Extended dicer substrate agents and methods for the specific inhibition of gene expression

Abstract

The invention provides compositions and methods for reducing expression of a target gene in a cell, involving contacting a cell with an isolated double stranded nucleic acid (dsNA) in an amount effective to reduce expression of a target gene in a cell. The dsNAs of the invention possess a pattern of deoxyribonucleotides (in most embodiments, the pattern comprises at least one deoxyribonucleotide-deoxyribonucleotide base pair) designed to direct the site of Dicer enzyme cleavage within the dsNA molecule. Deoxyribonucleotides of the dsNA molecules of the invention are located within a region of the dsNA that can be excised via Dicer cleavage to generate an active siRNA agent that no longer contains the deoxyribonucleotide pattern {e.g., deoxyribonucleotide-deoxyribonucleotide base pairs). Such DNA-extended Dicer-substrate siRNAs (DsiRNAs) were demonstrated to be more effective RNA inhibitory agents than corresponding double stranded RNA-extended DsiRNAs. DsiRNA agents were also found to tolerate guide strand mismatches.

Description

EXTENDED DJCER SUBSTRATE AGENTS AND METHODS FOR THE SPECIFIC INHIBITION OF GENE EXPRESSION CROSS-REFERENCE TO RELATED APPLICATIONS 5 The present application is related to and claims priority under 35 U.S.C. §119(e) to the following applications: U.S. provisional patent application No. 61 / 138,946, filed December 18, 2008; U.S. provisional patent application No. 61 / 166,227, filed April 2, 2009; U.S. provisional patent application No. 61 / 173,505, filed April 28, 2009; U.S. provisional patent application No. 61 / 173,514, filed April 28, 2009; U.S. provisional 10 patent application No. 61 / 173,52 I, filed April 28, 2009; U.S. provisional patent application No. 61 / 173,525, filed April 28, 2009; U.S. provisional patent application No. 61 / 173,532, filed April 28, 2009; U.S. provisional patent application No. 61 / 173,538, filed April 28, 2009; U.S. provisional patent application No. 61 / 173,544, filed April 28, 2009; U.S. provisional patent application No. 61 / 173,549, filed April 28, 2009; U.S. 15 provisional patent application No. 61 / 173,554, filed April 28, 2009; U.S. provisional patent application No. 61 / 173,556, filed April 28, 2009; U.S. provisional patent application No. 61 / 173,558, filed April 28, 2009; and U.S. provisional patent application No. 61 / 173,563, filed April 28, 2009. 20 BACKGROUND OF THE INVENTION Double-stranded RNA (dsRNA) agents possessing strand lengths of25 to 35 nucleotides have been described as effective inhibitors of target gene expression in mammalian cells (Rossi et al., U.S. Patent Publication Nos. 2005 / 0244858 and 2005 / 0277610). dsRNA agents of such length are believed to be processed by the 25 Dicer enzyme of the RNA interference (RNAi) pathway, leading such agents to be tenned "Dicer substrate siRNA" ("DsiRNA") agents. Certain modified structures of DsiRNA agents were previously described (Rossi et al., U.S. Patent Publication No. 2007 / 0265220). While robust, sequence-specific target gene silencing efficacy has been 30 identified for 25-35 nucleotide length dsRNA agents, a need exists for improved design of such agents, including design ofDsiRNA agents possessing enhanced in vitro and in vivo efficacy. Date Re9ue / Date Received 2022-03-14 BRIEF SUMMARY OF THE INVENTION The present invention is based, at least in part, upon the surprising discovery that double stranded nucleic acid agents having strand lengths in the range of 27-39 nucleotides in length that possess base paired deoxyribonucleotides either at or near 5 the 3' terminus of the sense strand / 5' terminus of the antisense strand or at or near the 5' terminus of the sense strand / 3' terminus of the antisense strand are effective RNA interference agents. Indeed, the instant invention relates to the demonstration that inclusion of base paired deoxyribonucleotides within a region of a Dicer substrate siRNA ("DsiRNAs") that is excised from a resultant active siRNA via Dicer enzyme IO cleavage, results in an effective inhibitory agent. Inclusion of one or more base paired deoxyribonucleotides within this region of a DsiRNA can impart certain advantages to such a modified DsiRNA molecule, including, e.g., enhanced efficacy (including enhanced potency and / or improved duration of effect), display of a recognition domain for DNA-binding molecules, and other attributes associated with a 15 DNA:DNA duplex region. Indeed, such double stranded DNA:DNA-extended DsiRNA agents were demonstrated to possess enhanced efficacy, especially including improved potency, relative to corresponding double stranded RNA:DNA- or RNA:RNA-extended DsiRNA agents. Among the advantages of the instant invention, the surprising discovery that 20 DNA-extended DsiRNA agents do not exhibit decreased efficacy as duplex length increases allows for the generation of DsiRNAs that remain effective RNA inhibitory agents while providing greater spacing for, e.g., attachment ofDsiRNAs to additional functional groups, inclusion / patterning of stabilizing modifications (e.g., PS-NA moieties) or other forms of modifications capable of adding further functionality 25 and / or enhancing, e.g., pharmacokinetics, pharmacodynamics or biodistribution of such agents, as compared to dsRNA agents of corresponding length that do not contain such double stranded DNA-extended domains. The effect of such dsDNAextension regions appears not to result from a stabilizing activity inherent in dsDNA regions, but rather appears to be attributable to the ability of specifically localized 30 deoxyribonucleotide residues (either located 3' of the projected Dicer cleavage site of the first strand and correspondingly 5' of the projected Dicer cleavage site of the second strand or located 5' of the projected Dicer cleavage site of the first strand and correspondingly 3' of the projected Dicer cleavage site of the second strand) to direct 2 Date Re9ue / Date Received 2022-03-14 Dicer cleavage such that a preferred cleavage product and / or population of cleavage products is generated and / or is made more prevalent. Thus, in certain aspects, the instant invention allows for design of RNA inhibitory agents possessing enhanced efficacies at greater length (via more precise 5 direction of the location of Dicer cleavage events) than previously described RNA inhibitory agents, thereby allowing for generation of dsRNA-containing agents possessing enhanced efficacy, delivery, pharmacokinetic, pharmacodynamic and biodistribution attributes, as well as improved ability, e.g., to be successfully formulated, to be attached to an active drug molecule and / or payload, to be attached to 10 another active nucleic acid molecule, to be attached to a detection molecule, to possess (e.g., multiple) stabilizing modifications, etc. In one aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the 15 first strand is 27 to 49 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that base pair with the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' 20 terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the second strand; and the second strand is sufficiently 25 complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In another aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' 30 terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that base pair with the 3 Date Re9ue / Date Received 2022-03-14 ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a 1-4 nucleotide 3' overhang; at least one of positions 24 to 5 the 3' terminal nucleotide residue of the first strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the second strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. 10 In an additional aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 27 to 49 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, 15 positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that base pair with the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' 20 terminus of the second strand form a blunt end and the ultimate and penultimate residues of the 3' terminus of the first strand and the ultimate and penultimate residues of the 5' terminus of the second strand form one or two mismatched base pairs, at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the 25 second strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In a further aspect, the invention provides an isolated double stranded nucleic 30 acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 27 to 49 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in 4 Date Re9ue / Date Received 2022-03-14 length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand 5 and the 5' terminus of the second strand form a blunt end; starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 9 of the first strand comprise a nucleotide that forms a mismatch with the second strand; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the second strand; 10 and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In another aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' 15 terminus and a second o Ii gonucl eotide strand having a 5' terminus and a 3 ' terminus, where the first strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently 20 complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a 1-4 nucleotide 3' overhang; starting from ,the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 25 to 9 of the first strand comprise a nucleotide that forms a mismatch with the second strand; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the second strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene 30 expression when the double stranded nucleic acid is introduced into a mammalian cell. In an additional aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' 5 Date Re9ue / Date Received 2022-03-14 terminus, where the first strand is 27 to 49 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are 5 sufficiently complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end and the ultimate and penultimate residues of the 3' terminus of the first strand and the ultimate and 10 penultimate residues of the 5' terminus of the second strand form one or two mismatched base pairs, starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 9 of the first strand comprise a nucleotide that forms a mismatch with the second strand; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a deoxyribonucleotide that base pairs 15 with a deoxyribonucleotide of the second strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In one embodiment, two or more, four or more, six or more, eight or more, ten 20 or more, twelve or more, fourteen or more, sixteen or more, eighteen or more, or twenty or more nucleotide residues of positions 24 to the 3' terminal nucleotide residue of the first strand are deoxyribonucleotides that base pair with deoxyribonucleotides of the second strand. Optionally, the deoxyribonucleotides of the first strand that base pair with the deoxyribonucleotides of the second strand are 25 consecutive deoxyribonucleotides. In one embodiment, two or more consecutive nucleotide residues of positions 24 to 27 of the first strand are deoxyribonucleotides that base pair with deoxyribonucleotides of the second strand. In another embodiment, each of positions 24 and 25 of the first strand is a deoxyribonucleotide that base pairs with a 30 deoxyribonucleotide of the second strand. In a further embodiment, each nucleotide residue of positions 24 to 27 of the first oligonucleotide strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the second strand. 6 Date Re9ue / Date Received 2022-03-14 In another embodiment, the first strand is 29 to 49 nucleotides in length. Optionally, each nucleotide residue of positions 24 to 27 of the first oligonucleotide strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the second strand. 5 In one embodiment, the first strand is 31 to 49 nucleotides in length. Optionally, each nucleotide residue of positions 24 to 29 of the first oligonucleotide strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the second strand. In a further embodiment, the first strand is 33 to 49 nucleotides in length. 10 Optionally, each nucleotide residue of positions 24 to 31 of the first oligonucleotide strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the second strand. In another embodiment, the first strand is 35 to 49 nucleotides in length. Optionally, each nucleotide residue of positions 24 to 33 of the first oligonucleotide 15 strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the second strand. In certain embodiments, the first strand is 37 to 49 nucleotides in length. Optionally, each nucleotide residue of positions 24 to 35 of the first oligonucleotide strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the 20 second strand. In one embodiment, the first strand is 39 to 49 nucleotides in length. Optionally, each nucleotide residue of positions 24 to 37 of the first oligonucleotide strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the second strand. 25 In an additional embodiment, the deoxyribonucleotides of the second strand that base pair with the deoxyribonucleotides of the first strand are not complementary to the target RNA. In certain embodiments, the second strand possesses a 3' overhang of 1-4 nucleotides in length. In a related embodiment, the nucleotides of the second strand 30 3' overhang comprise a modified nucleotide. Optionally, the modified nucleotide residue is 2 '-O-methyl, 2 '-methoxyethoxy, 2' -fluoro, 2 '-ally I, 2 '-O-[2- (methylamino )-2-oxoethyl], 4' -thio, 4' -CH2-O-2' -bridge, 4' -(CH2)2-O-2' -bridge, 2 ' - LNA, 2'-amino or 2'-O-(N-methlycarbamate). 7 Date Re9ue / Date Received 2022-03-14 In another embodiment, at least one of positions 24 to the 3' terminal nucleotide residue of the first strand further possess a modified nucleotide that is 2' - O-methyl, 2' -methoxyethoxy, 2' -fluoro, 2 '-allyl, 2 '-O-[2-(methylamino )-2-oxoethyl], 4'-thio, 4'-CH2-O-2'-bridge, 4'-(CH2)2-O-2'-bridge, 2'-LNA, 2'-amino or 2'-O-(N- 5 methlycarbamate). In one embodiment, the ultimate and penultimate residues of the 3' terminus of the first strand are deoxyribonucleotides and the ultimate and penultimate residues of the 5' terminus of the second strand are ribonucleotides. In another embodiment, the second strand, starting from the nucleotide residue 10 of the second strand that is complementary to the 5' terminal nucleotide residue of the first oligonucleotide strand (position 1 *), includes unmodified nucleotide residues at all positions from position 20* to the 5' terminal residue of the second strand. Optionally, starting from the first nucleotide (position 1 *) at the 3' terminus of the first strand, position 1 *, 2 * and / or 3 * is a deoxyribonucleotide. 15 In one embodiment, the first strand possesses a deoxyribonucleotide at position 1 * from the 3' terminus of the first strand. Optionally, the first strand possesses deoxyribonucleotides at positions 1 * and 2* from the 3' terminus of the first strand. In another embodiment, a nucleotide of the second or first oligonucleotide 20 strand is substituted with a modified nucleotide that directs the orientation of Dicer cleavage. 25 In an additional embodiment, starting from the first nucleotide (position 1 *) at the 3' terminus of the second strand, positions 1 *, 2*, and 3* from the 3' terminus of the second strand are modified nucleotides. Optionally, the first strand has a nucleotide sequence that is at least 60%, 70%, 80%, 90%, 95% or I 00% complementary to the second strand nucleotide sequence. In certain embodiments, the 3' terminal nucleotide residue of the first strand is attached to the 5' terminal nucleotide residue of the second strand by a nucleotide sequence. Optionally, the nucleotide sequence that attaches the 3' terminal nucleotide 30 residue of the first strand and the 5' terminal nucleotide residue of the second strand includes a tetraloop or a hairpin structure. In one embodiment, the first and second strands are joined by a chemical linker. Optionally, the 3' terminus of the first strand and the 5' terminus of the second strand are joined by a chemical linker. 8 Date Re9ue / Date Received 2022-03-14 In certain embodiments, at least one of positions 24 to the 3' terminal nucleotide residue of the first strand that is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the second strand is an unmodified deoxyribonucleotide. In a related embodiment, both the at least one of positions 24 to the 3' terminal 5 nucleotide residue of the first strand that is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the second strand and the deoxyribonucleotide of the second strand are unmodified deoxyribonucleotides. In one embodiment, the second oligonucleotide strand, starting from the nucleotide residue of the second strand that is complementary to the 5' terminal 10 nucleotide residue of the first oligonucleotide strand, includes alternating modified and unmodified nucleotide residues. In a further embodiment, a mismatch is present in the DsiRNA at any one or more of positions 3-9. In another embodiment embodiment, a mismatch is present at any one or more of positions positions 1-7. In a further embodiment, a mismatch is 15 present at any one or more of positions positions 3-7. Optionally, a mismatch is present at position 6. In another embodiment, starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 9 of the first strand possess two or more nucleotides that form mismatches with the second strand. Optionally, positions 2 and 20 6 are mismatches with the second strand. In an additional aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 27 to 49 nucleotide residues in length, where 25 starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the 30 second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end; starting from the nucleotide residue of the second strand that is complementary to the 5' terminal nucleotide residue of the first oligonucleotide strand (position 1 *), position 1 * to position 9* in the 5' direction of the second strand includes a nucleotide that forms a 9 Date Re9ue / Date Received 2022-03-14 mismatch with a sequence of the target RNA; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the second strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand 5 length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In another aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, IO where the first strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a 15 duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a 1-4 nucleotide 3' overhang; starting from the nucleotide residue of the second strand that is complementary to the 5' terminal nucleotide residue of the first oligonucleotide strand (position I*), position 20 1 * to position 9* in the 5' direction of the second strand includes a nucleotide that forms a mismatch with a sequence of the target RNA; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the second strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the 25 second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In a further aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, 30 where the first strand is 27 to 49 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions l to 23 of the first strand to form a 10 Date Re9ue / Date Received 2022-03-14 duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end and the ultimate and penultimate residues of the 3' terminus of the first strand and the ultimate and 5 penultimate residues of the 5' terminus of the second strand form one or two mismatched base pairs; starting from the nucleotide residue of the second strand that is complementary to the 5' terminal nucleotide residue of the first oligonucleotide strand (position 1 *), position 1 * to position 9* in the 5' direction of the second strand includes a nucleotide that forms a mismatch with a sequence of the target RNA; at 10 least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the second strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. 