein. The presence of a protein-coding gene in the filler DNA linearly correlates the nanorod length with the distance between the pII cleavage sites of (+)Ori1 and (+)Ori2, resulting in a nanorod's minimum length increasing proportionally with the number of added nucleotides. Each additional nucleotide in the ssDNA genome increases the nanorod's length by 0.133 nm (Newman et al., 1977). The upper limit of the length is one of the limits listed above, depending on the length of the filler DNA. The length of the filler DNA depends on the number of Ff proteins it encodes and the amount of other filler DNA, if any. Such nanorods can be generated from replica assembly cassettes with or without a (-)Ori between PS and (+)Ori2. If a (-)Ori is present, the location of filler2 will be between PS and (-)Ori. If a (-)Ori is present in the replica assembly cassette, it will also be present in the excised and replicated DNA contained in the nanorod. In some embodiments, the filler DNA encodes the Ff phage proteins pVII and / or pIX, which have been shown to increase nanorod production. The preferred length of such nanorods is approximately 95-125 nm. In some embodiments, pVIII is encoded by the filler nucleic acid sequence. In some embodiments, the filler nucleic acid sequence encodes the Ff phage proteins pVII, pVIII, and / or pIX, or encodes modified Ff phage proteins pVII, pVIII, and / or pIX, or a combination thereof. In some embodiments, the nucleic acid sequence encoding pVII, pVIII, and / or pIX, and / or modified pVII, pVIII, and / or pIX, is fused to a nucleic acid sequence encoding a heterologous polypeptide. In some embodiments, the filler nucleic acid sequence further encodes a heterologous polypeptide that may or may not be fused to the Ff phage protein or modified Ff phage protein. The preferred length of such nanorods is approximately 95-125 nm.

[0368] In another aspect, the present invention relates to a nanorod population encapsulating a circular single-stranded DNA, called a scaffold, excised by pII cleavage of a replica assembly cassette comprising a filamentous phage (+) OR1, a packaging signal (PS), a (-) OR2, and a filler nucleic acid sequence between (+) OR1 and PS or between PS and (+) OR2. The filler nucleic acid sequence encodes at least one filamentous phage protein, and at least 70% of the nanorods in the population are about 40 to about 800 nm in length. In one embodiment, the replica assembly cassette further comprises a (-) OR2 between the packaging signal and (+) OR2, and at least 70% of the nanorods in the population are about 60 to about 800 nm in length. In one embodiment, at least 70% of the nanorods in the population are about 60 to about 400 nm in length. In one embodiment, at least 70% of the nanorods in the population are about 60 to about 300 nm in length. In one embodiment, at least 70% of the nanorods in the population have a length of about 95 to about 125 nm.

[0369] In another aspect, the present invention relates to nanorods encapsulating circular single-stranded DNA, called scaffolds, excised by pII cleavage of a replication assembly cassette containing a filamentous phage (+)Ori1, a packaging signal (PS), and a (+)Ori2, but lacking a (-)Ori. Due to the lack of a (-)Ori, such nanorods have a smaller minimum size than previously described nanorods, e.g., less than 50 nm to approximately 40 nm. However, such nanorods can also have any of the upper size limits listed above, depending on the length of the filler DNA contained between the (+1)Ori1 and (+)Ori2. Accordingly, the present invention provides a nanorod population in which at least 70% of the nanorod population have lengths between 40 and 800 nm. The present invention also provides a nanorod population in which at least 70% of the nanorod population have lengths between 40 and 50 nm.

[0370] In another aspect, the present invention relates to nanorods approximately 35 to approximately 45 nm in length that encapsulate circular single-stranded DNA excised by pII cleavage of a replication assembly cassette containing a filamentous phage (+)Ori1, a packaging signal (PS), and a (+)Ori2, but lacking a (-)Ori.

[0371] In another aspect, the invention relates to a nanorod population comprising a plurality of nanorods about 35 to about 45 nm in length encapsulating circular single-stranded DNA excised by pII cleavage of a replication assembly cassette comprising a filamentous phage (+)Ori1, a packaging signal (PS), and a (+)Ori2, and lacking a (-)Ori, wherein at least 70% of the nanorods in the population are about 38 to about 42 nm in length. In one embodiment, at least 70% of the nanorods in the population are about 40 nm in length.

[0372] Specifically contemplated as embodiments of aspects of the invention relating to nanorods and / or nanorod populations are any and / or all of the embodiments described in other aspects of the invention relating to the nanorod generating systems (NPSs), nanorod conjugates, and methods of generating and / or making nanorods described herein, including, but not limited to, embodiments relating to nucleic acid expression constructs, scaffold and filler nucleic acid sequences, vectors, plasmids, nanorod replication assembly plasmids, helper plasmids, BSF nanoreplication assembly cassettes, origins of replication, Ff phage proteins, modified Ff phage proteins, Ff phage genes, modified Ff phage genes, promoters, selectable markers, scaffold and filler nucleic acid sequences, replicated +strand ccssDNA, binders, detection moieties, and fusion proteins.

[0373] In another aspect, the present invention provides a method for producing a medicament comprising administering to a subject a subject a medicament comprising administering to ... 13 The present invention relates to a method for producing a plurality of nanorods, comprising inducing production of nanorods / L in a host cell culture.

[0374] In one embodiment, the method comprises: 14 , preferably at least 1.0 x 1015 In one embodiment, the method comprises inducing the production of about 1.0×10 nanorods / L. 14 , preferably about 1.0 x 10 15 , preferably about 1.0 x 10 16 In one embodiment, the method comprises inducing the production of 1.0×10 nanorods / L. 14 , preferably 1.0 x 10 15 , preferably 1.0 x 10 16 This involves inducing the production of nanorods / L.

[0375] In one embodiment, the host cell culture is a eukaryotic cell culture or a prokaryotic cell culture. In one embodiment, the prokaryotic cell culture is a bacterial cell culture. In one embodiment, the bacterial cell culture is a Gram- bacterial cell culture. In one embodiment, the Gram- bacterial cell culture is an E. coli culture.

[0376] In one embodiment, the E. coli culture comprises at least 1.0 x 10 11 cells / L, preferably at least 1.0 x 10 12 / L, at least 2.0 × 10 12 cells / L, at least 3.0 × 10 12 At least 4.0 x 10 cells / L 12 cells / L, preferably at least 5.0 x 10 12 Contains cells / L.

[0377] In one embodiment, the E. coli culture comprises about 1.0 x 10 11 cells / L, preferably about 1.0 x 10< 12 / L, approx. 2.0×10 12 cells / L, approximately 3.0×10 12 cells / L, approximately 4.0×10 12 cells / L, preferably about 5.0 x 10 12 Contains cells / L.

[0378] In one embodiment, the E. coli cell comprises a mutation that allows suppression of a stop codon in at least one Ff phage coat protein. Preferably, the mutation is in the Ff phage gene gVIII described herein. Preferably, the coat protein is pVIII.

[0379] In one embodiment, the E. coli cell comprises a mutation that inhibits background expression from an inducible promoter. In one embodiment, the inducible promoter is any of the inducible promoters described herein for the above aspects of the invention. Preferably, the inducible promoter is a lac promoter, preferably lacUV5.

[0380] In one embodiment, the E. coli cells are strain K2091 (Table 1).

[0381] In one embodiment, the E. coli cells are strain K2485 (Table 1).

[0382] In one embodiment, the E. coli cell comprises at least one, and preferably two, auxotrophic mutations. In one embodiment, the auxotrophic mutations are ΔnadC727 and / 581>ΔmetE774.

[0383] The △nadC727 mutation allows auxotrophic selection of a plasmid expressing NadC on minimal medium supplemented with casamino acids (casein hydrolysate) in the absence of NAD.

[0384] The △metE774 mutation allows auxotrophic selection of MetE-expressing plasmids on minimal medium lacking methionine, and also allows in vivo incorporation of the artificial amino acid azidohomoalanine (Aha) into proteins at the ATG codon on minimal medium containing a specific mixture of methionine and Aha.

[0385] In one embodiment, inducing comprises contacting the E. coli cells with an inducer. In one embodiment, the inducer is an inducer of the lac promoter, preferably a mutant lac promoter, preferably lacUV5. In one embodiment, the inducer is IPTG.

[0386] In one embodiment, the method includes inducing nanorod production in E. coli cells during an optimal growth phase, which in one embodiment is determined by the optical density (OD600) of the E. coli cells in culture.

[0387] In one embodiment, the optimal growth phase is determined by an OD600 of at least 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, or 0.22, preferably at least 0.1. In one embodiment, the optimal growth phase is determined by an OD600 of about 0.09 to about 0.22, preferably about 0.1 to about 0.2, preferably 0.1 to 0.2.

[0388] In one embodiment, the optimal growth phase is determined by an OD600 of about 0.1. In one embodiment, the optimal growth phase is determined by an OD600 of about 0.15. In one embodiment, the optimal growth phase is determined by an OD600 of about 0.2. In one embodiment, the optimal growth phase is determined by an OD600 of 0.1. In one embodiment, the optimal growth phase is determined by an OD600 of 0.15. In one embodiment, the optimal growth phase is determined by an OD600 of 0.2 or 0.2.

[0389] In one embodiment, induction results in replication of a (+) strand circular ssDNA comprising the nucleic acid coding sequence for at least one, preferably two, Ff phage coat proteins or modified coat proteins, or both, hi one embodiment, induction results in expression of at least one, preferably two, Ff phage coat proteins or modified coat proteins that bind to the (+) strand circular ssDNA.

[0390] In one embodiment, the two Ff phage coat proteins or modified coat proteins are pVII and pIX.

[0391] Specifically contemplated as pVII, pIX and modified pVII and pIX embodiments in this method aspect of the invention are all of the pVII and pIX, modified pVII and pIX embodiments described in previous aspects of the invention relating to the NPS aspect of the invention.

[0392] In one embodiment, induction results in replication of a (+) strand circular ssDNA bound to at least one, preferably at least two, preferably at least three different Ff phage coat proteins and / or different modified Ff phage coat proteins, in one embodiment, the at least one, two, or three different Ff phage coat proteins and / or the one, two, or three different modified Ff phage coat proteins are selected from the group consisting of pVIII, pIII, pVII, pIX, and pVI.

[0393] In one embodiment, the E. coli cells contain a single nucleic acid construct that mediates the production of nanorods. In one embodiment, the single nucleic acid construct is a vector, preferably a plasmid, as described herein. In one embodiment, the single nucleic acid is a p-popup plasmid, as described herein.

[0394] In one embodiment, inducing production comprises a single transformation of only E. coli cells. In one embodiment, the single transformation comprises transforming the E. coli cells with only a single nucleic acid construct. In one embodiment, the single nucleic acid construct mediates production of nanorods. In one embodiment, the single nucleic acid construct is a vector, preferably a plasmid, as described herein. In one embodiment, the single plasmid is a p-popup plasmid, as described herein.

