JPWO2020191072A5 - - Google Patents

Claims (22)

A double-stranded artificial nucleic acid construct,
(a) a first strand and a second strand, each comprising 10-30 nucleotides or nucleosides in length, or a combination thereof;
(b) terminal overhangs on one or both of the 3' ends of said first strand and/or said second strand;
including
One or both of the 3' terminal overhangs comprise ribose or deoxyribose with 2'-O-R and/or 3'-O-R modifications, where the R groups are alkyl, aryl, haloalkyl, amino , methyl, acetyl, and halo; and
A double-stranded artificial nucleic acid construct, wherein at least 80% of said sugars in said double-stranded artificial nucleic acid construct comprise deoxyribose. a. both the 3' ends of said first strand and said second strand comprise terminal overhangs; and/or
b. one or both of the 3' terminal overhangs comprise a 2'-modified ribose unit having a methoxy group (-O-Me) at the 2'-position; and/or
c. one or both of said terminal overhangs is 1 nucleotide in length; and/or
d. all sugars in the double-stranded artificial nucleic acid construct are deoxyribose, except for the terminal 3' sugars of the first and second strands; and/or
e. 2. The double-stranded artificial nucleic acid construct of Claim 1, wherein said first strand and said second strand each comprise at least about 20 to about 30 nucleotides or nucleosides or combinations thereof in length. The first strand and the second strand have at least 80%, 85%, 90%, 95%, or 98%, or about 100% sequence identity with a portion of the transcriptional regulatory region of the gene of the agricultural product. and optionally,
(i) the transcriptional regulatory region is upstream of a transcription initiation site, TATA box, or activation sequence; and/or
(ii) a portion of said transcriptional regulatory region comprises at least 30% guanine-cytosine content; and/or
(iii) The double-stranded artificial nucleic acid construct of claim 1 or 2, wherein the agricultural product is soybean, corn, rice, tomato, alfalfa, wheat, potato, or pea. The double-stranded artificial nucleic acid construct according to any one of claims 1 to 3, wherein both said 3' terminal overhangs comprise a ribose unit having a methoxy group (-O-Me) at the 2' position. a. Upon contact with an organism, at least a portion of said artificial nucleic acid construct is configured to facilitate epigenetic modification of at least one base of a nucleotide or nucleoside in a nucleic acid sequence of said organism; Plants, seeds, fruits, leaves, stems, roots, flowers, archaebacteria, bacteria, fungi, viruses, virus-like particles, protists, algae, nematodes, parts of any of these, or any combination thereof and/or
b. said artificial nucleic acid construct promotes said epigenetic modification via a system comprising at least in part a DNA methyltransferase, a biologically active fragment thereof, or a derivative thereof, said system comprising an RNA-directed DNA methylation pathway; and/or
c. said epigenetic modification adds a methyl group to at least one cytosine of a nucleotide or nucleoside in a nucleic acid sequence of said organism; and/or
d. said artificial nucleic acid construct promotes said epigenetic modification in the absence of CRISPR, CRISPR-associated proteins (Cas), biologically active fragments thereof, derivatives thereof, fusion proteins thereof, or any combination thereof and/or
e. said artificial nucleic acid construct can promote said epigenetic modification in the absence of Cas, including Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, or any combination thereof; and/or
f. said modification increases the stability of said artificial nucleic acid construct; and/or
g. The artificial nucleic acid construct of any one of claims 1-4, wherein said modification increases uptake of said artificial nucleic acid construct by said organism. wherein each strand has a length of 24 nucleotides or nucleosides or a combination thereof and both 3' terminal overhangs comprise modified ribose with a methoxy group (-O-Me) at the 2' position. The artificial nucleic acid construct according to any one of 1-5. A plurality of at least about 2-100, about 2-50, or about 2-20 artificial nucleic acid constructs of any one of claims 1-6, wherein at least A plurality of artificial nucleic acid constructs, wherein about 10% to about 100% differ among said plurality of artificial nucleic acid constructs. A plant, seed, solution , microorganism, fertilizer or soil comprising an artificial nucleic acid construct according to any one of claims 1 to 6 or a plurality of artificial nucleic acid constructs according to claim 7 . ( 1 ) A formulation comprising the double-stranded artificial nucleic acid construct according to any one of claims 1 to 6 or the plurality of artificial nucleic acid constructs according to claim 7 , and (b) an excipient . 10. The formulation of claim 9, comprising an excipient and a plurality of double-stranded artificial nucleic acid constructs . 11. A formulation according to claim 9 or 10 , wherein said excipient is an agriculturally acceptable excipient. 12. The formulation of claim 11, wherein said excipient is water. A method comprising applying a substance to produce, said produce comprising seeds, plants, plant components, or any combination thereof, said substance comprising any one of claims 1-6. A method comprising an artificial nucleic acid construct according to claim 7, a plurality of artificial nucleic acid constructs according to claim 7, or a formulation according to any one of claims 9-12. 14. The method of claim 13, wherein a gene in said produce is at least partially silenced or activated, optionally said gene is at least partially silenced for at least one regeneration cycle. Where said applying is to: (a) prevent or reduce or delay enzymatic browning of said produce when comparing said produce to a comparable produce to which the material comprising said artificial nucleic acid construct is not applied; (b) (c) decreasing the growth rate, yield, or longevity of said produce; (d) pest resistance, salt tolerance, heat tolerance, heavy metal resistance, disease resistance of said produce; (e) an increase or at least a partial decrease in the amount or production of molecules made by said produce; (f) a discoloration of at least a portion of said produce; (g) flowering of said produce. (h) increased volume or weight of said produce; (i) improved flavor or texture of edible products of said produce; (j) increased shelf life of said produce; 15. The method of claim 13 or 14 , which results in one or more of: (k) reducing seed number and size of said produce; (j) increasing nutrient content of said produce. A method according to any one of claims 13-15, wherein said produce is a seed or a plant. 16. The method of any one of claims 13-15, wherein said applying causes methylation of at least one nucleotide or nucleoside base in the nucleic acid of said produce gene. wherein said first strand of said nucleic acid construct has 100% sequence identity with a portion of the transcriptional regulatory region of said gene, and said second strand is complementary to said first strand. Item 18. The method according to any one of items 13-17. A method according to any one of claims 13 to 18, wherein said produce is a plant and said substance is applied to leaves, stems, roots or flowers, or a combination thereof. A method according to any one of claims 13 to 19, wherein said produce is seed. 21. The method of any one of claims 13-20, wherein the agricultural product is soybean, corn, rice, tomato, alfalfa, wheat, potato, or pea. A method according to any one of claims 13-21, wherein the substance is applied as a formulation according to any one of claims 9-12.