Methods and compositions for delivery of polynucleotides
This disclosure provides compositions and methods for delivery of a polynucleotide to an organism. More specifically, this disclosure relates to compositions including a mixture of a polynucleotide and a cationic polysaccharide, and methods of providing such compositions to an organism, such as a pest (e.g., an insect, a nematode, a mollusk).
1. A composition comprising:
a. a cross-linked cationic polysaccharide, and
b. a polynucleotide,
wherein the polysaccharide is selected from the group consisting of starch and guar gum,
wherein the polynucleotide comprises at least 21 contiguous nucleotides that are complementary or identical to a target gene in a pest,
wherein the polynucleotide and the cross-linked cationic polysaccharide are bound by electrostatic attraction to form a complex,
wherein the complex has at least one dimension that exceeds 200 nanometers or has at least one dimension that exceeds 500 nanometers when hydrated, and
wherein the composition exhibits improved rain fastness for the polynucleotide compared with a composition without the cross-linked cationic polysaccharide.
2. The composition of claim 1 , wherein the complex is a porous, hydrophilic particle.
3. The composition of claim 1 , wherein the complex is non-soluble in an aqueous solution.
4. The composition of claim 1 , wherein the composition is provided as an aqueous suspension.
5. The composition of claim 1 , wherein the cross-linked cationic polysaccharide comprises one or more cationic functional groups.
6. The composition of claim 5 , wherein the one or more cationic functional groups comprises an amine or an ammonium cation.
7. The composition of claim 6 , wherein the amine or the ammonium cation
a). is one or more selected from the group consisting of a primary amine, a secondary amine, a tertiary amine, and a quaternary ammonium cation;
b). is one or more selected from the group consisting of an optionally substituted amine, a nitrogen-containing heterocyclyl, a quaternary ammonium, and a nitrogen-containing heteroaryl, each of which may be optionally independently substituted with one or more of hydroxyl, alkyl, cycloalkyl, alkenyl, alkoxy, amino, aryl, heterocyclyl, or heteroaryl; or
c). is one or more selected from the group consisting of —NH 2 , —NHR 1 , —NR 1 R 2 , —NR 1 R 2 R 3 , and —N + R 1 R 2 R 3 R 4 , wherein R 1 , R 2 , R 3 , and R 4 are each independently alkyl, each of which may be optionally independently substituted with one or more of hydroxyl, alkyl, alkenyl, alkoxy, amino, aryl, heterocyclyl, or heteroaryl.
8. The composition of claim 1 , wherein the cross-linked cationic polysaccharide is provided by reaction of a non-cross-linked polysaccharide with an alkylating agent.
9. The composition of claim 1 , wherein the cross-linked cationic polysaccharide is provided by reaction of
a). a non-cross-linked polysaccharide with diacid chlorides of dicarboxylic acids or with diglycidyl ethers;
b). a non-cross-linked polysaccharide with phosphoryl chloride (POCl 3 ), diglycidyl polyethylene glycol (PEG), diglycidyl bisphenol A, diglycidyl 1,4-butanediol, oxalyl chloride, phthaloyl chloride, or adipoyl chloride; or
c). a non-cross-linked starch with glycidyl trimethylammonium chloride (GTAC) and with phosphoryl chloride (POCl 3 ).
10. The composition of claim 1 , wherein the polynucleotide comprises RNA.
11. The composition of claim 1 , further comprising one or more selected from water, a biocide, an antimicrobial, an antifungal, a non-polynucleotide pesticide, a chelator, a buffer, a filler, a binder, a hardener, a sticking agent, a pH modifier, a dispersant, a non-ionic surfactant, a zwitterionic surfactant, a defoamer, a silicate, and a photo-protectant.
12. A method of controlling a pest infestation, comprising providing an insecticidally effective amount of the composition of claim 1 in the diet of the pest, wherein the polynucleotide comprises at least 21 contiguous nucleotides that are complementary or identical to a target gene in the pest.
13. The method of claim 12 , wherein the pest is a Colorado potato beetle ( Leptinotarsa decemlineata ), a potato leafhopper ( Empoasca fabae ), a potato aphid ( Macrosiphum euphorbiae ), or a green peach aphid ( Myzus persicae ).
14. The method of claim 12 , wherein the pest is a flea beetle.
15. The method of claim 12 , wherein the composition is applied to a plant surface.
16. The method of claim 12 , wherein the composition is applied as a foliar spray or to a seed.