[0395] In one embodiment, transformation of E. coli cells with a single nucleic acid construct results in at least 10-fold, preferably at least 100-fold, more transformed E. coli cells compared to transformation of E. coli cells with a double nucleic acid construct.

[0396] In one embodiment, the single nucleic acid construct is a vector, preferably a plasmid, preferably a ppopup plasmid as described herein.

[0397] Specifically contemplated as embodiments of a single nucleic acid expression construct are all of the embodiments of a single nucleic acid expression construct described above in the first NPS aspect of the invention that include a BSFnano replication assembly cassette, at least one auxotrophic marker, at least one inducible promoter operably linked to a nucleic acid sequence encoding at least one Ff phage protein, and at least one plasmid origin of replication that is not located within the BSFnano replication assembly cassette.

[0398] In one embodiment, inducing production involves double transformation of only E. coli cells. In one embodiment, the double transformation involves transforming the E. coli cells with a nucleic acid replication assembly construct and a helper nucleic acid expression construct described herein. In one embodiment, the double nucleic acid construct mediates nanorod production. In one embodiment, the double nucleic acid construct is a vector, preferably a plasmid described herein. In one embodiment, the two plasmids are the pBSF and pHP plasmid series described herein.

[0399] In one embodiment, the duplex nucleic acid construct is a vector, preferably a plasmid, preferably the pBSF and pHP series of plasmids described herein.

[0400] In one embodiment, the duplex nucleic acid constructs are different nucleic acid constructs.

[0401] In one embodiment, the double transformation is sequential transformation with different nucleic acid constructs, the first transformation being separated from the second transformation by at least 24 hours, preferably at least 32 hours, 40 hours, preferably at least 48 hours. In one embodiment, the double transformation is sequential transformation with different nucleic acid constructs, the first transformation being separated from the second transformation by about 24 hours, preferably about 32 hours, 40 hours, preferably about 48 hours.

[0402] In one embodiment, the method includes preparing transformation competent cells from cells that have undergone a first transformation.

[0403] In one embodiment, the first transformation comprises transformation with a helper nucleic acid expression construct comprising at least one selectable marker and at least one inducible promoter operably linked to a nucleic acid sequence encoding at least one Ff phage protein.

[0404] Specifically contemplated as embodiments of the helper nucleic acid expression construct are all of the embodiments relating to ii) the helper nucleic acid expression construct described above in the second NPS aspect of the invention.

[0405] In one embodiment, the second transformation comprises transformation with a nucleic acid replication assembly construct comprising a BSF nanoreplication assembly cassette, at least one auxotrophic marker, and at least one plasmid origin of replication that is not present within the BSF nanoreplication assembly cassette.

[0406] Specifically contemplated as embodiments of nucleic acid replicating assembly constructs are all of the embodiments relating to i) nucleic acid replicating assembly constructs described above in the second NPS aspect of the invention.

[0407] In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are between about 40 nm and about 1000 nm in length, preferably between about 40 nm and about 400 nm in length, and preferably between about 100 nm and 300 nm in length. In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are at least 40 nm, preferably at least 50 nm (Figure 17), 60 nm, 70 nm, 80 nm (Figure 19), 100 nm, 110 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 2000 nm, 3000 nm, 4000 nm, 5000 nm, or at least 6000 nm in length. In one embodiment, at least 70% of the nanorods have a length of about 40 nm, preferably about 50 nm (Figure 17), 60 nm, 70 nm, 80 nm (Figure 19), 100 nm, 110 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 2000 nm, 3000 nm, 4000 nm, 5000 nm, or 6000 nm. In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are 40 nm, 50 nm (FIG. 17), 60 nm, 70 nm, 80 nm (FIG. 19), 100 nm, 110 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 2000 nm, 3000 nm, 4000 nm, 5000 nm, or 6000 nm in length. In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are 40 nm in length.

[0408] In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods have a length of at least 40 nm, 50 nm (FIG. 17), 60 nm, 70 nm, or 80 nm (FIG. 19). In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods have a length of about 40 nm, 50 nm (FIG. 17), 60 nm, 70 nm, or 80 nm (FIG. 19). In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods have a length of 40 nm, 50 nm (FIG. 17), 60 nm, 70 nm, or 80 nm (FIG. 19). In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods have a length of at least 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or at least 1000 nm. In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are about 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or about 1000 nm in length. In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, or 1000 nm in length.

[0409] In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are about 80 nm in length (FIG. 19). In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are 80 nm in length. In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are about 100 nm, 110 nm, 200 nm, or 300 nm in length. In one embodiment, at least 70%, at least 75%, and preferably at least 80% of the nanorods are 100 nm, 110 nm, 200 nm, or 300 nm in length.

[0410] In one embodiment, the nanorods comprise a (+)-strand circular ssDNA containing an Ff phage origin of replication. In one embodiment, the nanorods comprise a (+)-strand circular ssDNA without a selectable marker. In one embodiment, the nanorods comprise a (+)-strand circular ssDNA without an antibiotic resistance marker.

[0411] In one embodiment, the nanorod comprises a (+) strand circular ssDNA encoding at least one, and preferably at least two, Ff phage coat proteins described herein.

[0412] In some embodiments, the nanorods comprise at least one modified Ff phage protein described herein. In one embodiment, the nanorods comprise at least one fusion protein described above in the first and second NPS aspects of the invention.

[0413] In another aspect, the present invention relates to a method for producing a plurality of nanorods comprising inducing replication of a circular ssDNA in a host cell culture from a single nucleic acid construct, the construct comprising a scaffold nucleic acid sequence encoding at least two Ff phage coat proteins or modified Ff phage coat proteins.

[0414] In one embodiment, the scaffold nucleic acid sequences (Figure 41, SEQ ID NO:53, SEQ ID NO:55; Figure 42, SEQ ID NO:59) encode pVII (SEQ ID NO:8, Figure 31) and pIX (SEQ ID NO:10, Figure 31).

[0415] In one embodiment, the amino acid sequence of pVII comprises, consists of, or consists essentially of SEQ ID NO:7 (Figure 31). In one embodiment, the nucleic acid sequence encoding pVII has at least 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:8 (Figure 31). In one embodiment, the nucleic acid sequence encoding pVII comprises, consists of, or consists essentially of SEQ ID NO:8 (Figure 31).

[0416] In one embodiment, the amino acid sequence of pIX comprises, consists of, or consists essentially of SEQ ID NO:9 (Figure 31). In one embodiment, the nucleic acid sequence encoding pIX has at least 70%, 80%, 90%, 95%, or 99% sequence identity to SEQ ID NO:10 (Figure 31). In one embodiment, the nucleic acid sequence encoding pIX comprises, consists of, or consists essentially of SEQ ID NO:10 (Figure 31).

[0417] Specifically contemplated as embodiments of this aspect of the invention are all of the embodiments described in the NPS, nanorod, composition, and method aspects herein, including, but not limited to, embodiments relating to nucleic acid expression constructs, scaffold and filler nucleic acid sequences, vectors, plasmids, nanorod replication assembly plasmids, helper plasmids, BSF nanoreplication assembly cassettes, origins of replication, Ff phage proteins, modified Ff phage proteins, Ff phage genes, modified Ff phage genes (including amino acid and nucleic acid sequences, and all possible mutations and modifications), promoters, inducible promoters and operable linkages, selectable markers, auxotrophic markers, (+)strand circular ssDNA, fusion proteins, induction of production, host cells and host cell culture, replicated ssDNA, single and double transformations, length of generated nanorods, and (+)strand circular ssDNA.

[0418] In another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a BSF nanoreplication assembly cassette; a scaffold nucleic acid sequence; two nucleic acid sequences each containing a pII cleavage site, each cleavage site located at the (+) oligonucleotide; and at least one nucleic acid sequence encoding at least one modified Ff phage coat protein.

[0419] In one embodiment, the nucleic acid construct comprises a nucleic acid sequence encoding at least two modified Ff phage proteins. In one embodiment, at least one modified Ff phage protein is a modified coat protein described herein. In one embodiment, at least one modified Ff phage protein is a modified replication protein described herein. In one embodiment, the single nucleic acid construct comprises the Ff phage protein pII operably linked to an inducible promoter.

[0420] As the skilled worker will appreciate, the scaffold nucleic acid sequence corresponds to the sequence between the vertical arrows shown in Figures 39, 41, 43, 45, 47.

[0421] In one embodiment, the single nucleic acid construct comprises SEQ ID NO:41, SEQ ID NO:43 (Figure 39), SEQ ID NO:52, SEQ ID NO:54 (Figure 41), SEQ ID NO:61, SEQ ID NO:63 (Figure 43), SEQ ID NO:70, SEQ ID NO:72 (Figure 45), SEQ ID NO:77, or SEQ ID NO:79 (Figure 47), SEQ ID NO:101 (Figure 56).

[0422] In one embodiment, the method comprises inducing replication of a (+) strand circular ssDNA, wherein the ssDNA is directed to replicate from a nucleic acid construct comprising SEQ ID NO:41, SEQ ID NO:43 (Figure 39), SEQ ID NO:52, SEQ ID NO:54 (Figure 41), SEQ ID NO:61, SEQ ID NO:63 (Figure 43), SEQ ID NO:70, SEQ ID NO:72 (Figure 45), SEQ ID NO:77, SEQ ID NO:79 (Figure 47), or SEQ ID NO:101 (Figure 56).

[0423] In one embodiment, the (+) strand circular ssDNA comprises, consists essentially of, or consists of SEQ ID NO:42, SEQ ID NO:44 (Figure 39), SEQ ID NO:53, SEQ ID NO:55 (Figure 41), SEQ ID NO:62, SEQ ID NO:64 (Figure 43); SEQ ID NO:71, SEQ ID NO:73 (Figure 45); SEQ ID NO:78, SEQ ID NO:80 (Figure 47), or SEQ ID NO:102 (Figure 56). These (+) strand circular ssDNAs are indicated by, defined by, and located between the arrows in Figures 39-47 and 56, respectively. The arrows indicate the pII cleavage site.

[0424] In one embodiment, the single nucleic acid construct is pPOPOP529LacYM (SEQ ID NO:94, Figure 51).

[0425] Specifically contemplated as embodiments of this method aspect of the invention are all embodiments described in the NPS, composition, nanorod, and method aspects herein, including, but not limited to, embodiments relating to nucleic acid expression constructs, scaffold and filler nucleic acid sequences, vectors, plasmids, nanorod replication assembly plasmids, helper plasmids, BSF nanoreplication assembly cassettes, origins of replication, Ff phage proteins, modified Ff phage proteins, Ff phage genes, modified Ff phage genes, amino acid and nucleic acid sequences, and all possible mutations and modifications, promoters, inducible promoters and operable linkages, selectable markers, auxotrophic markers, (+)strand circular ssDNA, fusion proteins, induction of production, host cells and host cell culture, replicated ssDNA, single and double transformations, length of generated nanorods, and (+)strand circular.

[0426] In another aspect, the invention relates to a method of producing a nanorod-binder conjugate, comprising conjugating a binder to a nanorod described herein or to a nanorod produced from an NPS described herein.