15 In one embodiment, a nucleotide of the 3' overhang of the second strand forms a mismatch with the target RNA. In another aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, 20 where the second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides; the first strand is 27 to 49 nucleotide residues in length and includes 23 consecutive ribonucleotides that base pair with the ribonucleotides of positions 1 to 23 of the second strand to form a duplex; the 3' 25 terminus of the first strand and the 5' terminus of the second strand form a blunt end; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the first strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the 30 double stranded nucleic acid is introduced into a mammalian cell. In one aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting from the first 11 Date Re9ue / Date Received 2022-03-14 nucleotide (position 1) at the 5' terminus of the second strand, positions I to 23 of the second strand are ribonucleotides; the first strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that base pair with the ribonucleotides of positions 1 to 23 of the second strand to form a duplex; the 3' 5 terminus of the first strand and the 5' terminus of the second strand form a 1-4 nucleotide 3' overhang; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the first strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand IO length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In an additional aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' 15 terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides; the first strand is 27 to 49 nucleotide residues in length and includes 23 consecutive ribonucleotides that base pair with the ribonucleotides of positions 1 to 23 of the second strand to form a 20 duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end and the ultimate and penultimate residues of the 3' terminus of the first strand and the ultimate and penultimate residues of the 5' terminus of the second strand form one or two mismatched base pairs; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a deoxyribonucleotide that base 25 pairs with a deoxyribonucleotide of the first strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In one aspect, the invention provides an isolated double stranded nucleic acid 30 (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position l) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides and positions 11 to 21 of the second strand 12 Date Re9ue / Date Received 2022-03-14 comprise a nucleotide that forms a mismatch with the first strand; the first strand is 27 to 49 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 1 to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus 5 of the second strand form a blunt end; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the first strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is 10 introduced into a mammalian cell. In another aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting 15 from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides and positions 11 to 21 of the second strand comprise a nucleotide that forms a mismatch with the first strand; the first strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that base pair with the ribonucleotides of positions 1 to 23 of the 20 second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a l-4 nucleotide 3' overhang; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the first strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the 25 second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In a further aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, 30 where the second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides and positions 11 to 21 of the second strand comprise a nucleotide that forms a mismatch with the first strand; the first strand is 27 to 49 nucleotide residues in length and includes 21 consecutive 13 Date Re9ue / Date Received 2022-03-14 ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 3 to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end and the ultimate and penultimate residues of the 3' terminus of the first strand and the ultimate and penultimate 5 residues of the 5' terminus of the second strand form one or two mismatched base pairs; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the first strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression 10 when the double stranded nucleic acid is introduced into a mammalian cell. In one embodiment, two or more, four or more, six or more, eight or more, ten or more, twelve or more, fourteen or more, sixteen or more, eighteen or more, or twenty or more nucleotide residues of positions 24 to the 3' terminal nucleotide residue of the second strand are deoxyribonucleotides that base pair with 15 deoxyribonucleotides of the first strand. In another embodiment, the deoxyribonucleotides of the second strand that base pair with the deoxyribonucleotides of the first strand are consecutive deoxyribonucleotides. Optionally, two or more consecutive nucleotide residues of positions 24 to 27 of the second strand are deoxyribonucleotides that base pair with 20 deoxyribonucleotides of the first strand. In one embodiment, each of positions 24 and 25 of the second strand of the DsiRNA is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the first strand. Optionally, each nucleotide residue of positions 24 to 27 of the second oligonucleotide strand is a deoxyribonucleotide that base pairs with a 25 deoxyribonucleotide of the first strand. 30 In one embodiment, the second strand of the DsiRNA is 29 to 53 nucleotides in length. Optionally, each nucleotide residue of positions 24 to 27 of the second oligonucleotide strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the first strand. In another embodiment, the second strand of the DsiRNA is 31 to 53 nucleotides in length. Optionally, each nucleotide residue of positions 24 to 29 of the second oligonucleotide strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the first strand. 14 Date Re9ue / Date Received 2022-03-14 In an additional embodiment, the second strand of the DsiRNA is 33 to 53 nucleotides in length. Optionally, each nucleotide residue of positions 24 to 31 of the second oligonucleotide strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the first strand. 5 In certain embodiments, the second strand of the DsiRNA is 35 to 53 nucleotides in length. Optionally, each nucleotide residue of positions 24 to 33 of the second oligonucleotide strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the first strand. In another embodiment, the second strand of the DsiRNA is 37 to 53 10 nucleotides in length. Optionally, each nucleotide residue of positions 24 to 35 of the second oligonucleotide strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the first strand. In a further embodiment, the second strand of the DsiRNA is 39 to 53 nucleotides in length. Optionally, each nucleotide residue of positions 24 to 37 of the 15 second oligonucleotide strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the first strand. In one embodiment, positions 24 to the 3' terminal nucleotide residue of the second strand comprise between one and 25 deoxyribonucleotide residues, where each of the deoxyribonucleotide residues of the second strand base pairs with a 20 deoxyribonucleotide of the first strand. In another embodiment, the second strand, starting from the nucleotide residue of the second strand (position I*) that is complementary to the residue of the first oligonucleotide strand that is located immediately 3' of the most 5' Dicer cleavage site of the first strand, includes unmodified nucleotide residues at all positions from 25 position 20* to the 5' terminal residue of the second strand. 30 In an additional embodiment, starting from the nucleotide located immediately 5' of the most 3' Dicer cleavage site of the second strand (position A) of the DsiRNA, positions A, B, and C from the residue in the 5' direction of the second strand are modified nucleotides. In another aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 15 Date Re9ue / Date Received 2022-03-14 I to 23 of the second strand are ribonucleotides; the first strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that base pair with the ribonucleotides of positions I to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a 1-4 5 nucleotide 3' overhang; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a deoxyribonucleotide that base pairs with a deoxyribonucleotide of the first strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is 10 introduced into a mammalian cell, where positions 11 to 21 of the second strand comprise a nucleotide that forms a mismatch with a nucleotide of the target RNA. In one embodiment, a mismatch is present at one or more of positions 13-21. Optionally, a mismatch is present at position 14. In another embodiment, starting from the first nucleotide (position 1) at the 5' 15 terminus of the second strand, positions 11 to 21 of the second strand possess two or more nucleotides that form mismatches with the first strand. Optionally, positions 14 and 18 of the second strand are mismatches with the first strand. Another aspect of the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' 20 terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 27 to 49 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that base pair with the 25 ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a phosphorothioate-modified nucleotide (PS- 30 NA) that base pairs with a deoxyribonucleotide of the second strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. 16 Date Re9ue / Date Received 2022-03-14 An additional aspect of the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 28 to 53 nucleotide residues in length, where 5 starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that base pair with the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a 10 blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a 1-4 nucleotide 3' overhang; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a phosphorothioate-modified nucleotide (PS-NA) that base pairs with a deoxyribonucleotide of the second strand; and the second strand is sufficiently 15 complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. A further aspect of the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' 20 terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 27 to 49 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that base pair with the 25 ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end and the ultimate and penultimate residues of the 3' terminus of the first strand and the ultimate and penultimate residues of the 5' 30 terminus of the second strand form one or two mismatched base pairs; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a phosphorothioate-modified nucleotide (PS-NA) that base pairs with a deoxyribonucleotide of the second strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand 17 Date Re9ue / Date Received 2022-03-14 length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In a further aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' 5 terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 27 to 49 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently 10 complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end; starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 9 of the 15 first strand comprise a nucleotide that forms a mismatch with the second strand; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a phosphorothioate-modified nucleotide (PS-NA) that base pairs with a deoxyribonucleotide of the second strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand 20 length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. An additional aspect of the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' 25 terminus, where the first strand is 27 to 49 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 1 to 23 of the first 30 strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end; starting from the nucleotide residue of the second strand that is complementary to the S' terminal nucleotide residue of the first oligonucleotide strand (position 1 *), position 1 * to 18 Date Re9ue / Date Received 2022-03-14 position 9* in the 5' direction of the second strand includes a nucleotide that forms a mismatch with a sequence of the target RNA; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a phosphorothioate-modified nucleotide (PS-NA) that base pairs with a deoxyribonucleotide of the second strand; 5 and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In one aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus 10 and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 27 to 49 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently 15 complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end; starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 9 of the 20 first strand comprise a nucleotide that forms a mismatch with the second strand; at least one nucleotide of the second strand base pairs with a deoxyribonucleotide of positions 24 to the 3' terminal nucleotide residue of the first strand and is a phosphorothioate-modified nucleotide (PS-NA); and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand 25 length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In another aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, 30 where the first strand is 27 to 49 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a 19 Date Re9ue / Date Received 2022-03-14 duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end; starting from the nucleotide residue of the second strand that is complementary to the 5' terminal nucleotide 5 residue of the first oligonucleotide strand (position 1 *), position I* to position 9* in the 5' direction of the second strand includes a nucleotide that forms a mismatch with a sequence of the target RNA; at least one nucleotide of the second strand base pairs with a deoxyribonucleotide of positions 24 to the 3' terminal nucleotide residue of the first strand and is a phosphorothioate-modified nucleotide (PS-NA); and the second IO strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In an additional aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 15 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 27 to 49 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are 20 sufficiently complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end; starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 25 to 9 of the first strand comprise a nucleotide that forms a mismatch with the second strand; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a deoxyribonucleotide that base pairs with a phosphorothioate-modified nucleotide (PS-NA) of the second strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand 30 length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In a further aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, 20 Date Re9ue / Date Received 2022-03-14 where the first strand is 27 to 49 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently 5 complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end; starting from the nucleotide residue of the second strand that is complementary to the 5' terminal nucleotide 10 residue of the first oligonucleotide strand (position 1 *), position 1 * to position 9* in the 5' direction of the second strand includes a nucleotide that forms a mismatch with a sequence of the target RNA; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a deoxyribonucleotide that base pairs with a phosphorothioate-modified nucleotide (PS-NA) of the second strand; and the second 15 strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In one aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus 20 and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 28 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently 25 complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a 1-4 nucleotide 3' overhang; starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 30 to 9 of the first strand comprise a nucleotide that forms a mismatch with the second strand; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a phosphorothioate-modified nucleotide (PS-NA) that base pairs with a deoxyribonucleotide of the second strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand 21 Date Re9ue / Date Received 2022-03-14 length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In another aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' 5 terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 28 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently 10 complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a 1-4 nucleotide 3' overhang; starting from the nucleotide residue of the second strand that is complementary to the 5' 15 terminal nucleotide residue of the first oligonucleotide strand (position 1 *), position 1 * to position 9* in the 5' direction of the second strand includes a nucleotide that forms a mismatch with a sequence of the target RNA; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a phosphorothioate-modified nucleotide (PS-NA) that base pairs with a deoxyribonucleotide of the second strand; 20 and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In an additional aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 25 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 28 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are 30 sufficiently complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a 1-4 nucleotide 3' overhang; starting from the first nucleotide (position 1) at the 5' terminus of the first 22 Date Re9ue / Date Received 2022-03-14 strand, positions 1 to 9 of the first strand comprise a nucleotide that forms a mismatch with the second strand; at least one nucleotide of the second strand base pairs with a deoxyribonucleotide of positions 24 to the 3' terminal nucleotide residue of the first strand and is a phosphorothioate-modified nucleotide (PS-NA); and the second strand 5 is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In a further aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' 10 terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 28 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently 15 complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a 1-4 nucleotide 3' overhang; starting from the nucleotide residue of the second strand that is complementary to the 5' 20 terminal nucleotide residue of the first oligonucleotide strand (position I*), position 1 * to position 9* in the 5' direction of the second strand includes a nucleotide that forms a mismatch with a sequence of the target RNA; at least one nucleotide of the second strand base pairs with a deoxyribonucleotide of positions 24 to the 3' terminal nucleotide residue of the first strand and is a phosphorothioate-modified nucleotide 25 (PS-NA); and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In another aspect, the invention provides an isolated double stranded nucleic 30 acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 28 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions I to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in 23 Date Re9ue / Date Received 2022-03-14 length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand 5 and the 5' terminus of the second strand form a 1-4 nucleotide 3' overhang; starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 9 of the first strand comprise a nucleotide that forms a mismatch with the second strand; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a deoxyribonucleotide that base pairs with a phosphorothioate-modified 10 nucleotide (PS-NA) of the second strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In an additional aspect, the invention provides an isolated double stranded 15 nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 28 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 20 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a 1-4 nucleotide 3' 25 overhang; starting from the nucleotide residue of the second strand that is complementary to the 5' terminal nucleotide residue of the first oligonucleotide strand (position 1 *), position 1 * to position 9* in the 5' direction of the second strand includes a nucleotide that forms a mismatch with a sequence of the target RNA; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a 30 deoxyribonucleotide that base pairs with a phosphorothioate-modified nucleotide (PSNA) of the second strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. 