Ff-nano, short functionalized nanorods derived from Ff (f1, fd, or M13) filamentous bacteriophage

Abstract

F-specific filamentous phage of Escherichia coli (Ff: f1, M13, or fd) are long thin filaments (860 nm × 6 nm). They have been a major workhorse in display technologies and bionanotechnology; however, some applications are limited by the high length-to-diameter ratio of Ff. Furthermore, use of functionalized Ff outside of laboratory containment is in part hampered by the fact that they are genetically modified viruses. We have now developed a system for production and purification of very short functionalized Ff-phage-derived nanorods, named Ff-nano, that are only 50 nm in length. In contrast to standard Ff-derived vectors that replicate in E. coli and contain antibiotic-resistance genes, Ff-nano are protein-DNA complexes that cannot replicate on their own and do not contain any coding sequences. These nanorods show an increased resistance to heating at 70(∘)C in 1% SDS in comparison to the full-length Ff phage of the same coat composition. We demonstrate that functionalized Ff-nano particles are suitable for application as detection particles in sensitive and quantitative "dipstick" lateral flow diagnostic assay for human plasma fibronectin.

Keywords: M13 phage; f1 phage; fd phage; fibronectin-binding protein; filamentous phage; lateral flow; nanorod; phage display.

FIGURE 1

The system for production of functionalized Ff-nano. Escherichia coli cells containing the Ff-nano production plasmid pNJB7 were infected with the helper phage Rnano3FnB containing the coding sequence of a “probe” or “detector” protein fused to pIII. Upon infection, pII from the helper phage induces positive strand replication from the pNJB7 Ff-nano origin of replication and also provides all other phage proteins and assembly machinery for production of the Ff-nano particles. All five copies of pIII are fusions to the probe (only three copies of pIII fusions are shown).

FIGURE 2

Transmission electron micrographs (TEM) of Ff-nano particles. (A) Cryo-negative TEM of particles obtained using the R777 helper phage. (B) Negative-stain TEM of particles obtained using the Rnano3 helper/vector. Arrow in (A) points to a double-length particle. Samples were prepared as described in Section “Materials and Methods.”

FIGURE 3

Ff-nano resistance to heating in SDS. (A) Intact (undamaged) particles and free ssDNA released from particles by the heat/SDS-treatment. Free ssDNA was visualized by staining the gel with ethidium bromide. Bands corresponding to the intact particles were visualized after soaking the gel in 0.4 M NaOH (see Materials and Methods for details). Gel sections containing the bands corresponding to the free ssDNA and intact full-length and Ff-nano particles are boxed. (B) Free ssDNA only, released by the heat/SDS treatment (visualized by direct ethidium bromide staining of the gel prior to NaOH treatment).

FIGURE 4

Confirmation of Ff-nano-FnB display by phage ELISA assay. (A) Schematic representation of the fibronectin-binding phage ELISA assay. (B) Assay result. Fibronectin was immobilized on a microtiter plate at a saturating concentration of 40 ng/μL (2 μg per well of a 96-well plate), whereas control wells for each assay were coated with PBS alone or BSA (1%) in PBS. The wells were exposed to (1 × 10⁸) Ff-nanoFnB or full-length phage Rnano3FnB that both displayed FnB as a pIII fusion, or control particles Ff-nano and Rnano3 that did not display FnB. After washing of the wells, bound Ff-nano or full-length phage were detected using a primary antibody to the major coat protein, then the secondary HRP-conjugated antibody, followed by detection of HRP through an enzymatic reaction according to the standard ELISA protocols. Data are presented as an average of three measurements. Error bars show standard deviation.

FIGURE 5

Fibronectin dipstick assay using Ff-nanoFnB. (A) Schematic representation of a lateral-flow dipstick assay; Fn-detection dipstick assay using: (B) unlabeled; (C) FITC-labeled particles. Each assay (50 μL) contained 1 × 10¹⁰ full-length Rnano3 (Rnano3FnB) or 1 × 10¹¹ Ff-nano (or Ff-nanoFnB) particles and 1 μg of Fn. The assay was performed and the unlabeled or FITC-labeled particles were detected as described in Section “Materials and Methods.” The test line, printed with collagen solution, appears as a triple band when the signal is high. This is due to secondary lines flanking the main line that form during printing of collagen on the card. The triple banding during printing is caused by the acidity of the solution, necessary to keep the collagen soluble (0.25% acetic acid).

FIGURE 6

Detection range of Fibronectin using Ff-nanoFnB. Series of Fn (analyte) dilutions analyzed using Ff-nanoFnB-based dipsticks were used for determination of lower detection limit (A) and quantitative range (B,C). Asterisk in (A) denotes the lowest Fn concentration at which the signal was detected on the test line. The graph in (C) corresponds to the Test/Control signal ratio vs. Fn concentration. Each assay contained Ff-nanoFnB particles (2 × 10¹¹) mixed with Fn at the indicated final concentrations in a total volume of 50 μL. The assay was performed and the signal quantified as described in the Section “Materials and Methods.”