[0427] In one embodiment, the conjugate comprises forming at least one covalent bond between an amino acid residue contained in the nanorod and a binding agent.

[0428] In one embodiment, the binding agent is selected from the group consisting of a small molecule or a polypeptide (e.g., biotin, an antibody, an antibody-derived single chain variable domain (scFv), a nanobody, a camelid heavy chain-only antibody or variable domain (VHH), or other type of analyte-binding polypeptide).

[0429] In one embodiment, the conjugate further comprises a detection agent.

[0430] In one embodiment, the detection agent is selected from the group consisting of a small molecule, biotin, a fluorophore, a quantum dot, an inorganic molecule, a metal alloy, a fluorescent or colored protein, and an enzyme that catalyzes a color-producing reaction.

[0431] In one embodiment, the nanorods comprise (+) strand ssDNA containing an Ff phage origin of replication. In one embodiment, the nanorods comprise (+) strand ssDNA without a selectable marker. In one embodiment, the nanorods comprise (+) strand ssDNA without an antibiotic resistance marker.

[0432] In one embodiment, the nanorod comprises a (+) strand ssDNA encoding at least one, preferably at least two, Ff phage coat proteins described herein. In some embodiments, the nanorod comprises at least one modified Ff phage protein described herein. In one embodiment, the nanorod comprises at least one fusion protein described herein.

[0433] In another aspect, the invention relates to a method of producing a nanorod-detection agent conjugate, comprising conjugating a detection agent to a nanorod described herein or to a nanorod produced from an NPS described herein.

[0434] In one embodiment, the conjugate comprises forming at least one covalent bond between an amino acid residue contained in the nanorod and the detection agent.

[0435] In one embodiment, the detection agent is selected from the group consisting of a small molecule, biotin, a fluorophore, a quantum dot, an inorganic molecule, a metal alloy, a fluorescent or colored protein, and an enzyme that catalyzes a color-producing reaction.

[0436] In one embodiment, the conjugate further comprises a binding agent.

[0437] In one embodiment, the binding agent is selected from the group consisting of a small molecule or a polypeptide (e.g., biotin, an antibody, a single chain variable domain derived from an antibody (scFv), a nanobody, a camelid heavy chain only antibody or variable domain (VHH), or other type of analyte-binding polypeptide).

[0438] In one embodiment, the nanorods comprise (+) strand ssDNA containing an Ff phage origin of replication. In one embodiment, the nanorods comprise (+) strand ssDNA without a selectable marker. In one embodiment, the nanorods comprise (+) strand ssDNA without an antibiotic resistance marker.

[0439] In one embodiment, the nanorod comprises a (+) strand ssDNA encoding at least one, preferably at least two, Ff phage coat proteins described herein. In some embodiments, the nanorod comprises at least one modified Ff phage protein described herein. In one embodiment, the nanorod comprises at least one fusion protein described herein.

[0440] The following aspects relate to both nanorod-binding and nanorod-detecting agent conjugates. In one embodiment, the nanorods contain modifications to the Ff phage coat protein that create functionalized handles. These modifications, known as "tag-and-modify" modifications, are made to enable targeted chemical or enzymatic modification of the Ff phage coat protein. For example, modifying mature coat proteins pVIII or pIII or pVII and pIX to include three or more extra glycines or two or more extra alanines at the N-terminus (the latter two proteins may require the addition of a heterologous signal sequence) creates a motif that can be used for enzymatic attachment of protein or nonprotein molecules linked to C-terminal LPXTA or LPXTG motifs. In this case, attachment of the molecule of interest is catalyzed by the enzyme sortase A (SrtA) from Streptococcus pyogenes (SrtA Sp) or Staphylococcus aureus (SrtA Sa), respectively (Hess et al., 2012). Interchangeable blocks (Figure 7, block iv; Figure 8, block iii) have been generated for NPSs that generate nanorods with pVIII displaying four Gly residues, or two Ala residues (Figures 30-32 and SEQ NOs: 19-23, 27-28) or five residues (Figure 54, SEQ NOs: 97-98) at the N-terminus.

[0441] In some embodiments, the amine group of the N-terminal residue or reactive groups of amino acids such as lysine, cysteine, tyrosine, aspartic acid, and glutamic acid can be used for chemical modification (Bernard and Francis, 2014). Alternatively, other motifs that allow enzymatic or chemical covalent attachment of non-protein molecules, such as SNAP tags, can be directly or indirectly inserted into nanorods to enable the attachment of diverse molecules. Also described herein is the generation of an exchangeable block displaying an unpaired Cys residue on pIII, enabling modification with maleimide-conjugated proteins and small molecules or other chemicals that target -SH groups (Figure 7, Block iv; Figure 8, Block iii; Figure 36, SEQ ID NOs: 33-34). A pVIII variant (Figure 33, SEQ ID NOs: 23 and 24) in which the exposed Met residue (Ala9 mutated to Met) and the buried Met28 residue are mutated to Leu allows the unnatural amino acid azidohomoalanine (Aha) to be incorporated into the surface-exposed position of pVIII in vivo without disrupting viral particle assembly and structure (Petrie, 2015). Aha contains an azide group in its side chain, allowing small molecules to be attached to viral particles using click chemistry targeting the azide group. In some embodiments, the nanorods described herein contain such modifications.

[0442] The skilled worker will understand that all known modifications applied in Ff-based phage display and materials science applications can also be applied to the nanorod functionalization described herein. All such modifications are contemplated as embodiments herein. In one embodiment, inserting four Gly residues at the N-terminus of our constructed mature pVIII (Figure 33, SEQ ID NO:27, SEQ ID NO:28) slightly reduced nanorod production. On the other hand, inserting an Ala followed by a Gly residue at the N-terminus of our constructed mature pVIII and deleting Pro6 from wild-type mature pVIII (Figure 32, SEQ ID NO:17, SEQ ID NO:18) prevented nanorod production. To overcome this latter issue, we "evolved" the gVIII sequence to increase the efficiency of this functionalized pVIII variant. This was achieved by transferring the coding sequence to the backbone of Ff phage (VCSM13). The resulting modified phage produced very small plaques and low titers; however, after three rounds of phage propagation in which host cells were infected at a low multiplicity of infection (1000 E. coli cells per phage), a "large plaque" mutant emerged. Sequencing of gVIII from two evolved phages identified two compensatory mutations, one in each mutant (D5A and L27S, as described above; Figures 32 and 33; SEQ ID NOs:19, 20, 21, and 22). These alleles were then restored to the inducible pPopUp and pHP plasmid backbone and shown to generate BSF nanorods. As those skilled in the art will appreciate, we believe it is possible to evolve the coding sequences of various Ff phage proteins to allow for other modifications that may interfere with BSF nanorod assembly.

[0443] In one enzymatic modification embodiment, evolved pVIII (SEQ ID NO: 19, 20) nanorods display an AlaAlaGlyGly motif on each pVIII copy. They were further enzymatically modified with LPETA-(LeuProGluThrAla)-tagged fluorescent dye FITC or the small molecule biotin via enzymatic attachment using Streptococcus pyogenes sortase (SrtA Sp, Figure 21). Analysis by native virus particle electrophoresis shows high-intensity fluorescence corresponding to the nanorod band after LPETA-FITC enzymatic conjugation (Figure 22A). Analysis of enzyme-biotinylated nanorods by transmission electron microscopy using avidin-coated gold beads demonstrates sortase-dependent binding along the nanorod length (Figure 23). For immunodetection assays, avidin-alkaline phosphatase can be attached to the nanorods (Figures 24A, 25-27). Enzymatic visualization of such avidin-alkaline phosphatase-labeled nanorods is performed by native agarose gel electrophoresis, blotted onto a membrane, and detected using a chromogenic substrate (Figure 24A).

[0444] In another embodiment, LPETG-β-glucosidase (GUS) was enzymatically attached directly to nanorods displaying an N-terminal 5-Gly peptide. The attachment of GUS to the nanorods was analyzed by agarose gel electrophoresis followed by an in-gel assay using a chromogenic substrate (Figure 24B).

[0445] The copy number and location of the displayed function vary depending on the coat protein used as the platform, whether it is a heterologous protein or a "handle" for modification. In some embodiments, using pVIII as a display platform results in a high copy number of peptides displayed along the nanorod shaft. The copy number of the displayed function depends on the number of pVIII subunits per nanorod, which in turn depends on the length of the ssDNA scaffold. The copy number of functional entities (fluorophores, small molecules, polymers, enzymes) varies with the length of the nanorod. For example, for a 1,000 nm long phage, approximately 400 copies of fluorophores or biotin are expected to be attached chemically or enzymatically per phage nanorod. Multiple different fluorescent dyes can be mixed for labeling purposes to enable barcoding and other more complex methods and detection.

[0446] In some embodiments, using minor coat proteins as a platform allows for the display of up to five copies per nanorod (each for pIII, pVII, and pIX; reviewed in (Rakonjac et al., 2017)). Furthermore, displaying both pVII and pIX allows for up to 10 copies per nanorod. In some embodiments discussed here, different fusion or attachment molecules are used to different minor Ff phage coat proteins. In this way, multiple different functions can be displayed on a single nanorod. For example, two functions can be displayed on one end of the nanorod (the pVII-pIX end) and one function on the other end (the pIII end). Such modifications have been demonstrated using various phage display methods using full-length Ff phage.

[0447] In another embodiment, the toxicity of the major coat protein pVIII is overcome by the introduction of an amber mutation. The major coat protein pVIII is toxic to E. coli when expressed in the absence of phage assembly. This toxicity results in poor growth of transformed E. coli cells expressing pVIII, even when the gVIII promoter is removed during cloning or when expression is controlled by an inducible promoter. To overcome this problem, a gVIII suppressible (nonsense) mutant was used to construct a helper plasmid. The construction was performed in an E. coli host that did not contain a suppressor mutation, preventing translation of most of the pVIII protein. Two different amber (TAG) mutants were used: one containing a G-to-T mutation, converting the GAG ​​codon 25 encoding glutamic acid at position 2 of the mature protein to TAG (SEQ ID NOS: 13-24, Figures 32-33), and the other in which the TCT codon 4 of the serine in the signal sequence was replaced by TAG (SEQ ID NOS: 25-28, Figure 33). A suppressor D mutation (supD) in serine tRNA is used to suppress these two amber mutations, and an E. coli strain containing this mutation is used to produce nanorods (Table 1).

[0448] An additional advantage of the gVIII suppressor amber mutant described here compared to E. coli cells expressing wild-type gVIII is that less pVIII is produced in the cells due to the lower translation efficiency of the suppressor compared to the cognate tRNA that reads the sense codon, resulting in a reduced ratio of shaft protein pVIII to end-capping proteins pIII, pVI, pVII, and pIX, favoring the assembly of short nanorods over long ones.