24 Date Re9ue / Date Received 2022-03-14 Another aspect of the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 27 to 49 nucleotide residues in length, where starting from the 5 first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand 10 form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end and the ultimate and penultimate residues of the 3' terminus of the first strand and the ultimate and penultimate residues of the 5' terminus of the second strand form one or two mismatched base pairs; starting from the first nucleotide (position 1) at the 5' 15 terminus of the first strand, positions 1 to 9 of the first strand comprise a nucleotide that forms a mismatch with the second strand; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a phosphorothioate-modified nucleotide (PS-NA) that base pairs with a deoxyribonucleotide of the second strand; and the second strand is sufficiently complementary to a target RNA along at least 19 20 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. An additional aspect of the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' 25 terminus, where the first strand is 27 to 49 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 1 to 23 of the first 30 strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end and the ultimate and penultimate residues of the 3' terminus of the first strand and the ultimate and penultimate residues of the 5' terminus of the second strand form one or two 25 Date Re9ue / Date Received 2022-03-14 mismatched base pairs; starting from the nucleotide residue of the second strand that is complementary to the 5' terminal nucleotide residue of the first oligonucleotide strand (position 1 *), position 1 * to position 9* in the 5' direction of the second strand includes a nucleotide that forms a mismatch with a sequence of the target RNA; at 5 least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a phosphorothioate-modified nucleotide (PS-NA) that base pairs with a deoxyribonucleotide of the second strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is 10 introduced into a mammalian cell. A further aspect of the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 27 to 49 nucleotide residues in length, where starting from the 15 first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand 20 form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end and the ultimate and penultimate residues of the 3' terminus of the first strand and the ultimate and penultimate residues of the 5' terminus of the second strand form one or two mismatched base pairs; starting from the first nucleotide (position 1) at the 5' 25 terminus of the first strand, positions 1 to 9 of the first strand comprise a nucleotide that forms a mismatch with the second strand; at least one nucleotide of the second strand base pairs with a deoxyribonucleotide of positions 24 to the 3' terminal nucleotide residue of the first strand and is a phosphorothioate-modified nucleotide (PS-NA); and the second strand is sufficiently complementary to a target RNA along 30 at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In another aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' 26 Date Re9ue / Date Received 2022-03-14 terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 27 to 49 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in 5 length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end and the ultimate and 10 penultimate residues of the 3' terminus of the first strand and the ultimate and penultimate residues of the 5' terminus of the second strand form one or two mismatched base pairs; starting from the nucleotide residue of the second strand that is complementary to the 5' terminal nucleotide residue of the first oligonucleotide strand (position 1 *), position 1 * to position 9* in the 5' direction of the second strand 15 includes a nucleotide that forms a mismatch with a sequence of the target RNA; at least one nucleotide of the second strand base pairs with a deoxyribonucleotide of positions 24 to the 3' terminal nucleotide residue of the first strand and is a phosphorothioate-modified nucleotide (PS-NA); and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand 20 length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In a further aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, 25 where the first strand is 27 to 49 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a 30 duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end and the ultimate and penultimate residues of the 3' terminus of the first strand and the ultimate and penultimate residues of the 5' terminus of the second strand form one or two 27 Date Re9ue / Date Received 2022-03-14 mismatched base pairs; starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions I to 9 of the first strand comprise a nucleotide that forms a mismatch with the second strand; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a deoxyribonucleotide that base pairs 5 with a phosphorothioate-modified nucleotide (PS-NA) of the second strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In an additional aspect, the invention provides an isolated double stranded 10 nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the first strand is 27 to 49 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the first strand, positions 1 to 23 of the first strand are ribonucleotides; the second strand is 27 to 53 15 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 1 to 23 of the first strand to form a duplex; the 5' terminus of the first strand and the 3' terminus of the second strand form a blunt end or a 1-4 nucleotide 3' overhang; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end and the ultimate 20 and penultimate residues of the 3' terminus of the first strand and the ultimate and penultimate residues of the 5' terminus of the second strand form one or two mismatched base pairs; starting from the nucleotide residue of the second strand that is complementary to the 5' terminal nucleotide residue of the first oligonucleotide strand (position I*), position I* to position 9* in the 5' direction of the second strand 25 includes a nucleotide that forms a mismatch with a sequence of the target RNA; at least one of positions 24 to the 3' terminal nucleotide residue of the first strand is a deoxyribonucleotide that base pairs with a phosphorothioate-modified nucleotide (PSNA) of the second strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce 30 target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In one embodiment, two or more nucleotide residues of positions 24 to the 3' terminal nucleotide residue of the first strand are PS-NA residues that base pair with deoxyribonucleotides of the second strand. 28 Date Re9ue / Date Received 2022-03-14 In another embodiment, the first strand PS-NA residues base pair with PS-NA deoxyribonucleotides of the second strand. Another aspect of the invention provides an isolated double stranded nucleic acid ( dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' 5 terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides; the first strand is 27 to 49 nucleotide residues in length and includes 23 consecutive ribonucleotides that base pair with the 10 ribonucleotides of positions 1 to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a phosphorothioate-modified nucleotide (PS-NA); and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the 15 second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In one aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the 20 second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides; the first strand is 27 to 49 nucleotide residues in length and includes 23 consecutive ribonucleotides that base pair with the ribonucleotides of positions 1 to 23 of the second strand to form a duplex; the 3' 25 terminus of the first strand and the 5' terminus of the second strand form a blunt end; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a deoxyribonucleotide that base pairs with a phosphorothioate-modified nucleotide (PS-NA) of the first strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce 30 target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In another aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, 29 Date Re9ue / Date Received 2022-03-14 where the second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides; the first strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently 5 complementary to the ribonucleotides of positions 1 to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a 1-4 nucleotide 3' overhang; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a phosphorothioate-modified nucleotide (PS-NA); and the second strand is sufficiently complementary to a target RNA along 10 at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In a further aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' 15 terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides; the first strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently 20 complementary to the ribonucleotides of positions 1 to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a 1-4 nucleotide 3' overhang; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a deoxyribonucleotide that base pairs with a phosphorothioate-modified nucleotide (PS-NA) of the first strand; and the second 25 strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In an additional aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 30 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides; the first strand is 27 to 49 nucleotide residues in length and includes 23 consecutive ribonucleotides that base 30 Date Re9ue / Date Received 2022-03-14 pair with the ribonucleotides of positions I to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end and the ultimate and penultimate residues of the 3' terminus of the first strand and the ultimate and penultimate residues of the 5' terminus of the second 5 strand form one or two mismatched base pairs; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a phosphorothioate-modified nucleotide (PS-NA); and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a 10 mammalian cell. In another aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting 15 from the first nucleotide (position I) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides; the first strand is 27 to 49 nucleotide residues in length and includes 23 consecutive ribonucleotides that base pair with the ribonucleotides of positions 1 to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end 20 and the ultimate and penultimate residues of the 3' terminus of the first strand and the ultimate and penultimate residues of the 5' terminus of the second strand form one or two mismatched base pairs; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a deoxyribonucleotide that base pairs with a phosphorothioate-modified nucleotide (PS-NA) of the first strand; and the second 25 strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. One aspect of the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus 30 and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides and positions 11 to 21 of the second strand comprise a nucleotide that forms a mismatch with the first strand; the first strand is 27 31 Date Re9ue / Date Received 2022-03-14 to 49 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 1 to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end; at least one of positions 24 to the 3' terminal 5 nucleotide residue of the second strand is a phosphorothioate-modified nucleotide (PS-NA); and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. 10 Another aspect of the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 15 1 to 23 of the second strand are ribonucleotides; the first strand is 27 to 49 nucleotide residues in length and includes 23 consecutive ribonucleotides that base pair with the ribonucleotides of positions 1 to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is 20 a phosphorothioate-modified nucleotide (PS-NA); and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell, where positions 11 to 21 of the second strand comprise a nucleotide that forms a mismatch with a nucleotide of the 2 5 target RN A. An additional aspect of the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the second strand is 27 to 53 nucleotide residues in length, where 30 starting from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides and positions 11 to 21 of the second strand comprise a nucleotide that forms a mismatch with the first strand; the first strand is 27 to 49 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 32 Date Re9ue / Date Received 2022-03-14 1 to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a deoxyribonucleotide that base pairs with a phosphorothioate-modified nucleotide (PS-NA) of the first strand; 5 and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. A further aspect of the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' 10 terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides; the first strand is 27 to 49 nucleotide residues in length and includes 23 consecutive ribonucleotides that base pair with the 15 ribonucleotides of positions 1 to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a deoxyribonucleotide that base pairs with a phosphorothioate-modified nucleotide (PS-NA) of the first strand; and the second strand is sufficiently complementary to a 20 target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell, where positions 11 to 21 of the second strand comprise a nucleotide that forms a mismatch with a nucleotide of the target RNA. In another aspect, the invention provides an isolated double stranded nucleic 25 acid ( dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides and positions 11 to 21 of the second 30 strand comprise a nucleotide that forms a mismatch with the first strand; the first strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 1 to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a 1-4 nucleotide 3' overhang; at least one of 33 Date Re9ue / Date Received 2022-03-14 positions 24 to the 3' terminal nucleotide residue of the second strand is a phosphorothioate-modified nucleotide (PS-NA); and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is 5 introduced into a mammalian cell. In one aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting from the first 10 nucleotide (position 1) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides; the first strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 1 to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand 15 form a 1-4 nucleotide 3' overhang; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a phosphorothioate-modified nucleotide (PS-NA); and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian 20 cell, where positions 11 to 21 of the second strand comprise a nucleotide that forms a mismatch with a nucleotide of the target RNA. In an additional aspect, the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' 25 terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides and positions 11 to 21 of the second strand comprise a nucleotide that forms a mismatch with the first strand; the first strand is 27 to 53 nucleotide residues in length and includes 23 consecutive 30 ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 1 to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a 1-4 nucleotide 3' overhang; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a deoxyribonucleotide that base pairs with a phosphorothioate-modified nucleotide (PS- 34 Date Re9ue / Date Received 2022-03-14 NA) of the first strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. 