Molecular nanotags

#This disclosure concerns nanoscale molecular tags and their use for the detection of targets, for example in a flow cytometer.

#Improved methods and devices for single nanoparticle detection, resolution and/or sorting would be advantageous for both clinical and research purposes. For example, they would be useful to identify and analyze extracellular vesicles (EVs) and other nanoscale particles released by cells that have important biological functions and significant biomedical potential for use as therapeutic agents, targets or biomarkers. It is generally accepted that the constituent components and biological function of EVs vary, based on the type of cell that produces them and the conditions under which they are produced (Raposo and Stoorvogel, J Cell Biol 200(4):373-383, 2013). However, it has not previously been possible to characterize subsets of these particles in the way that cellular lineages and subsets have been defined. Similarly, it has previously been difficult to detect, sort and count other nanoscale particles as well as individual molecular components of these nanoscale particles. An obstacle to this technology has been the lack of available tools and reagents to analyze, sort, and functionally study individual nanoscale particles, based on specific attributes.

#Fluorescent activated cell sorting (FACS) has been used since its introduction by Herzenberg and colleagues in 1972 to identify and sort labeled subsets of cells (Julius et al., Proc Natl Acacl Sci USA 69(7):1934-1938, 1972; Bonner et al, Rev Sci Instrum 43:404-409, 1972), but sorting submicron subpopulations has not been considered feasible for particles smaller than approximately 500 nm. Conventional wisdom in flow cytometry holds that the signal from particles smaller than one micrometer would be lost in the signal from sample debris and electronic noise and thus remain undetectable. Certain modern high resolution instruments have extended the sensitivity range to be able to detect EVs to the about 200 nm EV size and about 10 fluorescent molecule detection limits, but there are no instruments currently capable of detecting, analyzing, and sorting sub-200 nm EVs based on the detection of a single epitope. Therefore, a need exists for enhancements in reagents and methods, such as flow cytometry, that would allow for the detection and quantification of single molecules, such as a single receptor on the surface of an EV.

#At least some of the following figures are submitted in color:

#FIGS. 1A-1B: Schematic drawings of exemplary molecular nanotags. FIG. 1A illustrates molecular nanotags include a core particle 10 having a high complex refractive index (RI), such as a quantum nanocrystal or other high complex RI nanomaterial (such as silver or gold), and an “armor” 12, such as thiolated single-stranded DNA with a single functionalized DNA end. The functionalized DNA end 14 serves as a “Lock” (or a binding site for a “Lock”) which can bind a specific “Key,” such as a target ligand. Using the nanoFACS method, single particles can be resolved by side scattered (SSc) and/or fluorescent light and monovalent nanotags enable single molecule ligand enumeration. Alternatively, the single particles can be imaged by microscopy or other optical methods (e.g., nanoparticle tracking or SERS, see for example Stremersch et al., Small 12:3292-301, 2016). FIG. 1B shows an example of a monovalent armored quantum nanocrystal (such as a QDOT®) 20 with a thiolated single-stranded DNA armor 12 and single functionalized DNA end 14. The armored quantum nanocrystals can also be used in tandem using complementary DNA ends.

#FIG. 1C schematically illustrates monovalent quantum nanocrystals of the type that were disclosed in Farlow et al., Nat. Methods 10(12): 1203-1205 (2013), which is incorporated by reference. The monovalent quantum nanocrystals of Fowler et al. are not detectable as single particles by high resolution flow cytometry such as NanoFACS. The particles are only detected if they are run in massive excess such that they are detected as “swarms” of multiple coincident particles in the laser intercept. The figure also illustrates variations of that prior approach to produce molecular composite structures that can selectively bind to targets.

#The nucleic sequences are shown using standard letter abbreviations for nucleotide bases, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The sequence listing generated on Mar. 29, 2019 13 Kb, and submitted herewith, is herein incorporated by reference.

#SEQ ID NO: 1 is a A10-3.2 PSMA aptamer sequence.

#SEQ ID NO: 2 is a A9g PSMA aptamer sequence.