[0449] Specifically contemplated as embodiments of this aspect of the invention are all of the embodiments described in the NPS, composition, nanorod, and method aspects herein, including, but not limited to, embodiments relating to nucleic acid expression constructs, scaffold and filler nucleic acid sequences, vectors, plasmids, nanorod replication assembly plasmids, helper plasmids, BSF nanoreplication assembly cassettes, origins of replication, Ff phage proteins, modified Ff phage proteins, Ff phage genes, modified Ff phage genes (including amino acid and nucleic acid sequences, and all possible mutations and modifications), promoters, inducible promoters and operable linkages, selectable markers, auxotrophic markers, (+)strand circular ssDNA, fusion proteins, induction of production, host cells and host cell culture, replicated ssDNA, single and double transformations, length of nanorods generated, and (+)strand circular ssDNA.

[0450] In another aspect, the present invention relates to a nanorod-binding agent conjugate comprising a nanorod comprising at least one modified Ff phage coat protein, wherein the nanorod is produced from an NPS described herein, is a nanorod described herein, or is produced by a method for producing a nanorod described herein.

[0451] In one embodiment, the nanorod binding agent conjugate comprises at least one detection moiety that allows for detection of the nanorod binding agent conjugate.

[0452] In another aspect, the invention relates to a composition comprising the nanorod-binding agent conjugate described herein.

[0453] Specifically contemplated as embodiments of these aspects of the invention are all of the embodiments described in the NPS, composition, nanorod, and method aspects herein, including, but not limited to, embodiments relating to nucleic acid expression constructs, scaffold and filler nucleic acid sequences, vectors, plasmids, nanorod replication assembly plasmids, helper plasmids, BSF nanoreplication assembly cassettes, origins of replication, Ff phage proteins, modified Ff phage proteins, Ff phage genes, modified Ff phage genes (including amino acid and nucleic acid sequences, and all contemplated mutations and modifications), promoters, inducible promoters and operable linkages, selectable markers, auxotrophic markers, (+) strand circular ssDNA, fusion proteins, induction of production, host cells and host cell culture, replicated ssDNA, single and double transformations, length of generated nanorods, nanorod-binding agent conjugates, and (+) strand ssDNA.

[0454] In another aspect, the present invention relates to a nanorod-detector conjugate comprising a nanorod comprising at least one Ff phage coat protein comprising a covalently attached detection moiety, wherein the nanorod is a nanorod described herein, a nanorod produced from an NPS described herein, or a nanorod produced by a method described herein.

[0455] In one embodiment, the Ff phage protein is a modified coat protein as described herein.

[0456] In one embodiment, the detection moiety allows for detection of the nanorod detection agent conjugate.

[0457] In one embodiment, detection is performed by detecting a chemical, spectral, linear dichroic, fluorescent, visual, chemiluminescent, paramagnetic, sonic, electrical, surface plasmon resonance, isotopic, radioactive, or other chemical or physical signal.

[0458] In one embodiment, the nanorod detection agent conjugate comprises at least one detection moiety covalently attached to at least one modified Ff phage coat protein.

[0459] In one embodiment, the nanorod detection agent conjugate comprises a plurality of detection moieties covalently attached to a plurality of at least one modified Ff phage coat protein.

[0460] In one embodiment, the nanorod-detection-agent conjugate comprises at least two different types of modified Ff phage coat proteins.

[0461] In one embodiment, the nanorod detection agent conjugate comprises at least two different detection moieties.

[0462] In one embodiment, the nanorod detection agent conjugate comprises at least two different detection moieties, each covalently attached to a different type of modified Ff phage protein.

[0463] In one embodiment, the nanorod detection agent conjugate comprises a plurality of each of at least two different detection moieties covalently attached to a plurality of at least two different types of modified Ff phage proteins.

[0464] In one embodiment, the nanorod detection agent conjugate is included in a population of nanorod detection agent conjugates.

[0465] In one embodiment, the nanorod detection agent conjugate is included in a composition comprising a population of nanorod detection agent conjugates.

[0466] In one embodiment, at least a portion of the nanorod detection agent conjugates in a population or composition comprise different detection moieties.

[0467] In one embodiment, the detection moiety is selected from the group consisting of a fluorophore, a small molecule, a peptide, a protein, a polymer, a nucleic acid, an inorganic molecule, a dye, a radioisotope, a semiconductor, and a paramagnetic compound.

[0468] In one embodiment, the detection moiety is a fluorophore, a chromogenic substrate, a dye, a chemiluminescent, a paramagnetic, a molecule, a semiconductor, a conductor, a nucleic acid, a polypeptide, a polymer, a quantum dot, or a radioisotope.

[0469] In one embodiment, the fluorophore or chromogenic substrate is a fluorophore or chromogenic substrate.

[0470] In one embodiment, the nanorod detection agent conjugate comprises at least 3, 4, 5, 6, 7, 8, 9, or more different detection moieties.

[0471] In one embodiment, the nanorod-detection agent conjugate comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 200, 300, 400 or more detection moieties.

[0472] In one embodiment, the nanorod detection agent comprises one detection moiety for about every seven copies of Ff phage coat protein pVIII or modified pVIII contained in the nanorod.

[0473] In one embodiment, the nanorod detection agent conjugate further comprises a binding agent.

[0474] In one embodiment, the binding agent is covalently bound to at least one Ff phage coat protein. In one embodiment, the at least one Ff phage coat protein is a modified coat protein.

[0475] In another aspect, the present invention relates to a composition comprising at least one nanorod detection agent conjugate described herein.

[0476] In one embodiment, the composition comprises at least two nanorod detection agent conjugates, each nanorod detection agent conjugate comprising at least one different detection moiety.

[0477] In another aspect, the invention relates to a kit comprising one or more nanorod detection agent conjugates described herein.

[0478] Specifically contemplated as embodiments of these aspects of the invention are all of the embodiments described in the NPS, composition, nanorod, and method aspects herein, including, but not limited to, embodiments relating to nucleic acid expression constructs, scaffold and filler nucleic acid sequences, vectors, plasmids, nanorod replication assembly plasmids, helper plasmids, BSF nanoreplication assembly cassettes, origins of replication, Ff phage proteins, modified Ff phage proteins, Ff phage genes, modified Ff phage genes (including amino acid and nucleic acid sequences, and all contemplated mutations and modifications), promoters, inducible promoters and operable linkages, selectable markers, auxotrophic markers, (+) strand circular ssDNA, fusion proteins, induction of production, host cells and host cell culture, replicated ssDNA, single and double transformations, length of generated nanorods, nanorod-binding agent conjugates, and (+) strand ssDNA.

[0479] In another aspect, the present invention provides a method for detecting a target molecule in a sample, comprising the steps of: a) contacting a sample containing or suspected of containing a target molecule with a nanorod-binding agent conjugate; b) detecting binding of the nanorod-binding agent conjugate to the target molecule; The nanorod binder conjugate comprises at least one modified Ff phage coat protein, and the nanorods in the nanorod binder conjugate are generated from an NPS described herein, are nanorods described herein, are included in a nanorod population described herein, or are generated by a method for generating nanorods described herein.

[0480] In one embodiment, the nanorod-binding agent conjugate comprises at least one detection moiety covalently attached to at least one modified Ff phage coat protein.

[0481] In one embodiment, the nanorod-binding agent conjugate comprises one detection moiety for about every seven copies of Ff phage coat protein pVIII or modified pVIII contained in the nanorod.

[0482] In one embodiment, the nanorod-binding agent conjugate comprises a plurality of detection moieties covalently attached to a plurality of at least one modified Ff phage coat protein.

[0483] In one embodiment, the nanorod binding agent conjugate comprises at least 3, 4, 5, 6, 7, 8, 9, or more detection moieties.

[0484] In one embodiment, the nanorod-binding agent conjugate comprises at least two different types of modified Ff phage coat proteins.

[0485] In one embodiment, the nanorod-binding agent conjugate comprises at least two different types of detection moieties.

[0486] In one embodiment, the nanorod binder conjugate comprises at least 3, 4, 5, 6, 7, 8, 9 or more different types of detection moieties.

[0487] In one embodiment, the nanorod-binding agent conjugate comprises a plurality of each of at least two different detection moieties, each covalently attached to a plurality of at least two different types of modified Ff phage coat proteins.

[0488] In one embodiment, the nanorod binder conjugate is included in a population of nanorod binder conjugates.

[0489] In one embodiment, the binding agent is selected from the group consisting of a small molecule or a polypeptide.

[0490] In one embodiment, the polypeptide is selected from the group consisting of antibodies, antibody-derived single chain variable domains (scFv), camelid single chain antibody domains VHH, and other types of antibodies and analyte-binding polypeptides.

[0491] In one embodiment, the target molecule is immobilized on the solid support by binding to a capture molecule attached to the support.

[0492] In one embodiment, the target molecule is selected from the group consisting of a viral or bacterial protein, a disease marker, or other molecule of interest (analyte) in food, the environment, animals, or humans, hi one embodiment, the target molecule is a SARSCoV-2 molecule.

[0493] In one embodiment, detecting includes detecting a chemical signal, a spectral signal, a linear dichroism signal, a fluorescent signal, a visual signal, a chemiluminescent signal, a paramagnetic signal, an acoustic signal, an electrical signal, a surface plasmon resonance signal, an isotopic signal, a radioactive signal, or other chemical or physical signal.

[0494] In one embodiment, detection involves fluorescent signal detection or visual detection via an enzymatic reaction using a chromogenic or chemiluminescent substrate.

[0495] In one embodiment, the detection method is a dot blot assay, a lateral flow assay (LFA), or an enzyme-linked immunosorbent assay (ELISA).

[0496] In one embodiment, the detection method comprises flow cytometry or microfluidics.

[0497] In one embodiment, the nanorod-binding agent conjugate comprises a plurality of detection moieties covalently attached to a plurality of at least one modified Ff phage coat protein.

[0498] In one embodiment, the nanorod-binding agent conjugate comprises at least two different detection moieties, each covalently attached to a different type of modified Ff phage protein.

[0499] In one embodiment, the nanorod-binding agent conjugate comprises a plurality of each of at least two different detection moieties, each covalently attached to a plurality of at least two different types of modified Ff phage proteins. In one embodiment, the nanorod binder conjugate is included in a composition comprising a population of nanorod binder conjugates.

[0500] In one embodiment, at least a portion of the nanorod binding agent conjugates in a population or composition comprise different detection moieties.

[0501] In one embodiment, the detection moiety is a moiety that produces a detectable chemical, spectral, linear dichroic, fluorescent, visual, chemiluminescent, paramagnetic, acoustic, electrical, surface plasmon resonance, isotopic, radioactive or other chemical or physical signal.

[0502] In one embodiment, the detection moiety is a fluorophore, a chromogenic substrate, a dye, a chemiluminescent, a paramagnetic compound, a small molecule, a semiconductor, a conductor, a nucleic acid, a polypeptide, a polymer, a quantum dot, or a radioisotope.

[0503] In one embodiment, the fluorophore or chromogenic substrate is a fluorophore or chromogenic substrate described herein.