5 In a further aspect, the invention provides an isolated double stranded nucleic acid ( dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 10 1 to 23 of the second strand are ribonucleotides; the first strand is 27 to 53 nucleotide residues in length and includes 23 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 1 to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a 1-4 nucleotide 3' overhang; at least one of positions 24 to the 3' terminal 15 nucleotide residue of the second strand is a deoxyribonucleotide that base pairs with a phosphorothioate-modified nucleotide (PS-NA) of the first strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell, where positions 11 to 21 of the 20 second strand comprise a nucleotide that forms a mismatch with a nucleotide of the target RNA. In another aspect, the invention provides an isolated double stranded nucleic acid ( dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, 25 where the second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides and positions 11 to 21 of the second strand comprise a nucleotide that forms a mismatch with the first strand; the first strand is 27 to 49 nucleotide residues in length and includes 21 consecutive 30 ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 3 to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end and the ultimate and penultimate residues of the 3' terminus of the first strand and the ultimate and penultimate residues of the 5' terminus of the second strand form one or two mismatched base 35 Date Re9ue / Date Received 2022-03-14 pairs; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a phosphorothioate-modified nucleotide (PS-NA); and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded 5 nucleic acid is introduced into a mammalian cell. A further aspect of the invention provides an isolated double stranded nucleic acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting 10 from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides; the first strand is 27 to 49 nucleotide residues in length and includes 21 consecutive ribonucleotides that base pair with the ribonucleotides of positions 3 to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end 15 and the ultimate and penultimate residues of the 3' terminus of the first strand and the ultimate and penultimate residues of the 5' terminus of the second strand form one or two mismatched base pairs; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a phosphorothioate-modified nucleotide (PS-NA); and the second strand is sufficiently complementary to a target RNA along at least 19 20 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell, where positions 11 to 21 of the second strand comprise a nucleotide that forms a mismatch with a nucleotide of the target RNA. One aspect of the invention provides an isolated double stranded nucleic acid 25 (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides and positions 11 to 21 of the second strand 30 comprise a nucleotide that forms a mismatch with the first strand; the first strand is 27 to 49 nucleotide residues in length and includes 21 consecutive ribonucleotides that are sufficiently complementary to the ribonucleotides of positions 3 to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end and the ultimate and penultimate residues of the 36 Date Re9ue / Date Received 2022-03-14 3' terminus of the first strand and the ultimate and penultimate residues of the 5' terminus of the second strand form one or two mismatched base pairs; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a deoxyribonucleotide that base pairs with a phosphorothioate-modified nucleotide (PS- 5 NA) of the first strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell. In another aspect, the invention provides an isolated double stranded nucleic 10 acid (dsNA) having a first oligonucleotide strand having a 5' terminus and a 3' terminus and a second oligonucleotide strand having a 5' terminus and a 3' terminus, where the second strand is 27 to 53 nucleotide residues in length, where starting from the first nucleotide (position 1) at the 5' terminus of the second strand, positions 1 to 23 of the second strand are ribonucleotides; the first strand is 27 to 49 nucleotide 15 residues in length and includes 21 consecutive ribonucleotides that base pair with the ribonucleotides of positions 3 to 23 of the second strand to form a duplex; the 3' terminus of the first strand and the 5' terminus of the second strand form a blunt end and the ultimate and penultimate residues of the 3' terminus of the first strand and the ultimate and penultimate residues of the 5' terminus of the second strand form one or 20 two mismatched base pairs; at least one of positions 24 to the 3' terminal nucleotide residue of the second strand is a deoxyribonucleotide that base pairs with a phosphorothioate-modified nucleotide (PS-NA) of the first strand; and the second strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of the second strand length to reduce target gene expression when the double stranded 25 nucleic acid is introduced into a mammalian cell, where positions 11 to 21 of the second strand comprise a nucleotide that forms a mismatch with a nucleotide of the target RNA. In one embodiment, two or more nucleotide residues of positions 24 to the 3' terminal nucleotide residue of the second strand are phosphorothioate-modified 30 nucleotides (PS-NAs). Optionally, the second strand PS-NA residues base pair with PS-NA deoxyribonucleotides of the first strand. In one embodiment, the dsNA possesses two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or 37 Date Re9ue / Date Received 2022-03-14 more PS-NA residues in total. Optionally, the dsNA possesses two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, thirteen or more, fourteen or more, or fifteen or more PS-NA or other modified residues one either or both strands. 5 In a further embodiment, the dsNA possesses 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21, or more, 22 or more, 23 ore more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, or 30 or more PS-NA or other modified residues in total. In another embodiment, the dsNA is cleaved endogenously in a mammalian 10 cell by Dicer. In an additional embodiment, the dsNA is cleaved endogenously in a mammalian cell to produce a double-stranded nucleic acid of 19-23 nucleotides in length that reduces target gene expression. In a further embodiment, the isolated dsNA has a phosphonate, a phosphorothioate or a phosphotriester phosphate backbone modification. 15 In an additional embodiment, the dsNA reduces target gene expression in a mammalian cell in vitro by at least 10%, at least 50%, or at least 80-90%. Optionally, the dsNA reduces target gene expression in a mammalian subject, or in a cell or tissue of a mammalian subject, by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 20 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% or more. In certain embodiments, the duration of such levels of inhibition, following either single or multi-dose administration, is six hours, twelve hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days or more in the mammalian subject, tissue 25 or cell. In one embodiment, the dsNA, when introduced into a mammalian cell, reduces target gene expression in comparison to a reference dsRNA that does not possess a deoxyribonucleotide-deoxyribonucleotide base pair. In another embodiment, the dsNA, when introduced into a mammalian cell, 30 reduces target gene expression by at least 70% when transfected into the cell at a concentration selected from the group consisting of 1 nM or less, 200 pM or less, 100 pM or less, 50 pM or less, 20 pM or less, 10 pM or less, 5 pM or less, and 1 pM or less. 38 Date Re9ue / Date Received 2022-03-14 In a further embodiment, at least 50% of the ribonucleotide residues of the dsNA are unmodified ribonucleotides. In certain embodiments, at least 50% of the ribonucleotide residues of the second strand are unmodified ribonucleotides. Optionally, at least 50% of all deoxyribonucleotides of the dsNA are unmodified 5 deoxyribonucleotides. In one embodiment, the first strand has a nucleotide sequence that is at least 80%, 90%, 95% or 100% complementary to the second strand nucleotide sequence. In certain embodiments, the target RNA is KRAS. In one aspect, the invention provides a method for reducing expression of a 10 target gene in a cell, that includes contacting a cell with an isolated double stranded NA ( dsNA) as described herein in an amount effective to reduce expression of a target gene in a cell in comparison to a reference dsRNA. In another aspect, the invention provides a method for reducing expression of a target gene in an animal by treating an animal with an isolated double stranded NA 15 (dsNA) as described herein in an amount effective to reduce expression of a target gene in a cell of the animal in comparison to a reference dsRN A. In one embodiment, the dsNA possesses enhanced pharmacokinetics, enhanced pharmacodynamics, reduced toxicity, or enhanced intracellular uptake in comparison to an appropriate control DsiRNA. 20 In a further aspect, the invention provides a pharmaceutical composition for 25 reducing expression of a target gene in a cell of a subject that includes an isolated double stranded NA (dsNA) as described herein, present in an amount effective to reduce expression of a target gene in a cell in comparison to a reference dsRNA, and further including a pharmaceutically acceptable carrier. Another aspect of the invention provides a method of synthesizing a double stranded NA (dsNA) as described herein via either chemical or enzymatic synthesis of the dsNA. A further aspect of the invention provides a kit that includes a dsNA as described herein and instructions for its use. 30 An additional aspect of the invention provides an isolated double stranded nucleic acid (dsNA) as depicted in any one of Figures 30-43C. BRIEF DESCRIPTION OF THE DRAWINGS 39 Date Re9ue / Date Received 2022-03-14 Figure IA shows a schematic representation of the processing of a Dicer substrate inhibitory RNA agent ("DsiRNA"). The protein Dicer is represented by the large rectangle, with the PAZ (Piwi / Argonaute / Zwille) domain of Dicer also indicated. The PAZ domain binds the two-base overhang and the 3'-OH (hydroxyl group) at the 3' 5 end of the guide (antisense) strand, and each strand of the dsRNA duplex is cleaved by separate RNase III domains (black triangles). Substitution of 2 bases of DNA for RNA at the 3' end of the passenger (sense) strand forms a two-base long RNA / DNA duplex blunt end, which reduces or eliminates binding affinity for PAZ. Cleavage of the DsiRNA typically yields a 19mer duplex with 2-base overhangs at each end. 10 Figure JB shows that the addition of four bases of DNA duplex to the DsiRNA had no apparent inhibitory effect upon Dicer cleavage. The bases inserted into this example of an anti-HPRT DsiRNA (heavy black bars and arrows) were not complementary to the HPRT target sequence. Figure 2A presents histogram data showing the robust efficacy ofDsiRNA agents 15 possessing base paired deoxyribonucleotides in a duplexed region located 3' of the Dicer cleavage site of the sense strand / 5' of the Dicer cleavage site of the antisense strand ("Right-extended DsiRNA agents"). DsiRNA duplexes were transfected into HeLa cells at a fixed concentration of 20nM, and HPRT expression levels were measured 24 hours later. Transfections were performed in duplicate, and each 20 duplicate was assayed in triplicate for HPRT expression by qPCR. Error bars are the standard error. Duplex I targeted HPRT and was a 25 / 27mer configuration overhanging RNA / blunt two-DNA substitution as described in Rose et al. NAR 2005. All other duplexes were longer than Duplex 1 due to the insertion of bases that were not complementary to the HPRT target region. The length of the inserted sequence 25 ranged from two bases (Duplex 2) to eight bases (Duplexes 6, 7, and 8). Figure 2B shows duplex numbers, sequences and chemical modification patterns for agents for which data is presented in Figure 2A. UPPER case= unmodified RNA, Bold, underlined= 2'-O-methyl RNA, lower case= DNA, bold lower case= phosphorothioate-modified DNA (PS-DNA). A general description of each duplex 30 and the overall configuration is shown at right. Figures 3A and 3B show that DNA-extended DsiRNA agents were more effective than corresponding RNA-extended DsiRNA agents at low concentrations. An optimized 27 / 29mer DsiRNA duplex targeting HPRT was compared to a modified 40 Date Re9ue / Date Received 2022-03-14 duplex in a dose-response series at 10.0 nanomolar (nM), 1.0 nanomolar (nM) and 100 picomolar (100 pM or 0.1 nM), with efficacy of knockdown ofHPRT mRNA levels assessed in HeLa cells. Duplex concentrations shown represent the final concentration of oligonucleotides in the transfection mixture and culture medium as 5 described in the Examples. Duplex identities are indicated below the bars (1, 2, 3), with the "C" bar representing baseline HPRT expression in untreated cells. Figure 3B shows the sequences and chemical modification patterns of those duplexes depicted in Figure 3A. UPPER case = unmodified RNA, Bold, underlined = 2 '-0- methyl RNA, and lower case= DNA. DsiRNA 1 was a derivative of a previously 10 reported active 25 / 27mer DsiRNA duplex (HPRT-1, Rose et al. NAR 2005, Collingwood et al. 2008, see also Figure 2A above), but contained an insertion of two bases in each strand, which extended the oligonucleotide duplex to a 27 / 29mer (heavy black bars denote inserted base pairs). Duplex 2 was identical in sequence to duplex 1, but the two base pair insertion (heavy black bars), including two additional 15 nucleosides of both passenger strand (sense sequence) and guide strand (antisense sequence) were synthesized as DNA. Thus, duplex 2 terminated in 4 DNA bp (base pairs) at the 5' end of the guide strand, in contrast to previously reported two base DNA substitutions at the 3' end of the passenger (sense) strand (Rose et al, 2005). Duplex 3 (mismatch (MM) control) was derived from the optimized HPRT-1 duplex, 20 but synthesized with mismatches indicated by arrows. The base composition and chemical modification of each strand and the base sequences and overhang or blunt structure at the ends of duplex 3 were held constant relative to the optimized HPRT-1 duplex in order to control for non-targeted chemical effects (see Figure 5 below). Figures 4A-4D show that modified DsiRNA duplexes extended by two to eight base 25 paired deoxyribonucleosides were more effective at reducing HPRT target mRNA levels than corresponding ribonucleoside-extended DsiRNA agents. Figure 4A shows HPRT target gene mRNA levels for cells treated with lnM modified DsiRNA agents. Figure 4B shows HPRT target gene mRNA levels for cells treated with 1 OOpM modified DsiRNA agents. Figure 4C shows HPRT target gene mRNA levels 30 for cells treated with 1 OpM modified DsiRNA agents. Figure 4D shows the sequences and chemical modification patterns of those duplexes depicted in Figures 4A-4C. Inserted sequences (heavy bars beneath the duplexes) did not match the 41 Date Re9ue / Date Received 2022-03-14 HPRT mRNA target region. UPPER case= unmodified RNA, Bold, underlined = 2'-O-methyl RNA, lower case= DNA. U = untreated cells. Figure 5A shows HPRT target mRNA inhibition results for a series of modified 5 duplexes of increasing length administered at a fixed concentration of 1 00pM. Figure 5B shows duplex numbers, sequences and chemical modification patterns for agents for which data is presented in Figure 5A. Duplex 1 was an optimized 25 / 27mer DsiRNA containing chemical modifications, a two-base overhang at the 3 ' - end of the guide (antisense) strand and two DNA substitutions and a blunt end at the 10 3'-end of the passenger (sense) strand (Collingwood et al. 2008). Bases noncomplementary to HPRT mRNA were inserted two bases at a time as either RNA (duplexes 2 through 5) or DNA (duplexes 6 through 9), increasing total duplex configurations from 27 / 29mers to 33 / 35mers. UPPER case= unmodified RNA, Bold, underlined= 2'-O-methyl RNA, lower case= DNA. U = untreated cells. UPPER 15 case= unmodified RNA, Bold, underlined= 2'-O-methyl RNA, lower case= DNA. Figure 6 shows the structure and predicted Dicer-mediated processing of a "25 / 27mer DsiRNA" agent (top) and an exemplary "Left-extended" DsiRNA agent (bottom) which contains a mismatch residue (G:U) within the dsRNA duplex sequence. UPPER case= RNA residues; lower case= DNA residues. 20 Figure 7 shows the structures of a series of DNA-extended duplexes, with pictured duplexes alternately right- or left-extended with 5 base pair DNA sequences. Mismatches are introduced within both forms of extended DsiRNA agents as indicated, with numbering of such mismatches proceeding in the 3' direction from position 1 of the second strand, which is the predicted 5' terminal RNA residue of the 25 second strand after Dicer cleavage. Figure 8 depicts the results of an initial round of testing of the inhibitory activity of right- and left-extended agents shown in Figure 7. For comparisons between rightversus left-extended parent molecules, right- versus left-extended agents harboring a mismatch at position 14, right- versus left-extended agents possessing a mismatch at 30 position 16, and right- versus left-extended agents harboring a mismatch at both positions 14 and 18, left-extended agents were surprisingly observed to be more 42 Date Re9ue / Date Received 2022-03-14 effective at gene silencing than corresponding right-extended agents. (100 pM of each indicated duplex was transfected into HeLa cells for all such experiments and % of KRAS target mRNA remaining was assessed at 24 hours.) Figure 9 depicts the result of a second round of experiments performed with the 5 agents shown in Figure 7, showing that left-extended agents were reproducibly more effective target mRNA silencing agents than right-extended agents in three of the four instances which were initially observed to show such a bias in favor ofleft-extended agents. Inhibitory results for a non-extended 25 / 27mer DsiRNA are also shown ("Opt" 25 / 27mer). 10 Figure 10 shows the structure of a series ofDsiRNA agents designed to silence an HPRT target mRNA, and inhibitory efficacies of such agents in cell culture. Capital letters indicate ribonucleotides; lower case letters indicate deoxyribonucleotides; balded lower case letters indicate phosphorothioates (PS-NAs); balded and underlined uppercase letters indicate 2 '-O-methyl modified nucleotides; the bolded uppercase 15 letter of agent DP1065P / DP1067G indicates the site of a mismatched nucleotide (with respect to the sense strand) within the "seed" region sequence of the antisense strand of the DsiRNA agent. Figure 11 shows that phosphorothioate modified "right-extended" DsiRNAs retain target HPRTl gene inhibitory efficacy, and also indicates that passenger strand 20 extended residues might tolerate phosphorothioate modification better than guide strand extended residues while retaining target gene inhibitory activity. In vitro Dicer cleavage assays (left lane = untreated; right lane = Dicer enzyme treated) are also shown for all extended DsiRNAs. Capital letters indicate ribonucleotides; lower case letters indicate deoxyribonucleotides, while balded lower case letters indicate 25 phosphorothioate-modified deoxyribonucleotides. Figure 12 depicts the structures of control and "right-extended" DsiRNAs of the invention targeting the "KRAS-200" site within the KRAS transcript. Capital letters indicate ribonucleotides; lower case letters indicate deoxyribonucleotides. 