[0504] In one embodiment, the detection moiety is selected from the group consisting of a fluorophore, a small molecule, a peptide, a protein, a polymer, a nucleic acid, an inorganic molecule, a dye, a radioisotope, a semiconductor, and a paramagnetic compound.

[0505] In one embodiment, the nanorod-binding agent conjugate comprises at least 3, 4, 5, 6, 7, 8, 9, or more different detection moieties.

[0506] In one embodiment, the nanorod binder conjugate comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 200, 300, 400 or more detection moieties.

[0507] In one embodiment, the nanorod-binding agent comprises one detection moiety for about every seven copies of Ff phage coat pVIII contained in the nanorod.

[0508] It is understood that, using the approaches described herein, any amine-reactive fluorophore, or other amine-reactive dye, or other amine-reactive small molecule, should be suitable for attachment to the nanorods. In one non-limiting example, we demonstrated fluorescent labeling of the BSF nanorods described herein using the amine-reactive fluorescent dye DyLight 550 (Example 11, Figure 20C). In this example, the nanorods also displayed a binding molecule formed by fusion of pIII with FnB (the fibronectin-binding domain of S. pyogenes; SEQ ID NOs: 37, 38, Figure 37) and were used in a lateral flow assay to detect an analyte (fibronectin, Example 11, Figure 20C). Such labeling is contemplated as an embodiment of the present invention.

[0509] Because each pVIII subunit BSF nanorod has three surface-exposed amino acid residues, Glu2, Asp4, and Asp5, containing side-chain carboxyl groups, carboxyl-reactive molecules can also be chemically conjugated to the nanorods. Other reactive groups, such as the aromatic hydroxyl group of Tyr residues, can also be used to attach suitable reactive groups, as known in the art (Bernard and Francis, 2014). The attached molecule can be any type of organic molecule, including biotin, which is useful for binding commercially available or in-house fusions of biotin-binding proteins such as avidin. Thus, the wide range of avidin fusions to antibodies, dyes, or other functional molecules allows for multiple methods of indirectly visualizing nanorods. Nanorods displaying detection molecules can bind to analytes and be indirectly visualized via phage-specific antibodies or chemically attached fluorescent dyes (Figure 20). Such labeled nanorods are contemplated as embodiments herein.

[0510] Labeled nanorods that also display analyte-specific molecules, such as antibodies, can also be used in immunoassays. In one non-limiting example, nanorods displaying pIII fusion proteins that specifically bind to a SARS-CoV-2 spike-specific single-chain antibody (Figure 38, SEQ ID NOs: 39 and 40) or a SARS-CoV-2 nucleoprotein-specific camelid single-domain antibody VHH (Figure 55, SEQ ID NOs: 99 and 100) were generated. These pIII fusions were combined with pVIII, which displays an N-terminal Ala-Ala-Gly-Gly (AAGG), and evolved to efficiently assemble nanorods (Figure 32, SEQ ID NO: 18; Figure 33, SEQ ID NO: 20). LPETA-biotin was enzymatically attached to the nanorods, and an avidin-alkaline phosphatase fusion was further attached to the biotin-modified nanorods, allowing visualization of the nanorods by indirect labeling. Nanorods modified in this manner are used in dot blots, ELISAs, and lateral flow assays (Figures 25-27), as described in the Methods. All such modified nanorods and methods of use are contemplated as embodiments herein.

[0511] Specifically contemplated as embodiments of this aspect of the invention are all of the embodiments described in the NPS, composition, nanorod, and method aspects herein, including, but not limited to, nucleic acid expression constructs, scaffold and filler nucleic acid sequences, vectors, plasmids, nanorod replication assembly plasmids, helper plasmids, BSF nanoreplication assembly cassettes, origins of replication, Ff phage proteins, modified Ff phage proteins, Ff phage genes, modified Ff phage genes (including amino acid and nucleic acid sequences, and all possible mutations and modifications), promoters, inducible promoters and operable linkages, selectable markers, auxotrophic markers, (+) strand circular ssDNA, fusion proteins, induction of production, host cells and host cell culture, replicated ssDNA, single and double transformations, length of nanorods generated, nanorod binder conjugates, and (+) strand ssDNA.

[0512] Additional embodiments, set forth below as a set of potential claims, are provided to assist the reader in better understanding the invention and its practice and are merely exemplary.

[0513] Exemplary numbered embodiments: 1. A nanorod generating system (NPS) comprising a single nucleic acid expression construct, the construct comprising: a BSF nanoreplication assembly cassette; at least one auxotrophic marker; at least one inducible promoter operably linked to a nucleic acid sequence encoding at least one Ff phage protein; and at least one plasmid origin of replication that is not located within a BSF nanoreplication assembly cassette. 2. The NPS of embodiment 1, wherein the nucleic acid expression construct is a plasmid or is contained within a plasmid. 3. The NPS of embodiment 1 or embodiment 2, wherein the BSF nanoreplication assembly cassette comprises a packaging signal (PS), a (+)Ori1, a (+)Ori2, and a (-)Ori. 4. The NPS of any one of embodiments 1 to 3, wherein the BSF nanoreplication assembly cassette comprises a scaffold nucleic acid sequence. 5. The NPS of embodiment 4, wherein the scaffold nucleic acid sequence comprises 0, 1, or 2 filler nucleic acid sequences. 6. The NPS of embodiment 4 or embodiment 5, wherein the scaffold nucleic acid sequence comprises filler nucleic acid sequences encoding at least one, preferably at least two, Ff phage coat or Ff phage modified coat proteins, preferably comprising filler nucleic acid sequences encoding pVII and pIX or modified pVII and pIX. 7. The NPS of any one of embodiments 1 to 6, wherein the at least one auxotrophic marker is selected from the group consisting of metE, glyA, infA, thyA, argE, delta-thi-1, thi1, leuB, proAB, ara, and nadC, preferably nadC. 8. The NPS of any one of embodiments 1 to 7, wherein the at least one inducible promoter is selected from the group consisting of lac, tac, araC, or trp promoters, preferably the lac promoter. 9. The NPS of any one of embodiments 1 to 8, wherein the at least one inducible promoter is operably linked to a nucleic acid sequence encoding at least one Ff phage replication protein, or at least one Ff phage coat protein, or both. 10. The NPS of embodiment 9, wherein at least one Ff phage replication protein is pII or modified pII. 11. The NPS of embodiment 9, wherein at least one Ff phage coat protein is pVIII or modified pVIII. 12. The NPS of embodiment 11, wherein the modified pVIII comprises at least one amber mutation. 13. The NPS of any one of embodiments 1 to 12, wherein the single nucleic acid expression construct comprises a nucleic acid sequence encoding at least one additional Ff phage protein, preferably at least two additional Ff phage proteins. 14. The NPS of embodiment 13, wherein the additional Ff phage protein is selected from the group consisting of pill and pVI. In one embodiment, the additional Ff phage protein is pill or pVI or both. 15. The NPS of any one of embodiments 1 to 14, wherein the nucleic acid expression construct comprises a nucleic acid sequence encoding a fusion protein comprising at least one Ff phage protein or modified Ff phage protein or a functional portion thereof fused to a binding protein or a binding portion thereof. 16. The NPS of any one of embodiments 1 to 15, wherein at least one plasmid origin of replication (p-ori) is a theta origin of plasmid replication, preferably the p-ori is selected from the group consisting of ColE1, pMB1, pSC101, R6K, ColD and 15A, preferably the p-ori is 15A. 17. A nanorod generation system (NPS), comprising: i) a nucleic acid nanorod replication assembly construct, BSF nanoreplication assembly cassette, at least one auxotrophic marker, and a nucleic acid nanorod replication assembly construct comprising at least one plasmid origin of replication that is not present in the BSF nanoreplication assembly cassette; ii) a helper nucleic acid expression construct, at least one selectable marker, and a helper nucleic acid expression construct comprising at least one inducible promoter operably linked to a nucleic acid sequence encoding at least one Ff phage protein. 18. A method for producing a plurality of nanorods, comprising: generating at least 1.0 x 10 nanorods per 1 L of host cell culture; 13 This involves inducing the formation of nanorods. 19. The method of embodiment 18, comprising transforming E. coli cells with only a single nucleic acid construct. 20. The method of embodiment 19, wherein the single nucleic acid construct comprises a BSF nanoreplication assembly cassette, at least one auxotrophic marker, at least one inducible promoter operably linked to a nucleic acid sequence encoding at least one Ff phage protein, and at least one plasmid origin of replication not located in the BSF nanoreplication assembly cassette. 21. A method for producing a plurality of nanorods, comprising directing replication of (+) strand circular ssDNA from a single nucleic acid construct comprising a BSF nanoreplication assembly cassette, a scaffold nucleic acid sequence, two nucleic acid sequences each containing a pII cleavage site (each cleavage site within a (+) axis), and at least one nucleic acid sequence encoding at least one modified Ff phage coat protein (preferably at least two, preferably two Ff phage coat proteins).

[0514] example Methods and experimental procedures Bacteria, bacteriophages, and plasmids All bacterial strains used in this disclosure are derived from the non-pathogenic E. coli laboratory strain K12 (Table 1) containing one, two, or three compatible plasmids (Tables 2, 3, and 4). The bacteriophages used in various aspects of nanorod production are derived from Ff (f1 and M13, Table 5).

[0515] Media and growth conditions Liquid medium 2xYT was used at a concentration of 1x (16 g / L tryptone, 10 g / L yeast extract, 5.0 g / L NaCl, pH 7.4-7.6). 2xYT is a standard microbial growth medium used for culturing E. coli and Ff bacteriophage. This nutrient-rich microbial culture medium contains peptides, amino acids, and water-soluble vitamins in a low-salt formulation. When needed as a solid medium, 2xYT was used at a concentration of 1x (16 g / L tryptone, 10 g / L yeast extract, 5.0 g / L NaCl, 1-2% agar, pH 7.4-7.6). Agar (BDDifco) was used as the solidifying agent.

[0516] Minimal M9 medium contained 1x M9 salts (final concentrations: 15 g / L KH2PO4, 64 g / L Na2HPO4, 2.5 g / L NaCl, 5 g / L NH4Cl, pH 7.2), 2 g / L MgSO4, and 0.1 g / L CaCl2. This was supplemented with 2 g / L glucose and 2 g / L casamino acids. Casamino acids are a mixture of amino acids and oligopeptides obtained by acid hydrolysis of casein and are typically used in microbial growth media. They contain all essential amino acids except tryptophan, which is degraded during casein hydrolysis. Because casamino acids do not contain nicotinic acid (NAD), nadC can be used as a selective auxotrophic marker.

[0517] Bacteria is Difco TM Cultures were grown in 2xYT (Becton-Dickinson, BD) or M9 minimal medium containing glucose and casamino acids, supplemented with nicotinic acid (NA) as needed. Liquid cultures were incubated for 37 min with continuous shaking (200 rpm) unless otherwise noted. oThe plates were incubated at 4°C. To prepare the plates, Bacto-agar BD (1%) was added to the above medium and solidified. Antibiotics were supplemented as necessary at the following concentrations: ampicillin (Amp) 100 μg / mL, kanamycin (Kan) 50 μg / mL, and chloramphenicol (Cm) 25 μg / mL.

[0518] Recombinant DNA Technology Methods General molecular biology and recombinant DNA techniques, such as PCR, DNA restriction digestion, ligation, DNA sequencing, DNA agarose gel electrophoresis, preparation of competent cells, and transformation and purification of plasmid DNA, were performed as previously described (Sambrook and Russell, 2001). DNA fragments for construction of recombinant plasmids and phages were either custom synthesized or PCR amplified. Specific modifications are indicated in the protocols described below.

[0519] Titration of infectious Ff phage or phage-like particles Ff phages or phage-like particles containing antibiotic resistance were quantified by titration using the overlay plating method. Phage titration was performed on 2xYT plates, which were supplemented with the appropriate antibiotic for titrating phage-like particles containing antibiotic resistance markers. RFor titration of marker-containing particles, an intermediate layer (9 mL) of antibiotic-free 2xYT was poured immediately prior to titration, allowing bacterial growth for the several hours (before antibiotic diffusion) necessary for successful transfection. Once this layer solidified, an overnight culture (100 μL) of the appropriate indicator strain was mixed with 2.5 mL of molten (50°C) 2xYT soft agar (0.5% agar), and the mixture was poured onto the surface of the solidified intermediate layer. Once the overlay solidified, 5 μL of 100-fold serial dilutions of phage or phage-derived particles were spotted onto the surface. Plates were incubated overnight at 37°C, and the following day, phage titers were calculated based on plaque counts, while the number of particles containing infectious markers was determined from the number of antibiotic-resistant transductants. Titers are expressed as plaque-forming units (pfu) or transducing particles (tdp) / mL. Titers are expressed as plaque-forming units (pfu) or transducing particles (tdp) per mL.

[0520] The pVIIIAlaGly△P6 mutant was evolved to restore filamentous phage assembly.

[0521] Bacteriophage R786, encoding pVIII designed for enzymatic attachment of LPXTA-tagged proteins or small molecules, contains an AlaGly insertion between maturation positions 1 and 2 and a Pro deletion at position 6. The titer of this phage (R786) was approximately 10 10 The typical titer for the control phage R785 (Ff phage) was approximately 10 12), which is approximately 100-fold lower than the previous titer. Therefore, the difference in titer was likely due to the deletion of AlaGly and / or Pro at position 6 inserted between positions 1 and 2 of mature pVIII. To "evolve" R786 to a titer consistent with R785, the original R786 stock was grown three times in liquid culture and passaged into the host strain K2091. The stock was transferred in large quantities from one round to the next without plaque purification. In each round, phage was plated at a low moi (1,000 bacteria per phage). The phage stock from the third round of growth was diluted and plated on K2091 medium, yielding 100–300 plaques per plate. Large plaques resembling R785 were detected on these plates. Phage from three large, isolated plaques were clone-purified. The clone-purified phage was grown using standard methods and analyzed by titration. Three evolved mutants that showed increased titers consistent with R785 were shown to have acquired point mutations in pVIII. The mutated phages were tested and titered as helpers in the standard phagemid vector pUC118. The phage that showed the highest titers contained the mutation L27S. This phage was designated R788. The pVIII-containing sequence was amplified and inserted into the pHP backbone to obtain pHP1Aev and pHP1AevIIICM.

[0522] Agarose gel electrophoresis of native Ff phage and Ff-derived nanorods Agarose gel electrophoresis was used for rapid detection and characterization of native Ff phage and phage-derived nanorods (Nelson et al., 1981). The running buffer was 1x TAE (40 mM Tris, 2 mM EDTA, 20 mM acetic acid), pH 9.0 or 8.3.

[0523] The pH 9.0 buffer is used to detect mature pVIII (gVIII am25Nanorod variants containing Ser instead of Glu at the second position of the tRNA were used in the presence of supD tRNA from the host (Table 3). Before loading the samples onto the gel, they were mixed with native loading buffer (final concentration: 1x TAE, 5% glycerol, 0.05% BPB, pH 9.0 or pH 8.3). Electrophoresis was run at 20 V (1.5 V / cm) for 15 h, followed by staining with ethidium bromide (10 μg / mLEtBr, 1x TAE, pH 8.3) for 20 min to visualize free DNA and RNA within the samples. Native, intact nanorods are not visible at this stage because their DNA is still present within the intact nanorods. To visualize the nanorods, the coating protein was removed and the gel was immersed in 0.2 M NaOH for 45 min to expose the ssDNA. After rinsing with MiliQ water for 10 min, the gel was neutralized by immersion in 0.45 mM Tris (pH 7.1) and stained with EtBr for an additional 20 min. The gel was then destained in water and photographed with a CCD camera. Fluorescently labeled nanorods were visualized directly without staining.

[0524] Agarose gel electrophoresis of SDS-digested Ff phage and phage-derived nanorods The gels contained 0.8%–1.2% (w / v) agarose (depending on the size of the ssDNA to be analyzed) in 1x TAE buffer (pH 8.3 or 9.0). The agarose was mixed with SDS buffer (1% SDS, 1x TAE, 5% glycerol, 0.05% BPB) and heated at 99°C for 10–15 min to disintegrate the particles. After returning to room temperature, the samples were loaded onto the agarose gel. Electrophoresis was performed at 3.7 V / cm for 150 min. The gel was stained with EtBr for 20 min, then destained and visualized using GelDocXR.

[0525] Nanorod generation For nanorod production, highly efficient electrocompetent cells of the appropriate strain were transformed with the pPopUp single nanorod production plasmid in the case of the single-plasmid production system. For the two-plasmid nanorod production system, cells already containing the helper plasmid were transformed with the pBSF nanotemplate plasmid. After transformation, cells were allowed to recover in SOC medium for 1 hour. For antibiotic selection, transformed cells were suspended in 10 mL of liquid medium containing the appropriate antibiotic concentration, as needed, in 2xYT medium. For auxotrophic selection, after recovery in SOC medium, cells were washed twice with 0.5% NaCl to remove nutrients and resuspended in 10 mL of M9 glucose Cas medium containing the appropriate antibiotic concentration. The resuspended cells (5 mL) were added to 500 mL of prewarmed medium containing the same components in a 2 L flask and incubated for 37 minutes with aeration. o For pPOP or helper plasmids containing gII driven by the lacUV5 promoter, the OD 600 IPTG was added to the culture at 0.1% ribosomal ratio. After 16 hours of culture, the cells were centrifuged (4 o C) at 8000 × g, and the nanorods from the supernatant were concentrated by PEG precipitation (2x YT culture) or ultrafiltration (M9CasGlucose culture).

[0526] Concentration of nanorods by PEG precipitation The culture supernatant was poured into a sterile centrifuge bottle, and PEG8000 powder was added at 5% for nanorods ≥ 100 nm in length and up to 15% for nanorods ≤ 100 nm in length. After the PEG was dissolved, NaCl powder was added to 0.5 M to dissolve the mixture, and the suspension was incubated on ice for 2 hours. The mixture was then cooled to 4°C. o The nanorods were pelleted by centrifugation at 8000 x g for 30 min at 4°C. The supernatant was decanted, and the "empty" centrifuge bottle was centrifuged again under the same conditions for 5 min to allow the nanorod pellet to settle to the bottom of the bottle. This is necessary because the filamentous phage PEG pellet precipitates as a sticky film along the bottle wall during centrifugation. The pellet obtained after PEG precipitation was resuspended in 5 ml of 1x TBS (pH 7.6) and the remaining insoluble debris was removed by centrifugation. oThe nanorods were pelleted by centrifugation at 8000 x g for 30 min at 37°C. Next, a buffer containing DNAse and RNAse (final concentrations of 12 μg / mL DNase, 40 μg / mL RNase, 5 mM MgCl2, 10 mM TRIS pH 8.0) was added to the supernatant and incubated at room temperature for 1 h. A final concentration of 20 mM EDTA was then added to inactivate the DNAse and RNAse. The particles were repurified by precipitation with a 5%-15% PEG, 0.5 M NaCl solution as described above. The nanorod pellet was resuspended in 0.5 mL 1x TBS (pH 7.6) and centrifuged again at 4000 x g for 10 min at room temperature to remove insoluble debris.

[0527] Concentration of nanorods by ultrafiltration The culture supernatant was filtered through a bottle-top filter (0.22 μm) to remove remaining cells and cell debris. Nanorods from the filtered supernatant were concentrated by ultrafiltration using an Amicon Stirred Cell 400 mL pressure system, following the method outlined in (Rakonjac and Model, 1998), with an additional wash step (three times with 100 ml of TBS pH 7.3 each). The retentate was collected in a test tube, and free DNA and RNA were removed from the nanorod suspension by adding DNAse and RNAse, as described in the previous section. Nanorods were precipitated with PEG as described in the paragraph above.

[0528] Purification of nanorods by CsCl gradient ultracentrifugation Cesium chloride gradient centrifugation was used to separate the concentrated nanorods from fine cellular debris and bacterial proteins. Approximately 1 mL of 1000x concentrated nanorods was mixed with 2 mL of the same buffer in which the nanorods were resuspended, containing 1.5 g of solid CsCl, and the volume was adjusted to 4 mL with buffer to obtain a final concentration of 0.375 g / mL CsCl. Ultracentrifugation at 100,000 x g and 18 °C for 16 hours formed a density gradient, separating the nanorods from cellular debris and residual DNA and RNA (Sattar et al., 2015). Depending on the amount of nanorods, they were either visible as a gray band or visually undetectable. In both cases, the nanorods were collected using a hypodermic needle. If a visible band was observed, the tube was punctured directly below the band. Once the band was no longer visible, the bottom of the centrifuge tube was punctured and 100 μL (4 drops) fractions were collected.

[0529] Fractions were analyzed by agarose gel electrophoresis of SDS-digested nanorods to identify fractions containing nanorods but free of cellular DNA or RNA. Fractions containing the strongest nanorod ssDNA band and free of residual RNA and DNA were combined and dialyzed against 3,000 volumes of 1x PBS or TBS buffer or 50 mM Tris-HCl pH 8 at 4°C using a Slide-a-Lyzer® dialysis cassette with a 50 kDa cutoff. Alternatively, the fractions were concentrated and desalted by spin ultrafiltration, as described below.

[0530] Purification of nanorods by anion exchange chromatography If removal of residual proteins co-fractionated with the nanorods during CsCl gradient centrifugation was required, the samples were subjected to a separate purification step by anion exchange chromatography. For this purpose, a strong anion Q, -N+(CH3)3 column, SepFast TM(BioToolomics) was used. The column was equilibrated with 10 column volumes (CV) of binding buffer (Buffer A: 50 mM Tris-HCl pH 8). Next, the sample containing nanorods was passed through the column and washed with Buffer A. The bound nanorods were then eluted from the column with a 0 to 1.5 M NaCl gradient (50 mM Tris buffer, pH 8). Column fractions corresponding to the absorption peak at 280 nm were collected and analyzed by SDS-PAGE to identify fractions containing pure nanorods based on the known Ff protein pattern.