43 Date Re9ue / Date Received 2022-03-14 Figure 13 shows the KRAS inhibitory efficacies observed for the DsiRNA structures of Figure 12 in vitro. Figure 14 depicts the structures of control and "right-extended" DsiRNAs of the invention targeting the "KRAS-909" site within the KRAS transcript. Capital letters 5 indicate ribonucleotides; lower case letters indicate deoxyribonucleotides. Figure 15 shows the KRAS inhibitory efficacies observed for the DsiRNA structures of Figure 14 in vitro. Figure 16 depicts the structures of control and "right-extended" DsiRNAs of the invention targeting the "KRAS-249" site within the KRAS transcript, including 10 modification patterns of such DsiRNAs. Capital letters indicate ribonucleotides; lower case letters indicate deoxyribonucleotides, while balded lower case letters indicate phosphorothioate-modified deoxyribonucleotides. Underlined capital letters indicate 2'-O-methyl-modified ribonucleotides. Figure 17 depicts the structures of control and "right-extended" DsiRNAs of the 15 invention targeting the "KRAS-516" site within the KRAS transcript, including modification patterns of such DsiRNAs. Capital letters indicate ribonucleotides; lower case letters indicate deoxyribonucleotides, while balded lower case letters indicate phosphorothioate-modified deoxyribonucleotides. Underlined capital letters indicate 2 '-O-methyl-modified ribonucleotides. 20 Figure 18 depicts the structures of control and "right-extended" DsiRNAs of the invention targeting the "KRAS-909" site within the KRAS transcript, including modification patterns of such DsiRNAs. Capital letters indicate ribonucleotides; lower case letters indicate deoxyribonucleotides, while balded lower case letters indicate phosphorothioate-modified deoxyribonucleotides. Underlined capital letters 25 indicate 2' -O-methyl-modified ribonucleotides. Figure 19 shows in vitro KRAS inhibitory efficacy results obtained for the "rightextended" DsiRNAs of Figures 16-18. Results were obtained in HeLa cells contacted with the indicated DsiRNAs at 0. lnM concentration, assayed at 24 hours post- 44 Date Re9ue / Date Received 2022-03-14 RNAiMAX™ treatment. Capital letters indicate ribonucleotides; lower case letters indicate deoxyribonucleotides, while bolded lower case letters indicate phosphorothioate-modified deoxyribonucleotides. Figure 20 depicts the structures of 25 / 27mer "KRAS-249" site targeting DsiRNAs 5 which were assessed for mismatch residue tolerance. Closed arrows indicate projected Dicer enzyme cleavage sites, while open arrow indicates projected Ago2 cleavage site within target strand sequence corresponding to passenger strand DsiRNA sequence shown. Capital letters indicate ribonucleotides; lower case letters indicate deoxyribonucleotides. Bolded capital letters indicate sites of target- 10 mismatched residues of guide strand (and complementary residues of passenger strand, where applicable), with such target-mismatched residues obtained by "flipping" individual residues between guide and passenger strand during DsiRNA design. Horizontal bracket within DP1301P / DP1302G duplex indicates "seed region" of this duplex (with seed regions of all other DsiRNA structures occurring in the same 15 vertically-aligned position). Figure 21 shows in vitro KRAS inhibitory efficacy results obtained for the DsiRNAs of Figure 20. Results were obtained in HeLa cells contacted with the indicated DsiRNAs at 0.lnM concentration, assayed at 24 hours post-RNAiMAX™ treatment. Figure 22 shows single dose (10 mg / kg) in vivo KRAS inhibitory efficacy results in 20 liver tissue for an unmodified 25 / 27mer "KRAS-249" site targeting DsiRNA ("K.249"), a 2'-O-methyl-modified form of this 25 / 27mer ("KRAS-249M") and a DNA-extended form of this modified DsiRNA ("K.249DNA", shown in Figure 16 as "K.249D"; "5% Glu" = 5% glucose control). Figure 23 shows single dose (10 mg / kg) in vivo KRAS inhibitory efficacy results in 25 kidney tissue for an unmodified 25 / 27mer "KRAS-249" site targeting DsiRNA ("K.249"), a 2' -O-methyl-modified form of this 25 / 27mer ("KRAS-249M") and a DNA-extended form of this modified DsiRNA ("K.249DNA", shown in Figure 16 as "K.249D"; "5% Glu" = 5% glucose control). 45 Date Re9ue / Date Received 2022-03-14 Figure 24 shows single dose (10 mg / kg) in vivo KRAS inhibitory efficacy results in spleen tissue for an unmodified 25 / 27mer "KRAS-249" site targeting DsiRNA ("K249"), a 2'-O-methyl-modified form of this 25 / 27mer ("KRAS-249M") and a DNA-extended form of this modified DsiRNA ("K249DNA", shown in Figure 16 as 5 "K249D"; "5% Glu" = 5% glucose control). Figure 25 shows single dose (10 mg / kg) in vivo KRAS inhibitory efficacy results in lymph node tissue for an unmodified 25 / 27mer "KRAS-249" site targeting DsiRNA ("K249"), a 2'-O-methyl-modified form of this 25 / 27mer ("KRAS-249M") and a DNA-extended form of this modified DsiRNA ("K249DNA", shown in Figure 16 as 10 "K249D"; "5% Glu" = 5% glucose control). Figure 26 shows multi-dose (2 mg / kg, administered a total of four times, with each administration performed at three day intervals) in vivo KRAS inhibitory efficacy results in liver tissue for a 2'-O-methyl-modified form of a 25 / 27mer "KRAS-249" site targeting DsiRNA ("KRAS-249M") and a DNA-extended form of this modified 15 DsiRNA ("K249D", as shown in Figure 16). Figure 27 shows multi-dose (2 mg / kg, administered a total of four times, with each administration performed at three day intervals) in vivo KRAS inhibitory efficacy results in lung tissue for a 2'-O-methyl-modified form of a 25 / 27mer "KRAS-249" site targeting DsiRNA ("KRAS-249M") and a DNA-extended form of this modified 20 DsiRNA ("K249D", as shown in Figure 16). Figure 28 shows multi-dose (2 mg / kg, administered a total of four times, with each administration performed at three day intervals) in vivo KRAS inhibitory efficacy results in spleen tissue for a 2 '-O-methyl-modified form of a 25 / 27mer "KRAS-249" site targeting DsiRNA ("KRAS-249M") and a DNA-extended form of this modified 25 DsiRNA ("K249D", as shown in Figure 16). Figure 29 shows multi-dose (2 mg / kg, administered a total of four times, with each administration performed at three day intervals) in vivo KRAS inhibitory efficacy results in kidney tissue for a 2 '-O-methyl-modified form of a 25 / 27mer "KRAS-249" 46 Date Re9ue / Date Received 2022-03-14 site targeting DsiRNA ("KRAS-249M") and a DNA-extended form of this modified DsiRNA ("K249D", as shown in Figure 16). Figure 30 shows exemplary structures of "right extended" DsiRNA agents that form a blunt end between the 3' terminus of the first strand and 5' terminus of the second 5 strand. Upper case letters indicate ribonucleotides; lower case characters denote deoxyribonucleotides; open triangle denotes a site within the sequence of the first strand (here, the sense strand) corresponding to the Ago2 cleavage site within the target RNA; filled triangles indicate projected sites of Dicer cleavage; and[#] denotes a duplex region of four to sixteen or more base pairs in length which comprises at 10 least one deoxyribonucleotide-deoxyribonucleotide base pair. (In alternative embodiments,[#] indicates a duplex region of four to sixteen or more base pairs in length which comprises at least four deoxyribonucleotides but is not required to possess a deoxyribonucleotide-deoxyribonucleotide base pair.) Nucleotide position numbering is also shown. 15 Figure 31 shows an exemplary structure of a "right extended" DsiRNA agent that possesses a 3 '-terminal overhang of the first strand relative to the 5' terminus of the second strand. Upper case letters indicate ribonucleotides; lower case characters. denote deoxyribonucleotides; open triangle denotes a site within the sequence of the first strand (here, the sense strand) corresponding to the Ago2 cleavage site within the 20 target RNA; filled triangles indicate projected sites of Dicer cleavage; and[#] denotes a duplex region of four to sixteen or more base pairs in length which comprises at least one deoxyribonucleotide-deoxyribonucleotide base pair. (In alternative embodiments,[#] indicates a duplex region of four to sixteen or more base pairs in length which comprises at least four deoxyribonucleotides but is not required to 25 possess a deoxyribonucleotide-deoxyribonucleotide base pair.) Nucleotide position numbering is also shown. Figure 32 shows an exemplary structure of a "right extended" DsiRNA agent that forms a fray at the 3'-terminus of the first strand and corresponding 5' terminus of the second strand. Upper case letters indicate ribonucleotides; lower case characters 30 denote deoxyribonucleotides; open triangle denotes a site within the sequence of the first strand (here, the sense strand) corresponding to the Ago2 cleavage site within the 47 Date Re9ue / Date Received 2022-03-14 target RNA; filled triangles indicate projected sites of Dicer cleavage; and[#] denotes a duplex region of four to sixteen or more base pairs in length which comprises at least one deoxyribonucleotide-deoxyribonucleotide base pair. (In alternative embodiments, [#] indicates a duplex region of four to sixteen or more base pairs in 5 length which comprises at least four deoxyribonucleotides but is not required to possess a deoxyribonucleotide-deoxyribonucleotide base pair.) Nucleotide position numbering is also shown. Figure 33 shows exemplary structures of "right extended" DsiRNA agents that form a blunt end between the 3' terminus of the first strand and 5' terminus of the second 10 strand, and that also possess mismatched residues within antisense strand sequences which are projected to be retained within the interference agent following Dicer cleavage. Upper case letters indicate ribonucleotides; lower case characters denote deoxyribonucleotides; open triangle denotes a site within the sequence of the first strand (here, the sense strand) corresponding to the Ago2 cleavage site within the 15 target RNA; filled triangles indicate projected sites of Dicer cleavage; and[#] denotes a duplex region of four to sixteen or more base pairs in length which comprises at least one deoxyribonucleotide-deoxyribonucleotide base pair. (In alternative embodiments,[#] indicates a duplex region of four to sixteen or more base pairs in length which comprises at least four deoxyribonucleotides but is not required to 20 possess a deoxyribonucleotide-deoxyribonucleotide base pair.) Seed and mismatch regions of the antisense strand, as well as nucleotide position numbering of each strand is also shown. The underlined antisense residue of the bottom agent indicates a nucleotide which base pairs with the sense strand of the DsiRNA agent, yet is projected to form a mismatch with the target RNA. 25 Figure 34 shows exemplary structures of "right extended" DsiRNA agents that possess a 3'-terminal overhang of the first strand relative to the 5' terminus of the second strand, and that also possess mismatched residues within antisense strand sequences which are projected to be retained within the interference agent following Dicer cleavage. Upper case letters indicate ribonucleotides; lower case characters 30 denote deoxyribonucleotides; open triangle denotes a site within the sequence of the first strand (here, the sense strand) corresponding to the Ago2 cleavage site within the target RNA; filled triangles indicate projected sites of Dicer cleavage; and[#] denotes 48 Date Re9ue / Date Received 2022-03-14 a duplex region of four to sixteen or more base pairs in length which comprises at least one deoxyribonucleotide-deoxyribonucleotide base pair. (In alternative embodiments,[#] indicates a duplex region of four to sixteen or more base pairs in length which comprises at least four deoxyribonucleotides but is not required to 5 possess a deoxyribonucleotide-deoxyribonucleotide base pair.) Seed and mismatch regions of the anti sense strand, as well as nucleotide position numbering of each strand is also shown. The underlined antisense residue of the bottom agent indicates a nucleotide which base pairs with the sense strand of the DsiRNA agent, yet is projected to form a mismatch with the target RNA. IO Figure 35 shows exemplary structures of"right extended" DsiRNA agents that form a fray at the 3'-terminus of the first strand and corresponding 5' terminus of the second strand, and that also possess mismatched residues within antisense strand sequences which are projected to be retained within the interference agent following Dicer cleavage. Upper case letters indicate ribonucleotides; lower case characters denote 15 deoxyribonucleotides; open triangle denotes a site within the sequence of the first strand (here, the sense strand) corresponding to the Ago2 cleavage site within the target RNA; filled triangles indicate projected sites of Dicer cleavage; and[#] denotes a duplex region of four to sixteen or more base pairs in length which comprises at least one deoxyribonucleotide-deoxyribonucleotide base pair. (In alternative 20 embodiments,[#] indicates a duplex region of four to sixteen or more base pairs in length which comprises at least four deoxyribonucleotides but is not required to possess a deoxyribonucleotide-deoxyribonucleotide base pair.) Seed and mismatch regions of the antisense strand, as well as nucleotide position numbering of each strand is also shown. The underlined antisense residue of the bottom agent indicates a 25 nucleotide which base pairs with the sense strand of the DsiRNA agent, yet is projected to form a mismatch with the target RNA. Figure 36 shows exemplary structures of "left extended" DsiRNA agents that form a blunt end between the 3' terminus of the first strand and 5' terminus of the second strand. Upper case letters indicate ribonucleotides; lower case characters denote 30 deoxyribonucleotides; open triangle denotes a site within the sequence of the first strand (here, the sense strand) corresponding to the Ago2 cleavage site within the target RNA; filled triangles indicate projected sites of Dicer cleavage; and[#] denotes 49 Date Re9ue / Date Received 2022-03-14 a duplex region of four to sixteen or more base pairs in length which comprises at least one deoxyribonucleotide-deoxyribonucleotide base pair. (In alternative embodiments,[#] indicates a duplex region of four to sixteen or more base pairs in length which comprises at least four deoxyribonucleotides but is not required to 5 possess a deoxyribonucleotide-deoxyribonucleotide base pair.) Nucleotide position numbering is also shown. Figure 37 shows exemplary structures of"left extended" DsiRNA agents that possess a 3'-terminal overhang of the first strand relative to the 5' terminus of the second . strand. Upper case letters indicate ribonucleotides; lower case characters denote 10 deoxyribonucleotides; open triangle denotes a site within the sequence of the first strand (here, the sense strand) corresponding to the Ago2 cleavage site within the target RNA; filled triangles indicate projected sites of Dicer cleavage; and[#] denotes a duplex region of four to sixteen or more base pairs in length which comprises at least one deoxyribonucleotide-deoxyribonucleotide base pair. (In alternative 15 embodiments, [ #] indicates a duplex region of four to sixteen or more base pairs in length which comprises at least four deoxyribonucleotides but is not required to possess a deoxyribonucleotide-deoxyribonucleotide base pair.) Nucleotide position numbering is also shown. Figure 38 shows an exemplary structure of a "left extended" DsiRNA agent that 20 forms a fray at the 3 '-terminus of the first strand and corresponding 5' terminus of the second strand. Upper case letters indicate ribonucleotides; lower case characters denote deoxyribonucleotides; open triangle denotes a site within the sequence of the first strand (here, the sense strand) corresponding to the Ago2 cleavage site within the target RNA; filled triangles indicate projected sites of Dicer cleavage; and [ #] denotes 25 a duplex region of four to sixteen or more base pairs in length which comprises at least one deoxyribonucleotide-deoxyribonucleotide base pair. (In alternative embodiments, [#] indicates a duplex region of four to sixteen or more base pairs in length which comprises at least four deoxyribonucleotides but is not required to possess a deoxyribonucleotide-deoxyribonucleotide base pair.) Nucleotide position 30 numbering is also shown. 