[0531] Concentration of purified nanorods by spin ultrafiltration If necessary, the purified nanorods were concentrated and desalted by filtration through a 50 kDa cutoff filter using centrifugal force in a Vivaspin system (GE Healthcare) according to the manufacturer's instructions. If buffer exchange or desalting was required, the filter unit was washed up to six times with the desired buffer. o The nanorods were removed from the filter by storing it overnight at C. The filter was covered with buffer solution. The next day, the buffer solution was gently pipetted up and down onto the filter and collected in an appropriate sterile vial or tube.

[0532] Quantification of nanorods Because the nanorods were unlabeled, they were separated by agarose gel electrophoresis and quantified by densitometry of SDS-digested ssDNA (Rakonjac and Model, 1998). Each quantification gel was loaded with a series of known amounts of purified ssDNA extracted from similarly sized nanorods to generate a densitometric standard curve. Images of the EtBr-stained gels were analyzed using ImageJ software and Microsoft Excel.

[0533] Alternatively, highly purified nanorods (after CsCl gradient centrifugation or ion exchange chromatography) were quantified by spectrophotometer using a value of ε = 3.84 ml / (mg*cm) at a wavelength of 269 nm ( Day, 1969 ).

[0534] Staining of nanorods and transmission electron microscopy All transmission electron microscopy images (micrographs) were collected at the Manawatu Microscopy and Imaging Centre (MMIC), School of Basic Sciences, Manawatu Campus, Massey University. Purified phage or nanorod samples were diluted with Milli-Q water to a final concentration of 10. 10 Nanorods / mL. An 80 μL drop of the sample was placed on a Parafilm TM The sample was placed in a glass Petri dish lined with a film (Bemis Company Inc., USA). A Formvar / carbon-coated 200-mesh copper grid (Agar Scientific, coated in the lab) was placed film-side down on top of the sample drop and left for 4 minutes to allow the phage to adsorb to the grid. The grid was carefully lifted and placed on a sheet of Whatman No. 1 filter paper to remove excess liquid.

[0535] The phage nanorod-adsorbed film was stained by placing it on a drop of 2% uranyl acetate in Milli-Q and incubating it at room temperature for 4 minutes. The excess liquid was again drained off, and the film was placed on Whatman No. 1 paper to dry. Images were collected in a 100 kV TEM (FEI Tecnai G2 Spirit BioTWIN, Czech Republic).

[0536] Fibronectin lateral flow assay The pre-prepared dipsticks (containing collagen and pVIII-specific mouse monoclonal antibodies printed on the T and C lines, respectively) were stored in Ziploc® bags protected from light. Before use, the dipsticks were blocked overnight at 4°C using Odyssey™ blocking buffer supplemented with a 1:1,500 monoclonal anti-Fn antibody to minimize nonspecific binding of FnB-displayed nanorods to collagen that may be contaminated with Fn on the T line. Under the same conditions, the 96-well microtiter plate used for the reaction mixture was blocked using the same buffer without the Fn-specific antibody. After blocking, the dipsticks were rinsed twice with PBST buffer and dried at 37°C for 2 hours. A total of 10 dipsticks were used per assay. 11 Nanorods were mixed with serial dilutions of analyte in 1× PBS in a 96-well plate in a total volume of 50 μL and incubated at room temperature for 30 min.

[0537] The dried, blocked dipsticks were immersed in wells containing the reaction mixture for 15 minutes at room temperature, then removed from the wells, placed on filter paper, and dried at 37°C for 1 hour. Unlabeled nanorods bound to the dipsticks were visualized using a rabbit M13-specific antibody, followed by visualization using a secondary AP-conjugated antibody. Fluorescently labeled nanorods (DyLight® 550) were directly visualized using an Azurec 600 fluorescent imager.

[0538] High-density sortase-mediated labeling of BSF nanorods His-tagged sortase A (SrtA Sp) from Streptococcus pyogenes was expressed from the plasmid pET28a-SpySrtA (Table 4) and affinity-purified using Ni-NTA agarose. Sortase reactions were performed in a 500 μL volume in a microfuge tube. For biotin labeling, the reaction mixture contained 50 μM SrtA Sp, 200 μM K(biotin)-LPETAA (GenScript), and 5 nM nanorods displaying the Spike-specific antibody C121 (BSF nano728Aev1C121, approximately 3 × 10) in sortase buffer (50 mM Tris pH 7.5, 150 mM NaCl). 12 The mixture contained 1000 μM (1000 μM / mL) nanorods. The mixture was incubated at 37°C for 3 hours with continuous shaking. After incubation, 1 mL of sortase buffer was added to the microtube containing the reaction mixture to dilute the substrate and enzyme. The mixture was then transferred to an equilibrated VivaSpin tube (GE Health Care, 100 kDa cutoff, 2 mL volume) and centrifuged at 4000 × g for ≥ 10 minutes at 4°C until a residual volume of approximately 150 μL was reached. The flow-through was discarded, and the solution in the concentrator was replenished with TBS (25 mM Tris pH 7.6, 150 mM NaCl) to a volume of 1.5 mL. This procedure of centrifugation, removal of flow-through, and volume replenishment was repeated two more times. The Vivaspin tube was then centrifuged at 4000 × g for ≥ 10 minutes at 4°C to reach the desired concentration (approximately 150 μM). The concentrate containing the biotin-labeled nanorods was transferred to a microtube and stored for ≥ 4 minutes until further characterization and use. o Stored at C.

[0539] Sortase-mediated labeling of BSF nanorods with fluorescein isothiocyanate (FITC) was performed as described for biotin labeling, with some modifications. The reaction mixture contained 50 μM SrtA Sp, 200 μM FITC(Ahx)-LPETAA (Mimotopes, Australia), and 5 nM BSF nanorods (approximately 3 × 10 ) in sortase buffer (50 mM Tris pH 7.5, 150 mM NaCl). 12The mixture was then incubated in the dark.

[0540] Dot blot assay for detection of SARS-CoV-2 spike ectodomain (ECD) using nanorods displaying spike-specific scFv On a nitrocellulose membrane strip (2x9cm, 0.2µm pore size, Advantec), 2µL of each sample was gently pipetted onto a predefined area in the following order: SARS-CoV-2 spike ectodomain (ECD, SinoBiological Cat: 40589-V08B1) at 50ng / µL, 5ng / µL, and 0.5ng / µL; biotinylated BSF nano728Aev1C121 (10 11 10 μL of blocking buffer (3% bovine serum albumin, 20 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween 20) was added to the tube and incubated for 2 hours with continuous rotation at room temperature. The blocking buffer was then discarded. Biotinylated BSF Nano728Aev1C121 nanorods (10 11 Two mL of nanorods / mL (diluted from a stock in TBST (20 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween 20)) was pipetted into the tube and incubated at room temperature for 1 hour with continuous rotation. The membrane was then washed three times with 5 mL of TBST for 5 minutes each, followed by labeling with 5 mL of a 1:5000 dilution of streptavidin-alkaline phosphatase conjugate (Sigma) for 1 hour at room temperature. The membrane was then washed five times with 5 mL of TBST for 5 minutes each. Visualization was performed by incubating 2 mL of SIGMAFAST® BCIP™ / NBT working solution at room temperature for 15 minutes.

[0541] Enzyme-linked immunosorbent assay (ELISA) using unlabeled BSF nanorods ELISA was performed in a 96-well microplate (F96 Maxisorp Nunc-Immuno, ThermoFisher Scientific). First, plates were coated with 100 μL of CR3022 antibody (Abcam) per well at a concentration of 1 mg / mL in PBS (pH 7.4) overnight at 4°C. Afterwards, plates were washed once with 200 μL of TBST buffer (20 mM Tris pH 7.5, 150 mM NaCl, 0.05% Tween-20) per well and incubated with 200 μL of blocking buffer (5% low-fat milk powder in TBST) per well for 2 hours at room temperature. The blocking buffer was discarded, and the plates were washed once with TBST (200 μL / well). Next, SARS-CoV-2 ECD solution (100 μL / well) was added to the predefined wells at 10-fold dilutions from 10 ng / μL to 0.001 ng / μL prepared in blocking buffer and incubated for 1 hour at room temperature. Each ECD concentration treatment was performed in triplicate. Wells incubated with 100 μL of blocking buffer were included as negative controls. The plate was then washed five times with TBST (200 μL / well). The 10 ECD concentrations prepared in blocking buffer were added to the predefined wells and incubated for 1 hour at room temperature. 10A 100 μL solution of BSF Nano728Aev1C121 nanorods / mL was added per well and incubated at room temperature for 1 hour. The plate was then washed five times with TBST (200 μL / well). A 1:1000 dilution of M13-specific rabbit polyclonal antibody solution in blocking buffer (Invitrogen PA1-26758) was added at 100 μL / well and incubated at room temperature for 1 hour. The plate was then washed five times with TBST (200 μL / well). A 1:5000 dilution of HRP-conjugated anti-rabbit monoclonal antibody (NA934vs, Cytiva) in blocking buffer was added at 100 μL / well. The plate was then incubated at room temperature for 30 minutes and washed five times with TBST (200 μL / well). Signal development was achieved by adding 100 μL of 1-Step® UltraTMB-ELISA substrate solution (ThermoFisher Scientific) and incubating for 30 minutes at room temperature. The reaction was stopped by adding 100 μL of H2SO4 2M. Absorbance was measured at 450 nm.

[0542] ELISA assay using biotinylated BSF nanorods ELISAs were performed in 96-well microplates, F96 Maxisorp Nunc-Immuno and NUNC Immobilized Amino plates, which were used for assays in which antibodies and aptamers were immobilized as capture molecules, respectively (both from Thermo Fisher Scientific). For SARS-CoV-2 spike protein ELISAs, plates were first coated with 100 μL per well of SARS-CoV spike-specific capture antibody CR3022 (Abcam) at 1 mg / mL in PBS (pH 7.4). Alternatively, for SARS-CoV-2 nucleocapsid protein (NC) ELISAs, 100 μL of custom-synthesized aminated cognate aptamer (Cho et al., 2011) was added at 50 ng / mL. The plates were incubated overnight at 4°C or room temperature, respectively, and then washed once with 200 μL of PBST wash buffer (PBS pH 7.4, 0.05% Tween-20) per well and incubated with 200 μL of Odyssey blocking buffer (Licor) for 2 hours at room temperature. The blocking buffer was discarded, and the plates were washed once with PBST (200 μL / well). Next, antigen solutions prepared in PBST were added to predefined wells (100 μL / well). Spiked ECD was added at 10-fold serial dilutions ranging from 10 to 0.001 ng / μL, and recombinant NC was added at dilutions ranging from 10 to 0.0000001 ng / μL and incubated for 1 hour at room temperature. Each antigen concentration was assayed in triplicate. Wells incubated with 100 μL of PBST buffer were included as negative controls. The plates were then washed five times with PBST (200 μL / well). 10 prepared in PBST 9Biotinylated BSF Nano728Aev1C121 (Spike ECD ELISA) or BSF Nano728AevN3 (NC ELISA) nanorod solution was added at 100 μL per well and incubated at room temperature for 1 hour. The plat...