50 Date Re9ue / Date Received 2022-03-14 Figure 39 shows exemplary structures of "left extended" DsiRNA agents that form a blunt end between the 3' terminus of the first strand and 5' terminus of the second strand, and that also possess mismatched residues within antisense strand sequences which are projected to be retained within the interference agent following Dicer 5 cleavage. Upper case letters indicate ribonucleotides; lower case characters denote deoxyribonucleotides; open triangle denotes a site within the sequence of the first strand (here, the sense strand) corresponding to the Ago2 cleavage site within the target RNA; filled triangles indicate projected sites of Dicer cleavage; and[#] denotes a duplex region of four to sixteen or more base pairs in length which comprises at 10 least one deoxyribonucleotide-deoxyribonucleotide base pair. (In alternative embodiments,[#] indicates a duplex region of four to sixteen or more base pairs in length which comprises at least four deoxyribonucleotides but is not required to possess a deoxyribonucleotide-deoxyribonucleotide base pair.) Seed and mismatch regions of the antisense strand, as well as nucleotide position numbering of each 15 strand is also shown. The underlined antisense residue of the lower two agents indicates a nucleotide which base pairs with the sense strand of the DsiRNA agent, yet is projected to form a mismatch with the target RNA. Figure 40 shows exemplary structures of "left extended" DsiRNA agents that possess a 3'-terminal overhang of the first strand relative to the 5' terminus of the second 20 strand, and that also possess mismatched residues within antisense strand sequences which are projected to be retained within the interference agent following Dicer cleavage. Upper case letters indicate ribonucleotides; lower case characters denote deoxyribonucleotides; open triangle denotes a site within the sequence of the first strand (here, the sense strand) corresponding to the Ago2 cleavage site within the 25 target RNA; filled triangles indicate projected sites of Dicer cleavage; and[#] denotes a duplex region of four to sixteen or more base pairs in length which comprises at least one deoxyribonucleotide-deoxyribonucleotide base pair. (In alternative embodiments, [ #] indicates a duplex region of four to sixteen or more base pairs in length which comprises at least four deoxyribonucleotides but is not required to 30 possess a deoxyribonucleotide-deoxyribonucleotide base pair.) Seed and mismatch regions of the antisense strand, as well as nucleotide position numbering of each strand is also shown. The underlined antisense residue of the middle agent indicates a 51 Date Re9ue / Date Received 2022-03-14 nucleotide which base pairs with the sense strand of the DsiRNA agent, yet is projected to form a mismatch with the target RNA. Figure 41 shows exemplary structures of "left extended" DsiRNA agents that form a fray at the 3 '-terminus of the first strand and corresponding 5' terminus of the second 5 strand, and that also possess mismatched residues within antisense strand sequences which are projected to be retained within the interference agent following Dicer cleavage. Upper case letters indicate ribonucleotides; lower case characters denote deoxyribonucleotides; open triangle denotes a site within the sequence of the first strand (here, the sense strand) corresponding to the Ago2 cleavage site within the 10 target RNA; filled triangles indicate projected sites of Dicer cleavage; and[#] denotes a duplex region of four to sixteen or more base pairs in length which comprises at least one deoxyribonucleotide-deoxyribonucleotide base pair. (In alternative embodiments,[#] indicates a duplex region of four to sixteen or more base pairs in length which comprises at least four deoxyribonucleotides but is not required to 15 possess a deoxyribonucleotide-deoxyribonucleotide base pair.) Seed and mismatch regions of the antisense strand, as well as nucleotide position numbering of each strand is also shown. The underlined antisense residue of the bottom agent indicates a nucleotide which base pairs with the sense strand of the DsiRNA agent, yet is projected to form a mismatch with the target RNA. 20 Figures 42A-42C show exemplary structures of "right extended" DsiRNA agents. Upper case letters indicate ribonucleotides; lower case characters denote deoxyribonucleotides; open triangle denotes a site within the sequence of the top strand (here, the sense strand) corresponding to the Ago2 cleavage site within the target RNA; filled triangles indicate projected sites of Dicer cleavage; and nucleotide 25 position numbering is also shown. Figures 43A-43C show exemplary structures of"left extended" DsiRNA agents. Upper case letters indicate ribonucleotides; lower case characters denote deoxyribonucleotides; open triangle denotes a site within the sequence of the top strand (here, the sense strand) corresponding to the Ago2 cleavage site within the 30 target RNA; filled triangles indicate projected sites of Dicer cleavage; and nucleotide position numbering is also shown. 52 Date Re9ue / Date Received 2022-03-14 DETAILED DESCRIPTION The invention provides compositions and methods for reducing expression of a target gene in a cell, involving contacting a cell with an isolated double stranded nucleic acid ( dsNA) in an amount effective to reduce expression of a target gene in a 5 cell. The dsNAs of the invention possess a pattern of deoxyribonucleotides (in most embodiments, the pattern comprises at least one deoxyribonucleotidedeoxyribonucleotide base pair) designed to direct the site of Dicer enzyme cleavage within the dsNA molecule. The deoxyribonucleotide pattern of the dsNA molecules of the invention is located within a region of the dsNA that can be excised via Dicer 10 cleavage to generate an active siRNA agent that no longer contains the deoxyribonucleotide pattern (e.g., in most embodiments, the deoxyribonucleotide pattern comprises one or more deoxyribonucleotide-deoxyribonucleotide base pairs). Surprisingly, as demonstrated herein, DNA:DNA-extended Dicer-substrate siRNAs (DsiRNAs) were more effective RNA inhibitory agents than corresponding 15 RNA:DNA- or RNA:RNA-extended DsiRNAs. It was also surprising to discover that DsiRNAs comprising DNA:DNA extensions which were positioned at the 5' end of the first strand and corresponding 3' end of the second strand of a dsRNA DsiRNA agent (where the second strand is complementary to a sufficient region of target RNA sequence to serve as an effective 20 guide strand sequence of an RNAi agent (antisense to the target RNA)) constituted effective - and in many instances enhanced - inhibitory agents. The surprising discovery that DNA-extended DsiRNA agents do not exhibit decreases in efficacy as duplex length increases allows for the generation of DsiRNAs that remain effective while providing greater spacing for, e.g., attachment of 25 DsiRNAs to additional and / or distinct functional groups, inclusion / patterning of stabilizing modifications (e.g., PS-NA moieties) or other forms of modifications capable of adding further functionality and / or enhancing, e.g., pharmacokinetics, pharmacodynamics or biodistribution of such agents, as compared to dsRNA agents of corresponding length that do not contain such double stranded DNA-extended 30 domains. The advantage provided by the newfound ability to lengthen DsiRNAcontaining dsNA duplexes while retaining activity of a post-Dicer-processed siRNA agent at levels greater than dsRNA duplexes of similar length is emphasized by the 53 Date Re9ue / Date Received 2022-03-14 results presented herein, which show that complete phosphorothioate (PS) modification of all nucleotides of a double-stranded DNA:DNA region of an extended DsiRNA agent completely abolished silencing activity (see duplex #8 of Figures 2A and 2B). The ability to extend DsiRNA agents without observing a corresponding 5 reduction in RNA silencing activity can also allow for inclusion of, e.g., more modified nucleotides within a single molecule that still retains RNA silencing activity than could otherwise be achieved were such modified nucleotides not allowed such spacing (in view of the inhibitory effect associated with certain modifications when present in a tandem series- e.g., tandem PS or 2'-O-methyl modifications). 10 Similarly, the ability to include longer duplex extensions in such DsiRNA-containing agents while retaining RNA inhibitory function can also allow for certain functional groups to be attached to such agents that would otherwise not be possible, because of the ability of such functional groups to interfere with RNA silencing activity when present in tighter configurations. 15 Definitions Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of 20 Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991 ). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. 25 As used herein, the term "nucleic acid" refers to deoxyribonucleotides, ribonucleotides, or modified nucleotides, and polymers thereof in single- or doublestranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the 30 reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). 54 Date Re9ue / Date Received 2022-03-14 As used herein, "nucleotide" is used as recognized in the art to include those with natural bases (standard), and modified bases well known in the art. Such bases are generally located at the l' position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group. The nucleotides can be 5 unmodified or modified at the sugar, phosphate and / or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see, e.g., Usman and McSwiggen, supra; Eckstein, et al., International PCT Publication No. WO 92 / 07065; Usman et al, International PCT Publication No. WO 93 / 15187; Uhlman & Peyman, supra). 10 There are several examples of modified nucleic acid bases known in the art as summarized by Limbach, ct al, Nucleic Acids Res. 22:2183, 1994. Some of the non-limiting examples of base modifications that can be introduced into nucleic acid molecules i11clude, bypoxanthine, purine, pyridin- 4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl 15 uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcyti<lines (e.g., 5- methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5- bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne, and others (Burgin, et al., Biochemistry 35:14090, 1996; Uhlman & Pcyman, supra). By "modified bases" in this aspect is meant nucleotide bases other 20 than adenine, guanine, cytosine and uracil at l' position or their equivalents. As used herein, a "double-stranded nucleic acid" or "dsNA" is a molecule comprising two oligonuclcotidc strands which fonn a duplex. A dsNA may contain ribonucleotidcs, deoxyribonuclcotidcs, modified nucleotides, and combinations thereat: The double-stranded NAs of the instant invention are substrates for proteins 25 and protein complexes in the RNA interference pathway, e.g., Dicer and RISC. An ex.emplary structure of one form of dsNA ofthc invention is shown in Figure lA, and such structures characteristically comprise an RNA duplex in a region that is capable of functioning as a Dicer substrate siRNA (DsiRNA) and a DNA duplex comprising at least one deoxyribonucleotide, which is located at a position 3' of the projected 30 Dicer cleavage site of the first strand of the DsiRN NON A agent, and is base paired with a cognate deoxyribonucleotide of the second strand, which is located at a position 5' of the projected Dicer cleavage site of the second strand of the DsiRNA / DNA agent. In alternative embodiments, the instant invention provides a structure that characteristically comprises an RNA duplex within a region that is Date Re9ue / Date Received 2022-03-14 capable of functioning as a Dicer substrate siRNA (DsiRNA) and a DNA duplex comprising at least one deoxyribonucleotide, which is located at a position 5' of the projected Dicer cleavage site of the first strand of the DsiRNNDNA agent, and is base paired with a cognate deoxyribonucleotide of the second strand, which is located 5 at a position 3' of the projected Dicer cleavage site of the second strand of the DsiRNNDNA agent (see, e.g., "Left-Extended" DsiRNA agent of Figure 6). In certain embodiments, the DsiRNAs of the invention can possess deoxyribonucleotide residues at sites immediately adjacent to the projected Dicer enzyme cleavage site(s). For example, in the second, fourth and sixth DsiRNAs 10 shown in Figure 12, deoxyribonucleotides can be found (starting at the 5' terminal residue of the first strand as position 1) at position 22 and sites 3' of position 22 (e.g., 23, 24, 25, etc.).Correspondingly, deoxyribonucleotides can also be found on the second strand commencing at the nucleotide that is complementary to position 20 of the first strand, and also at positions on the second strand that are located in the 5' 15 direction of this nucleotide.Thus, certain effective DsiRNAs of the invention possess only 19 duplexed ribonucleotides prior to commencement of introduction of deoxyribonucleotides within the first strand, second strand, and / or both strands of such DsiRNAs. While the preceding statements regarding placement of deoxyribonucleotides immediately adjacent to a projected Dicer enzyme cleavage site 20 of the DsiRNAs of the invention explicitly contemplates "right-extended" DsiRNAs of the invention, parallel placement of deoxyribonucleotides can be performed within "left-extended" DsiRNAs of the invention (e.g., deoxyribonucleotides can be placed immediately adjacent to the projected Dicer enzyme cleavage site within "leftextended" DsiRNAs - e.g., immediately 5' on the sense strand of the most 5' 25 projected Dicer cleavage site on the sense strand of such a "left-extended" DsiRNA and / or immediately 3' on the antisense strand of the most 3' projected Dicer cleavage site on the antisense strand of such a "left-extended" DsiRNA). As used herein, "duplex" refers to a double helical structure formed by the interaction of two single stranded nucleic acids. According to the present invention, a 30 duplex may contain first and second strands which are sense and antisense, or which are target and antisense. A duplex is typically formed by the pairwise hydrogen bonding of bases, i.e., "base pairing", between two single stranded nucleic acids which are oriented antiparallel with respect to each other. Base pairing in duplexes generally occurs by Watson-Crick base pairing, e.g., guanine (G) forms a base pair 56 Date Re9ue / Date Received 2022-03-14 with cytosine (C) in DNA and RNA (thus, the cognate nucleotide of a guanine deoxyribonucleotide is a cytosine deoxyribonucleotide, and vice versa), adenine (A) forms a base pair with thymine (T) in DNA, and adenine (A) forms a base pair with uracil (U) in RNA. Conditions under which base pairs can form include physiological 5 or biologically relevant conditions (e.g., intracellular: pH 7 .2, 140 mM potassium ion; extracellular pH 7.4, 145 mM sodium ion). Furthermore, duplexes are stabilized by stacking interactions between adjacent nucletotides. As used herein, a duplex may be established or maintained by base pairing or by stacking interactions. A duplex is formed by two complementary nucleic acid strands, which may be substantially 10 complementary or fully complementary (see below). By "complementary" or "complementarity" is meant that a nucleic acid can form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick or Hoogsteen base pairing. In reference to the nucleic acid molecules of the present disclosure, the binding free energy for a nucleic acid molecule with its 15 complementary sequence is sufficient to allow the relevant function of the nucleic acid to proceed, e.g., RNAi activity. Determination of binding free energies for nucleic acid molecules is well known in the art (see, e.g., Turner, et al., CSH Symp. Quant. Biol. LIi, pp. 123-133, 1987; Frier, et al., Proc. Nat. Acad. Sci. USA 83:9373- 9377, 1986; Turner, et al., J. Am. Chem. Soc. 109:3783-3785, 1987). A percent 20 complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence ( e.g., 5, 6, 7, 8, 9, or 10 nucleotides out of a total of 10 nucleotides in the first oligonucleotide being based paired to a second nucleic acid sequence having 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100% 25 complementary, respectively). To determine that a percent complementarity is of at least a certain percentage, the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence is calculated and rounded to the nearest whole number ( e.g., 12, 13, 14, 15, 16, or 17 nucleotides out of a total of 23 nucleotides in the first 30 oligonucleotide being based paired to a second nucleic acid sequence having 23 nucleotides represents 52%, 57%, 61 %, 65%, 70%, and 74%, respectively; and has at least 50%, 50%, 60%, 60%, 70%, and 70% complementarity, respectively). As used herein, "substantially complementary'' refers to complementarity between the strands such that they are capable of hybridizing under biological conditions. Substantially 57 Date Re9ue / Date Received 2022-03-14 5 complementary sequences have 60%, 70%, 80%, 90%, 95%, or even I 00% complementarity. Additionally, techniques to determine if two strands are capable of hybridizing under biological conditions by examining their nucleotide sequences are well known in the· art. The first and second strands of the agents of the invention (antisense and sense oligonucleotides) are not required to be completely complementary. In one embodiment, the RNA sequence of the antisense strand contains one or more mismatches or modified nucleotides with base analogs. In an exemplary embodiment, such mismatches occur within the 3' region of RNA sequence of the antisense strand 10 (e.g., within the RNA sequence of the antisense strand that is complementary to the target RNA sequence that is positioned 5' of the projected Argonaute 2 (Ago2) cut site within the target RNA - see, e.g., Figure 6 for illustration of exemplary location of such a mismatch-containing region). In one aspect, about two mismatches or modified nucleotides with base analogs are incorporated within the RNA sequence of 15 the antisense strand that is 3' in the antisense strand of the projected Ago2 cleavage site of the target RNA sequence when the target RNA sequence is hybridized. The use of mismatches or decreased thermodynamic stability (specifically at or near the 3 '-terminal residues of sense / 5'-terminal residues of the antisense region of siRNAs) has been proposed to facilitate or favor entry of the antisense strand into 20' RISC (Schwarz et al., 2003; Khvorova et al., 2003), presumably by affecting some rate-limiting unwinding steps that occur with entry of the siRNA into RISC. Thus, terminal base composition has been included in design algorithms for selecting active 2lmer siRNA duplexes (Ui-Tei et al., 2004; Reynolds et al., 2004). In certain embodiments, mismatches ( or modified nucleotides with base 25 analogs) can be positioned within a parent DsiRNA (optionally a right- or leftextended DsiRNA agent) at or near the predicted 3'-terminus of the sense strand of the siRNA projected to be formed following Dicer cleavage. In such embodiments, the small end-terminal sequence which contains the mismatch(es) will either be left unpaired with the antisense strand (become part of a 3'-overhang) or be cleaved 30 entirely off the final 21-mer siRNA. In such embodiments, mismatches in the original (non-Dicer-processed) agent do not persist as mismatches in the final RNA component of RISC. It has been found that base mismatches or destabilization of segments at the 3'-end of the sense strand of Dicer substrate improved the potency of synthetic duplexes in RN Ai, presumably by facilitating processing by Dicer 58 Date Re9ue / Date Received 2022-03-14 (Collingwood et al., 2008). In some embodiments, one or more mismatches are positioned within a DsiRNA agent of the invention (optionally a right- or left-extended DsiRNA agent) at a location within the region of the antisense strand of the DsiRNA agent that 5 hybridizes with the region of the target mRNA that is positioned 5' of the predicted Ago2 cleavage site within the target mRNA (see, e.g., location(s) of mismatches within the agents of Figure 7). Optionally, two or more mismatches are positioned within the right- or left-extended DsiRNA agents of the instant invention within this relatively 3' region of the anti sense strand that hybridizes to a sequence of the target 10 RNA that is positioned 5' of the projected Ago2 cleavage site of the target RNA (were target RNA cleavage to occur). Inclusion of such mismatches within the DsiRNA agents of the instant invention can allow such agents to exert inhibitory effects that resemble those of naturally-occurring miRNAs, and optionally can be directed against not only naturally-occurring miRNA target RNAs (e.g., 3' UTR 15 regions of target transcripts) but also against RNA sequences for which no naturallyoccurring antagonistic miRNA is known to exist. For example, DsiRNAs of the invention possessing mismatched base pairs which are designed to resemble and / or function as miRNAs can be synthesized to target repetitive sequences within genes / transcripts that might not be targeted by naturally-occurring miRNAs (e.g., 20 repeat sequences within the Notch protein can be targeted, where individual repeats within Notch can differ from one another (e.g., be degenerate) at the nucleic acid level, but which can be effectively targeted via a miRNA mechanism that allows for mismatch(es) yet also ·allows for a more promiscuous inhibitory effect than a corresponding, perfect match siRNA agent). In such embodiments, target RNA 25 cleavage may or may not be necessary for the mismatch-containing DsiRNA agent to exert an inhibitory effect. In one embodiment, a double stranded nucleic acid molecule of the invention comprises or functions as a microRNA (miRNA). By "microRNA" or "miRNA" is meant a small double stranded RNA that regulates the expression of target messenger 30 RNAs either by mRNA cleavage, translational repression / inhibition or heterochromatic silencing (see for example Ambros, 2004, Nature, 431, 350-355; Bartel, 2004, Cell, 116, 281-297; Cullen, 2004, Virus Research., 102, 3-9; He et al., 2004, Nat. Rev. Genet., 5, 522-531; and Ying et al., 2004, Gene, 342, 25-28). In one embodiment, the microRNA of the invention, has partial complementarity (i.e., less 59 Date Re9ue / Date Received 2022-03-14 than 100% complementarity) between the sense strand (e.g., first strand) or sense region and the antisense strand (e.g., second strand) or antisense region of the miRNA molecule or between the antisense strand or anti sense region of the miRNA and a corresponding target nucleic acid molecule (e.g., target mRNA). For example, partial 5 complementarity can include various mismatches or non-base paired nucleotides (e.g., 1, 2, 3, 4, 5 or more mismatches or non-based paired nucleotides, such as nucleotide bulges) within the double stranded nucleic acid molecule structure, which can result in bulges, loops, or overhangs that result between the sense strand or sense region and the antisense strand or antisense region of the miRNA or between the antisense strand 10 or antisense region of the miRNA and a corresponding target nucleic acid molecule. Single-stranded nucleic acids that base pair over a number of bases are said to "hybridize." Hybridization is typically determined under physiological or biologically relevant conditions (e.g., intracellular: pH 7.2, 140 mM potassium ion; extracellular pH 7.4, 145 mM sodium ion). Hybridization conditions generally 15 contain a monovalent cation and biologically acceptable buffer and may or may not contain a divalent cation, complex anions, e.g. gluconate from potassium gluconate, uncharged species such as sucrose, and inert polymers to reduce the activity of water in the sample, e.g. PEG. Such conditions include conditions under which base pairs can form. 20 Hybridization is measured by the temperature required to dissociate single stranded nucleic acids forming a duplex, i.e., (the melting temperature; Tm). Hybridization conditions are also conditions under which base pairs can form. Various conditions of stringency can be used to determine hybridization (see, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. 25 (1987) Methods Enzymol. 152:507). Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 3 7° C, and most preferably of at least about 42° C. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10°C less than the melting temperature (Tm} of the hybrid, where Tm is determined according to the 30 following equations. For hybrids less than 18 base pairs in length, Tm(0 C)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(0 C)=81.5+ 16.6(1og IO(Na+])+0.41 (% G+C)-(600 / N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ((Na+] for lxSSC=0.165 M). For example, a hybridization determination 60 Date Re9ue / Date Received 2022-03-14 buffer is shown in Table 1. Table 1. final cone. Vender Cat# Lot# m.wJStock To make50 ml solution 5 NaCl 100 mM Siama S-5150 41K8934 SM 1 KCI B0mM Sioma P-9541 70K0002 74.55 0.298 MoCl2 8mM Siama M-1028 120K8933 1M 0.4 sucrose 2%w / v Fisher BP220- 212 907105 342.3 1 Tris-HCI 16mM Fisher BP510705 7- 12419 1M 0.8 NaH2PO4 1 mM Sigma S-3193 52H- 120.0 0.006 029515 EDTA 0.02 mM Siama E-7889 110K89271 0.5M 2 H2O Siama W-4502 51K2359 to 50 pH= 7.0 adjust with at 20°c HCI Useful variations on hybridization conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art 15 and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Antisense to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, 20 Cold Spring Harbor Laboratory Press, New York. As used herein, "oligonucleotide strand" is a single stranded nucleic acid molecule. An oligonucleotide may comprise ribonucleotides, deoxyribonucleotides, modified nucleotides (e.g., nucleotides with 2' modifications, synthetic base analogs, etc.) or combinations thereof. Such modified oligonucleotides can be preferred over 25 native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases. Certain dsNAs of this invention are chimeric dsNAs. "Chimeric dsNAs" or "chimeras", in the context of this invention, are dsNAs which contain two or more chemically distinct regions, each made up of at least one nucleotide. These dsNAs 30 typically contain at least one region primarily comprising ribonucleotides (optionally including modified ribonucleotides) that form a Dicer substrate siRNA ("DsiRNA") molecule. This DsiRNA region is covalently attached to a second region comprising base paired deoxyribonucleotides (a "dsDNA region") which confers one or more beneficial properties (such as, for example, increased efficacy, e.g., increased potency 61 Date Re9ue / Date Received 2022-03-14 ml g ml g ml g ul ml and / or duration of DsiRNA activity, function as a recognition domain or means of targeting a chimeric dsNA to a specific location, for example, when administered to cells in culture or to a subject, functioning as an extended region for improved attachment of functional groups, payloads, detection / detectable moieties, functioning 5 as an extended region that allows for more desirable modifications and / or improved spacing of such modifications, etc.). This second region comprising base paired deoxyribonucleotides may also include modified or synthetic nucleotides and / or modified or synthetic deoxyribonucleotides. As used herein, the term "ribonucleotide" encompasses natural and synthetic, 10 unmodified and modified ribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and / or to the linkages between ribonucleotides in the oligonucleotide. As used herein, the term "ribonucleotide" specifically excludes a deoxyribonucleotide, which is a nucleotide possessing a single proton group at the 2' ribose ring position. 15 As used herein, the term "deoxyribonucleotide" encompasses natural and synthetic, unmodified and modified deoxyribonucleotides. Modifications include changes to the sugar moiety, to the base moiety and / or to the linkages between deoxyribonucleotide in the oligonucleotide. As used herein, the term "deoxyribonucleotide" also includes a modified ribonucleotide that does not permit 20 Dicer cleavage of a dsNA agent, e.g., a 2'-O-methyl ribonucleotide, a phosphorothioate-modified ribonucleotide residue, etc., that does not permit Dicer cleavage to occur at a bond of such a residue. As used herein, the term "PS-NA" refers to a phosphorothioate-modified nucleotide residue. The term "PS-NA" therefore encompasses both phosphorothioate- 25 modified ribonucleotides ("PS-RNAs") and phosphorothioate-modified deoxyribonucleotides ("PS-DNAs"). In certain embodiments, a chimeric DsiRNA / DNA agent of the invention comprises at least one duplex region of at least 23 nucleotides in length, within which at least 50% of all nucleotides are unmodified ribonucleotides. As used herein, the 30 term "unmodified ribonucleotide" refers to a ribonucleotide possessing a hydroxyl (OH) group at the 2' position of the ribose sugar. In certain embodiments, a chimeric DsiRNA / DNA agent of the invention comprises at least one region, located 3' of the projected Dicer cleavage site on the first strand and 5' of the projected Dicer cleavage site on the second strand, having a 62 Date Re9ue / Date Received 2022-03-14 length of at least 2 base paired nucleotides in length, wherein at least 50% of all nucleotides within this region of at least 2 base paired nucleotides in length are unmodified deoxyribonucleotides. As used herein, the term "unmodified deoxyribonucleotide" refers to a ribonucleotide possessing a single proton at the 2' 5 position of the ribose sugar. 10 As used herein, "antisense strand" refers to a single stranded nucleic acid molecule which has a sequence complementary to that of a target RNA. When the antisense strand contains modified nucleotides with base analogs, it is not necessarily complementary over its entire length, but must at least hybridize with a target RNA. As used herein, "sense strand" refers to a single stranded nucleic acid molecule which has a sequence complementary to that of an antisense strand. When the antisense strand contains modified nucleotides with base analogs, the sense strand need not be complementary over the entire length of the antisense strand, but must at least duplex with the antisense strand. 15 As used herein, "guide strand" refers to a single stranded nucleic acid molecule of a dsRNA or dsRNA-containing molecule, which has a sequence sufficiently complementary to that of a target RNA to result in RNA interference. After cleavage of the dsRNA or dsRNA-containing molecule by Dicer, a fragment of the guide strand remains associated with RISC, binds a target RNA as a component of 20 the RISC complex, and promotes cleavage of a target RNA by RISC. As used herein, the guide strand does not necessarily refer to a continuous single stranded nucleic acid and may comprise a discontinuity, preferably at a site that is cleaved by Dicer. A guide strand is an antisense strand. As used herein, "target RNA" refers to an RNA that would be subject to 25 modulation guided by the antisense strand, such as targeted cleavage or steric blockage. The target RNA could be, for example genomic viral RNA, mRNA, a premRNA, or a non-coding RNA. The preferred target is mRNA, such as the mRNA encoding a disease associated protein, such as ApoB, Bcl2, Hif-lalpha, Survivin or a p21 ras, such as Ha. ras, K-ras or N-ras. 30 As used herein, "passenger strand" refers to an oligonucleotide strand of a dsRNA or dsRNA-containing molecule, which has a sequence that is complementary to that of the guide strand. As used herein, the passenger strand does not necessarily refer to a continuous single stranded nucleic acid and may comprise a discontinuity, preferably at a site that is cleaved by Dicer. A passenger strand is a sense strand. 63 Date Re9ue / Date Received 2022-03-14 As used herein, "Dicer" refers to an endoribonuclease in the RNasc III family that cleaves a dsRNA or dsRNA-containing molecule, e.g., double-stranded RNA (dsRNA) or pre-microRNA (miRNA), into double-stranded nucleic acid fragments about 19-25 nucleotides Jong, usually with a two-base overhang on the 3' end. With 5 respect to the dsNAs of the invention, the duplex formed by a dsRNA region of a dsNA of the invention is recognized by Dicer and is a Dicer substrate on at least one strand of the duplex. Dicer catalyzes the first step in the RNA interference pathway, which consequently results in the degradation of a target RNA. The protein sequence of human Dicer is provided at the NCBI database under accession number 10 NP 085124. Dicer "cleavage" is detenuined as follows (e.g., see Collingwood el al., Oligonucleotides l 8:187-200 (2008)). In a Dicer cleavage assay, RNA duplexes (100 pmol) are incubated in 20 μL of20 mM Tris pH 8.0, 200 mM NaCl, 2.5 mM MgCl2 with or without 1 unit of recombinant human Dicer (Stratagene, La Jolla, CA) at 37°C 15 for 18-24 hours. Samples are desalted using a Perforrna SR 96-well plate (Edge Biosystems, Gaithersburg, MD). Electrospray-ionization liquid chromatography ma<;s spectroscopy (ESI-LCMS) of duplex RNAs pre- and post-treatment with Dicer is done using an Oligo HTCS system (Novatia, Princeton, NJ; Hail et al., 2004), which consists of a TherrnoFinnigan TSQ7000, Xcalibur data system, ProMass data 20 processing software and Paradigm MS4 HPLC (Michrom BioResources, Auburn, CA}. In this assay, Dicer cleavage occurs where at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even l 00% of the Dicer substrate dsRNA, (i.e., 25-35 bp dsRNA, preferably 26-30 bp dsRNA, optionally ex.tended as described herein) is cleaved to a shortt.T dsRNA (e.g., 19-23 bp dsRNA, preferably, 21-23 bp 25 dsRNA). As used herein, "Dicer cleavage site" refers to the sites at which Dicer cleaves a dsRNA (e.g., the dsRNA region of a dsNA of the invention). Dicer contains two RNase III domains which typically cleave both the sense and antisensc strands of a dsRNA. The average distance between the RNase Ill domains and the PAZ domain 30 determines the length of the short double-stranded nucleic acid fragments it produces and this distance can vary (Macrae I, ct al. (2006). "Structural basis for doublestranded RNA processing by Dicer". Science 311 (5758): 195-8.). As shown in Figure I A, Dicer is projected to cleave certain double-stranded nucleic acids of the instant invention that possess an antiscnsc strand having a 2 nucleotide 3' overhang at (,.j. Date Re9ue / Date Received 2022-03-14 a site between the 21 st and 22nd nucleotides removed from the 3' terminus of the antisense strand, and at a corresponding site between the 21 st and 22nd nucleotides removed from the 5' terminus of the sense strand. The projected and / or prevalent Dicer cleavage site(s) for dsNA molecules distinct from those depicted in Figure IA 5 may be similarly identified via art-recognized methods, including those described in Macrae et al. While the Dicer cleavage event depicted in Figure IA generates a 21 nucleotide siRNA, it is noted that Dicer cleavage of a dsNA (e.g., DsiRNA) can result in generation of Dicer-processed siRNA lengths of 19 to 23 nucleotides in length. Indeed, in one aspect of the invention that is described in greater detail below, a 10 double stranded DNA region is included within a dsNA for purpose of directing prevalent Dicer excision of a typically non-preferred 19mer siRNA. As used herein, "overhang" refers to unpaired nucleotides, in the context of a duplex having two or four free ends at either the 5' terminus or 3' terminus of a dsNA. In certain embodiments, the overhang is a 3' or 5' overhang on the anti sense 15 strand or sense strand. As used herein, "target" refers to any nucleic acid sequence whose expression or activity is to be modulated. In particular embodiments, the target refers to an RNA which duplexes to a single stranded nucleic acid that is an antisense strand in a RISC complex. Hybridization of the target RNA to the antisense strand results in 20 processing by the RISC complex. Consequently, expression of the RNA or proteins encoded by the RNA, e.g., mRNA, is reduced. As used herein, the term "RNA processing" refers to processing activities performed by components of the siRNA, miRNA or RNase H pathways (e.g., Drosha, Dicer, Argonaute2 or other RISC endoribonucleases, and RNaseH), which 25 are described in greater detail below (see "RNA Processing" section below). The term is explicitly distinguished from the post-transcriptional processes of 5' capping of RNA and degradation of RNA via non-RISC- or non-RNase H-mediated processes. Such "degradation" of an RNA can take several forms, e.g. deadenylation (removal of a 3' poly(A) tail), and / or nuclease digestion of part or all of the body of the RNA by 30 any of several endo- or exo-nucleases (e.g., RNase III, RNase P, RNase Tl, RNase A (1, 2, 3, 4 / 5), oligonucleotidase, etc.). As used herein, "reference" is meant a standard or control. As is apparent to one skilled in the art, an appropriate reference is where only one element is changed 65 Date Re9ue / Date Received 2022-03-14 in order to determine the effect of the one element. As used herein, "modified nucleotide" refers to a nucleotide that has one or more modifications to the nucleoside, the nucleobase, pentose ring, or phosphate group. For example, modified nucleotides exclude ribonucleotides containing 5 adenosine monophosphate, guanosine monophosphate, uridine monophosphate, and cytidine monophosphate and deoxyribonucleotides containing deoxyadenosine monophosphate, deoxyguanosine monophosphate, deoxythymidine monophosphate, and deoxycytidine monophosphate. Modifications include those naturally occuring that result from modification by enzymes that modify nucleotides, such as 10 methyltransferases. Modified nucleotides also include synthetic or non-naturally occurring nucleotides. Synthetic or non-naturally occurring modifications in nucleotides include those with 2' modifications, e.g., 2'-methoxyethoxy, 2'-fluoro, 2'allyl, 2'-O-[2-(methylamino)-2-oxoethyl], 4'-thio, 4'-CHi-O-2'-bridge, 4'-(CH2) 2-O-2'bridge, 2'-LNA, and 2'-O-(N-methylcarbamate) or those comprising base analogs. In 15 connection with 2'-modified nucleotides as described for the present disclosure, by "amino" is meant 2'-NH2 or 2'-O-NH2, which can be modified or unmodified. Such modified groups are described, e.g., in Eckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S. Pat. No. 6,248,878. The term "in vitro" has its art recognized meaning, e.g., involving purified 20 reagents or extracts, e.g., cell extracts. The term "in vivo" also has its art recognized meaning, e.g., involving living cells, e.g., immortalized cells, primary cells, cell lines, and / or cells in an organism. In reference to the nucleic acid molecules of the present disclosure, the modifications may exist in patterns on a strand of the dsNA. As used herein, 25 "alternating positions" refers to a pattern where every other nucleotide is a modified nucleotide or there is an unmodified nucleotide (e.g., an unmodified ribonucleotide) between every modified nucleotide over a defined length of a strand of the dsNA (e.g., 5'-MNMNMN-3'; 3'-MNMNMN-5'; where Mis a modified nucleotide and N is an unmodified nucleotide). The modification pattern starts from the first nucleotide 30 position at either the 5' or 3' terminus according to any of the position numbering conventions described herein (in certain embodiments, position 1 is designated in reference to the terminal residue of a strand following a projected Dicer cleavage event of a DsiRNA agent of the invention; thus, position 1 does not always constitute a 3' terminal or 5' terminal residue of a pre-processed agent of the invention). The 66 Date Re9ue / Date Received 2022-03-14 pattern of modified nucleotides at alternating positions may run the full length of the strand, but in certain embodiments includes at least 4, 6, 8, 10, 12, 14 nucleotides containing at least 2, 3, 4, 5, 6 or 7 modified nucleotides, respectively. As used herein, "alternating pairs of positions" refers to a pattern where two consecutive 5 modified nucleotides are separated by two consecutive unmodified nucleotides over a defined length ofa strand of the dsNA (e.g., 5'-MMNNMMNNMMNN-3'; 3'MMNNMMNNMMNN- 5'; where M is a modified nucleotide and N is an unmodified nucleotide). The modification pattern starts from the first nucleotide position at either the 5' or 3' tenninus according to any of the position numbering conventions IO described herein. The pattern of modified nucleotides at alternating positions may run the full length of the strand, but preferably includes at least 8, 12, 16, 20, 24, 28 nucleotides containing at least 4, 6, 8, IO, 12 or 14 modified nucleotides, respectively. lt is emphasized that the above modification patterns are exemplary and are not intended as limitations on the scope of the invention. 