Claims

1. 1. A nanorod generating system (NPS) comprising a nucleic acid expression construct, a replication assembly cassette comprising a filamentous phage (+)Ori1, a packaging signal (PS), and a (+)Ori2; at least one plasmid origin of replication that is not present in the replication assembly cassette and that allows replication of the construct in bacteria; at least one inducible promoter operably linked to a nucleic acid sequence encoding at least one Ff phage replication protein; The expression construct expresses Ff phage replication proteins to generate excised and replicated DNA sequences from the replication assembly cassette, forming circular single-stranded DNA encapsulated within nanorods, the nanorod generating system (NPS).

2. The NPS of claim 1 , wherein at least one Ff phage replication protein is pII.

3. The NPS of claim 1, wherein the replicating assembly cassette further comprises a (-) ori between PS and (+) ori 2.

4. The NPS of claim 1 , wherein the nucleic acid expression construct is a plasmid.

5. The NPS of claim 1 , wherein the NPS lacks a second nucleic acid construct encoding one or more filamentous phage proteins.

6. The NPS of claim 1, wherein the nucleic acid expression construct comprises nucleic acid sequences encoding each of the Ff phage pI to pXI proteins.

7. The NPS of claim 6, wherein any or all of the nucleic acid sequences encoding each of the Ff phage pI to pXI proteins encode modified Ff phage proteins.

8. The NPS of claim 1, wherein the nucleic acid construct comprises a nucleic acid sequence encoding a modified Ff phage protein comprising mutations that allow chemical or enzymatic conjugation of small molecules, synthetic polymers, or biological polymers, and optionally the modified Ff phage protein is pIII and / or pVIII.

9. The NPS of claim 1 , wherein the nucleic acid construct comprises a nucleic acid sequence encoding a modified Ff phage protein pVIII containing an amber mutation.

10. The NPS of claim 1 , wherein the nucleic acid construct comprises a nucleic acid sequence encoding at least one of pIII, pVI, pVII, pVIII, and pIX fused to a nucleic acid sequence encoding a heterologous polypeptide.

11. The NPS of claim 1 , wherein the nucleic acid expression construct further comprises a nucleic acid sequence encoding an auxotrophic marker.

12. The NPS of claim 1, wherein the nucleic acid sequence between (+)Ori1 and PS or between PS and (+)Ori2 is a filler nucleic acid sequence encoding at least one Ff phage protein.

13. The NPS of claim 12 , wherein the filler nucleic acid sequence encodes pVII, pVIII, and / or pIX.

14. The NPS of claim 12 , wherein the filler nucleic acid sequence further encodes a prokaryotic or eukaryotic protein of interest.

15. The NPS of claim 1 , wherein the bacterium is Escherichia coli.

16. An isolated host cell comprising the NPS of claim 1.

17. A method for producing nanorods, comprising culturing isolated host cells containing the NPS of claim 1 and supplying the host cells with an inducer for the inducible promoter during an optimal growth phase, whereby Ff phage replication proteins are expressed intracellularly to produce excised and replicated DNA sequences, forming circular single-stranded DNA encapsulated within the nanorods.

18. 18. The method of claim 17, wherein the optimal growth phase is determined by the optical density (OD600) of the host cells.

19. 18. The method of claim 17, wherein the Ff phage replication protein is pII.

20. A nanorod having a length of 60 to 800 nm encapsulating a circular single-stranded DNA excised by pII cleavage of a replication assembly cassette comprising a filamentous phage (+)Ori1, a packaging signal (PS), and a (+)Ori2, and a filler nucleic acid sequence between the (+)Ori1 and the PS or between the PS and the (+)Ori2, wherein the filler nucleic acid sequence encodes at least one Ff phage protein.

21. The nanorod of claim 20 , wherein the replica assembly cassette further comprises a (−) Ori between PS and (+) Ori 2.

22. 21. The nanorod of claim 20, wherein the filler nucleic acid sequence encodes pVII, pVIII and / or pIX, or encodes modified pVII, pVIII and / or pIX, or a combination thereof.

23. 23. The nanorod of claim 22, wherein the nucleic acid sequence encoding pVII, pVIII and / or pIX and / or modified pVII, pVIII and / or pIX is fused to a nucleic acid sequence encoding a heterologous polypeptide.

24. 21. The nanorod of claim 20, wherein the filler nucleic acid sequence further encodes a heterologous polypeptide that may or may not be fused to the Ff phage protein or modified Ff phage protein.

25. 21. The nanorod of claim 20, having a length of about 95 to 125 nm.

26. A nanorod population encapsulating circular single-stranded DNA excised by pII cleavage of a replication assembly cassette comprising a phage (+)Ori1, a packaging signal (PS), and a (+)Ori2, and a filler nucleic acid sequence between the (+)Ori1 and the PS or between the PS and the (+)Ori2, wherein the filler nucleic acid sequence encodes at least one Ff phage protein, and at least 70% of the nanorods in the population have a length of about 40 to about 800 nm.

27. 27. The nanorod population of claim 26, wherein the replica assembly cassette further comprises a (-) Ori between the packaging signal and the (+) Ori 2, and wherein at least 70% of the nanorods in the population have a length of about 60 to about 800 nm.

28. 28. The nanorod population of claim 27, wherein at least 70% of the nanorods in the population have a length between about 60 and about 400 nm.

29. 28. The nanorod population of claim 27, wherein at least 70% of the nanorods in the population have a length between about 60 and about 300 nm.

30. 28. The nanorod population of claim 27, wherein at least 70% of the nanorods in the population have a length between about 95 and about 125 nm.

31. Nanorods approximately 35 to 45 nm in length encapsulating circular single-stranded DNA excised by pII cleavage of a replication assembly cassette containing a filamentous phage (+)Ori1, a packaging signal (PS), and (+)Ori2, but lacking a (-)Ori.

32. 32. A nanorod population comprising a plurality of nanorods according to claim 31, wherein at least 70% of the nanorods in the population have a length between about 38 and about 42 nm.

33. 33. The nanorod population of claim 32, wherein at least 70% of the nanorods in the population have a length of about 40 nm.

34. 1. A nanorod generation system (NPS), comprising: i) a nucleic acid nanorod replication assembly construct, a replication assembly cassette comprising a filamentous phage (+)Ori1, a packaging signal (PS), and a (+)Ori2; and a nucleic acid nanorod replication assembly construct that includes at least one plasmid origin of replication that is not present in the replication assembly cassette and that allows replication of the construct in bacteria; ii) a helper nucleic acid expression construct, at least one selectable marker, and a helper nucleic acid expression construct comprising at least one inducible promoter operably linked to a nucleic acid sequence encoding at least one Ff phage replication protein; The helper nucleic acid construct expresses Ff phage replication proteins to generate DNA sequences that are excised and replicated from the replication assembly cassette to form circular single-stranded DNA encapsulated within nanorods, the nanorod generating system (NPS).

35. The NPS of claim 34, wherein the replicating assembly cassette further comprises a (-) Ori between PS and (+) Ori 2.

36. 35. The NPS of claim 34, wherein the Ff phage replication protein is pII.

37. 35. An isolated host cell comprising the NPS of claim 34.

38. 1. A method for producing nanorods, comprising: Culturing an isolated host cell comprising the NPS of claim 34; and supplying an inducer to the inducible promoter to the host cell during an optimal growth phase, thereby expressing the Ff phage replication protein pII to produce a DNA sequence that is excised and replicated from the replication assembly cassette, forming a circular single-stranded DNA that is encapsulated within the nanorod.

39. 1. A method for detecting a target molecule in a sample, comprising: c) contacting a sample containing or suspected of containing a target molecule with a nanorod-binding agent conjugate; d) detecting binding of the nanorod-binding agent conjugate to the target molecule; The nanorod binder conjugate comprises at least one modified Ff phage coat protein, and the nanorods in the nanorod binder conjugate are generated from the NPS of claim 1 or 34, are nanorods of any one of claims 20 or 31, are one of the nanorod populations of claim 26 or 32, or are made by the method of any one of claims 17 or 38.

40. 40. The method of claim 39, wherein the nanorod-binding agent conjugate comprises at least one detection moiety covalently attached to at least one modified Ff phage coat protein.

41. 40. The method of claim 39, wherein the nanorod-binding agent conjugate comprises one detection moiety for about every seven copies of Ff phage coat protein pVIII or modified pVIII contained in the nanorod.

42. 40. The method of claim 39, wherein the nanorod-binding agent conjugate comprises a plurality of detection moieties covalently attached to a plurality of at least one modified Ff phage coat protein.

43. 40. The method of claim 39, wherein the nanorod binding agent conjugate comprises at least 3, 4, 5, 6, 7, 8, 9, or more detection moieties.

44. 40. The method of claim 39, wherein the nanorod-binding agent conjugate comprises at least two different types of modified Ff phage coat proteins.

45. 40. The method of claim 39, wherein the nanorod-binding agent conjugate comprises at least two different types of detection moieties.

46. 40. The method of claim 39, wherein the nanorod-binding agent conjugate comprises at least 3, 4, 5, 6, 7, 8, 9 or more different types of detection moieties.

47. 40. The method of claim 39, wherein the nanorod-binding agent conjugate comprises a plurality of each of at least two different detection moieties, each detection moiety being covalently attached to a plurality of at least two different types of modified Ff phage coat proteins.

48. 40. The method of claim 39, wherein the nanorod binder conjugate is included in a population of nanorod binder conjugates.

49. 49. The method of claim 48, wherein at least a portion of the nanorod-binding agent conjugates in the population comprise different detection moieties.

50. 40. The method of claim 39, wherein the target molecule is immobilized on the solid support by binding to a capture molecule attached to the support.

51. 51. The method of claim 50, wherein the binding agent is selected from the group consisting of a small molecule or a polypeptide.

52. 52. The method of claim 51 , wherein the polypeptide is selected from the group consisting of antibodies, antibody-derived single chain variable domains (scFv), camelid single chain antibody domains VHH, and other types of antibodies and analyte-binding polypeptides.

53. 40. The method of claim 39, wherein detecting comprises detecting a chemical signal, a spectral signal, a linear dichroism signal, a fluorescent signal, a visual signal, a chemiluminescent signal, a paramagnetic signal, a sonic signal, an electrical signal, a surface plasmon resonance signal, an isotopic signal, a radioactive signal, or other chemical or physical signal.

54. 40. The method of claim 39, wherein the detection comprises visual detection or fluorescent signal detection via an enzymatic reaction using a chromogenic or chemiluminescent substrate.

55. 40. The method of claim 39, which is a dot blot assay, a lateral flow assay (LFA), or an enzyme-linked immunosorbent assay (ELISA).

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