15 As used herein, "base analog" refors to a heterocyclic moiety which is located at the I' position of a nucleotide sugar moiety in a modified nucleotide that can be incorporated into a nucleic acid duplex (or the equivalent position in a nucleotide sugar moiety substitution that can be incorporated into a nucleic acid duplex). In the dsNAs of the invention, a base analog is general1y either a purine or pyrimidine base 20 excluding the common bases guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U). Base analogs can duplex with other bases or base analogs in dsRNAs. Base analogs include those useful in the compounds and methods of the invention., e.g., those disclosed in US Pat. Nos. 5,432,272 and 6,001,983 to Benner and US Patent Publication No. 20080213891 to Manoharan. 25 Non-limiting examples of bases include hypoxanthine (I), xanthine (X), 3P·D-ribofuranosyl-(2,6-diaminopyrimidine) (K), 3-f}-D-ribofuranosyl-(]-methylpyrazolo[ 4,3-d]pyrirnidine-5, 7( 4H,6H)-dione) (P), iso-cytosine (iso-C), iso-guanine (iso-G), 1-~-D-ribofuranosyl-(5-nitroindole), l-f}-D-ribofuranosyl-(3-nitropyrrole), 5- bromouracil, 2-aminopurine, 4-thio-dT, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Os) and 30 pyrrole-2-carbaldehyde (Pa), 2-amino-6-(2-thienyl)purine (S), 2-oxopyridine (Y), difluorotolyl, 4-ftuoro-6-methylbenzimidazole, 4-methylbcnzimidazole, '.>-methyl isocarbostyrilyl, 5-methyl isocarbostyrily], and 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl, 6-melhyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isoc!lfbostyrilyl, propynyl-7-azaindolyl, 67 Date Re9ue / Date Received 2022-03-14 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenzyl, tetracenyl, pentacenyl, and structural derivates thereof (Schweitzer et al., J. Org. Chem., 59:7238-7242 (1994); Berger et al., Nucleic Acids Research, 28(15):2911-2914 (2000); Moran et al., J. Am. 5 Chem. Soc., 119:2056-2057 (1997); Morales et al., J. Am. Chem. Soc., 121 :2323- 2324 (1999); Guckian et al., J. Am. Chem. Soc., 118:8182-8183 (1996); Morales et al., J. Am. Chem. Soc., 122(6):1001-1007 (2000); McMinn et al., J. Am. Chem. Soc., 121 :11585-11586 (1999); Guckian et al., J. Org. Chem., 63:9652-9656 (1998); Moran et al., Proc. Natl. Acad. Sci., 94:10506-10511 (1997); Das et al., J. Chem. Soc., Perkin 10 Trans., 1:197-206 (2002); Shibata et al., J. Chem. Soc., Perkin Trans., 1: 1605-1611 (2001); Wu et al., J. Am. Chem. Soc., 122(32):7621-7632 (2000); O'Neill et al., J. Org. Chem., 67:5869-5875 (2002); Chaudhuri et al., J. Am. Chem. Soc., 117:10434- 10442 (1995); and U.S. Pat. No. 6,218,108.). Base analogs may also be a universal base. 15 As used herein, ''universal base" refers to a heterocyclic moiety located at the 1' position of a nucleotide sugar moiety in a modified nucleotide, or the equivalent position in a nucleotide sugar moiety substitution, that, when present in a nucleic acid duplex, can be positioned opposite more than one type of base without altering the double helical structure (e.g., the structure of the phosphate backbone). Additionally, 20 the universal base does not destroy the ability of the single stranded nucleic acid in which it resides to duplex to a target nucleic acid. The ability of a single stranded nucleic acid containing a universal base to duplex a target nucleic can be assayed by methods apparent to one in the art (e.g., UV absorbance, circular dichroism, gel shift, single stranded nuclease sensitivity, etc.). Additionally, conditions under which 25 duplex formation is observed may be varied to determine duplex stability or formation, e.g., temperature, as melting temperature (Tm) correlates with the stability of nucleic acid duplexes. Compared to a reference single stranded nucleic acid that is exactly complementary to a target nucleic acid, the single stranded nucleic acid containing a universal base forms a duplex with the target nucleic acid that has a 30 lower Tm than a duplex formed with the complementary nucleic acid. However, compared to a reference single stranded nucleic acid in which the universal base has been replaced with a base to generate a single mismatch, the single stranded nucleic acid containing the universal base forms a duplex with the target nucleic acid that has a higher Tm than a duplex formed with the nucleic acid having the mismatched base. 68 Date Re9ue / Date Received 2022-03-14 Some universal bases are capable of base pairing by forming hydrogen bonds between the universal base and all of the bases guanine (G), cytosine (C), adenine (A), thymine (T), and uracil (U) under base pair forming conditions. A universal base is not a base that forms a base pair with only one single complementary base. In a 5 duplex, a universal base may form no hydrogen bonds, one hydrogen bond, or more than one hydrogen bond with each of G, C, A, T, and U opposite to it on the opposite strand of a duplex. Preferably, the universal bases does not interact with the base opposite to it on the opposite strand of a duplex. In a duplex, base pairing between a universal base occurs without altering the double helical structure of the phosphate 10 backbone. A universal base may also interact with bases in adjacent nucleotides on the same nucleic acid strand by stacking interactions. Such stacking interactions stabilize the duplex, especially in situations where the universal base does not form any hydrogen bonds with the base positioned opposite to it on the opposite strand of the duplex. Non-limiting examples of universal-binding nucleotides include inosine, 15 1-P-D-ribofuranosyl-5-nitroindole, and / or 1-P-D-ribofuranosyl-3-nitropyrrole (US Pat. Appl. Puhl. No. 20070254362 to Quay et al.; Van Aerschot et al., An acyclic 5- nitroindazole nucleoside analogue as ambiguous nucleoside. Nucleic Acids Res. 1995 Nov 11 ;23(21 ):4363-70; Loakes et al., 3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR. Nucleic Acids Res. 1995 Jul 20 11;23(13):2361-6; Loakes and Brown, 5-Nitroindole as an universal base analogue. Nucleic Acids Res. 1994 Oct 11 ;22(20):4039-43). As used herein, "loop" refers to a structure formed by a single strand of a nucleic acid, in which complementary regions that flank a particular single stranded nucleotide region hybridize in a way that the single stranded nucleotide region 25 between the complementary regions is excluded from duplex formation or WatsonCrick base pairing. A loop is a single stranded nucleotide region of any length. Examples ofloops include the unpaired nucleotides present in such structures as hairpins, stem loops, or extended loops. As used herein, "extended loop" in the context of a dsRNA refers to a single 30 stranded loop and in addition I, 2, 3, 4, 5, 6 or up to 20 base pairs or duplexes flanking the loop. In an extended loop, nucleotides that flank the loop on the 5' side form a duplex with nucleotides that flank the loop on the 3' side. An extended loop may form a hairpin or stem loop. As used herein, "tetraloop" in the context of a dsRNA refers to a loop (a single 69 Date Re9ue / Date Received 2022-03-14 stranded region) consisting of four nucleotides that forms a stable secondary structure that contributes to the stability of an adjacent Watson-Crick hybridized nucleotides. Without being limited to theory, a tetraloop may stabilize an adjacent Watson-Crick base pair by stacking interactions. In addition, interactions among the four 5 nucleotides in a tetraloop include but are not limited to non-Watson-Crick base pairing, stacking interactions, hydrogen bonding, and contact interactions (Cheong et al., Nature 1990 Aug 16;346(6285):680-2; Heus and Pardi, Science 1991 Jul l 2;253(5016): 191-4). A tetraloop confers an increase in the melting temperature (Tm} of an adjacent duplex that is higher than expected from a simple model loop 10 sequence consisting of four random bases. For example, a tetraloop can confer a melting temperature of at least 55°C in 1 OmM NaHPO4 to a hairpin comprising a duplex of at least 2 base pairs in length. A tetraloop may contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Examples of RNA tetraloops include the UNCG family oftetraloops (e.g., UUCG), the GNRA 15 family of tetraloops ( e.g., GAAA}, and the CUUG tetraloop. (Woese et al., Proc Natl Acad Sci US A. 1990 Nov;87(21):8467-71; Antao et al., Nucleic Acids Res. 1991 Nov 11;19(21):5901-5). Examples of DNA tetraloops include the d(GNNA) family oftetraloops (e.g., d(GTTA), the d(GNRA)) family oftetraloops, the d(GNAB) family of tetraloops, the d(CNNG) family oftetraloops, the d(TNCG) family of 20 tetraloops (e.g., d(TTCG)). (Nakano et al. Biochemistry, 41 (48), 14281 -14292, 2002.; SHINJI et al. Nippon Kagakkai Koen Yokoshu VOL.78th; NO.2; PAGE.731 (2000).) As used herein, "increase" or "enhance" is meant to alter positively by at least 5% compared to a reference in an assay. An alteration may be by 5%, 10%, 25%, 25 30%, 50%, 75%, or even by I 00% compared to a reference in an assay. By "enhance Dicer cleavage," it is meant that the processing of a quantity of a dsRNA or dsRNAcontaining molecule by Dicer results in more Dicer cleaved dsRNA products, that Dicer cleavage reaction occurs more quickly compared to the processing of the same quantity of a reference dsRNA or dsRNA-containing molecule in an in vivo or in vitro 30 assay of this disclosure, or that Dicer cleavage is directed to cleave at a specific, preferred site within a dsNA and / or generate higher prevalence of a preferred population of cleavage products (e.g., by inclusion of DNA residues as described herein). In one embodiment, enhanced or increased Dicer cleavage of a dsNA molecule is above the level of that observed with an appropriate reference dsNA 70 Date Re9ue / Date Received 2022-03-14 molecule. In another embodiment, enhanced or increased Dicer cleavage of a dsNA molecule is above the level of that observed with an inactive or attenuated molecule. As used herein "reduce" is meant to alter negatively by at least 5% compared to a reference in an assay. An alteration may be by 5%, 10%, 25%, 30%, 50%, 75%, 5 or even by 100% compared to a reference in an assay. By "reduce expression," it is meant that the expression of the gene, or level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits, or level or activity of one or more proteins or protein subunits encoded by a target gene, is reduced below that observed in the absence of the nucleic acid molecules (e.g., dsRNA molecule or 10 dsRNA-containing molecule) in an in vivo or in vitro assay of this disclosure. In one embodiment, inhibition, down-regulation or reduction with a dsNA molecule is below that level observed in the presence of an inactive or attenuated molecule. In another embodiment, inhibition, down-regulation, or reduction with dsNA molecules is below that level observed in the presence of, e.g., a dsNA molecule with scrambled sequence 15 or with mismatches. In another embodiment, inhibition, down-regulation, or reduction of gene expression with a nucleic acid molecule of the instant disclosure is greater in the presence of the nucleic acid molecule than in its absence. As used herein, "cell" is meant to include both prokaryotic (e.g., bacterial) and eukaryotic ( e.g., mammalian or plant) cells. Cells may be of somatic or germ line 20 origin, may be totipotent or pluripotent, and may be dividing or non-dividing. Cells can also be derived from or can comprise a gamete or an embryo, a stem cell, or a fully differentiated cell. Thus, the term "cell" is meant to retain its usual biological meaning and can be present in any organism such as, for example, a bird, a plant, and a mammal, including, for example, a human, a cow, a sheep, an ape, a monkey, a pig, 25 a dog, and a cat. Within certain aspects, the term "cell" refers specifically to mammalian cells, such as human cells, that contain one or more isolated dsNA molecules of the present disclosure. In particular aspects, a cell processes dsRNAs or dsRNA-containing molecules resulting in RNA intereference of target nucleic acids, and contains proteins and protein complexes required for RNAi, e.g., Dicer and RISC. 30 As used herein, "animal" is meant a multicellular, eukaryotic organism, including a mammal, particularly a human. The methods of the invention in general comprise administration of an effective amount of the agents herein, such as an agent of the structures of formulae herein, to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably 71 Date Re9ue / Date Received 2022-03-14 administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, or a symptom thereof. By "pharmaceutically acceptable carrier" is meant, a composition or formulation that allows for the effective distribution of the nucleic acid molecules of 5 the instant disclosure in the physical location most suitable for their desired activity. The present invention is directed to compositions that comprise both a double stranded RNA ("dsRNA") duplex and DNA-containing extended region - in most embodiments, a dsDNA duplex - within the same agent, and methods for preparing them, that are capable of reducing the expression of target genes in eukaryotic cells. 10 One of the strands of the dsRNA region contains a region of nucleotide sequence that has a length that ranges from about 15 to about 22 nucleotides that can direct the destruction of the RNA transcribed from the target gene. The dsDNA duplex region of such an agent is not necessarily complementary to the target RNA, and, therefore, in such instances does not enhance target RNA hybridization of the region of 15 nucleotide sequence capable of directing destruction of a target RNA. Double stranded NAs of the invention can possess strands that are chemically linked, or can also possess an extended loop, optionally comprising a tetraloop, that links the first and second strands. In some embodiments, the extended loop containing the tetraloop is at the 3' terminus of the sense strand, at the 5' terminus of the antisense strand, or 20 both. In one embodiment, the dsNA of the invention comprises a double stranded RNA duplex region comprising 18-30 nts (for example, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 nts) in length. The DsiRNNdsDNA agents of the instant invention can enhance the 25 following attributes of such agents relative to DsiRNAs lacking such dsDNA regions: in vitro efficacy (e.g., potency and duration of effect), in vivo efficacy (e.g., potency, duration of effect, pharmacokinetics, pharmacodynamics, intracellular uptake, reduced toxicity). In certain embodiments, the dsDNA region of the instant invention can optionally provide an additional agent (or fragment thereof), such as an aptamer 30 or fragment thereof; a binding site (e.g., a "decoy" binding site) for a native or exogenously introduced moiety capable of binding to dsDNA in either a nonsequence- selective or sequence-specific manner (e.g., the dsDNA-extended region of an agent of the instant invention can be designed to comprise one or more transcription factor recognition sequences and / or the dsDNA-extended region can 72 Date Re9ue / Date Received 2022-03-14 provide a sequence-specific recognition domain for a probe, marker, etc.). As used herein, the term "pharmacokinetics" refers to the process by which a drug is absorbed, distributed, metabolized, and eliminated by the body. In certain embodiments of the instant invention, enhanced pharmacokinetics of a 5 DsiRNA / dsDNA agent relative to an appropriate control DsiRNA refers to increased absorption and / or distribution of such an agent, and / or slowed metabolism and / or elimination of such a DsiRNA / dsDNA agent from a subject administered such an agent. As used herein, the term "pharmacodynamics" refers to the action or effect of 10 a drug on a living organism. In certain embodiments of the instant invention, enhanced pharmacodynamics of a DsiRNA / dsDNA agent relative to an appropriate control DsiRNA refers to an increased (e.g., more potent or more prolonged) action or effect of a DsiRNA / dsDNA agent upon a subject administered such agent, relative to an appropriate control DsiRNA. 15 As used herein, the term "stabilization" refers to a state of enhanced persistence of an agent in a selected environment ( e.g., in a cell or organism). In certain embodiments, the DsiRNA / dsDNA chimeric agents of the instant invention exhibit enhanced stability relative to appropriate control DsiRNAs. Such enhanced stability can be achieved via enhanced resistance of such agents to degrading enzymes 20 (e.g., nucleases) or other agents. DsiRNA Design / Synthesis It was previously shown that longer dsRNA species of from 25 to about 30 nucleotides (DsiRNAs} yield unexpectedly effective RNA inhibitory results in terms of potency and duration of action, as compared to 19-23mer siRNA agents. Without 25 wishing to be bound by the underlying theory of the dsRNA processing mechanism, it is thought that the longer dsRNA species serve as a substrate for the Dicer enzyme in the cytoplasm of a cell. In addition to cleaving the dsNA of the invention into shorter segments, Dicer is thought to facilitate the incorporation of a single-stranded cleavage product derived from the cleaved dsNA into the RISC complex that is responsible for 30 the destruction of the cytoplasmic RNA of or derived from the target gene. Prior studies (Rossi et al., U.S. Patent Application No. 2007 / 0265220) have shown that the cleavability of a dsRNA species (specifically, a DsiRNA agent) by Dicer corresponds with increased potency and duration of action of the dsRNA species. The instant 73 Date Re9ue / Date Received 2022-03-14 invention, at least in part, provides for design of RNA inhibitory agents that direct the site of Dicer cleavage, such that preferred species of Dicer cleavage products are thereby generated. A model of DsiRNA processing is presented in Figure IA. Briefly, Dicer 5 enzyme binds to a DsiRNA agent, resulting in cleavage of the DsiRNA at a position 19-23 nucleotides removed from a Dicer PAZ domain-associated 3' overhang sequence of the antisense strand of the DsiRNA agent. This Dicer cleavage event results in excision of those duplexed nucleic acids previously located at the 3' end of the passenger (sense) strand and 5' end of the guide (antisense) strand. (Cleavage of 10 the DsiRNA shown in Figure IA typically yields a 19mer duplex with 2-base overhangs at each end.) As presently modeled in Figure IA, this Dicer cleavage event generates a 21-23 nucleotide guide (antisense) strand capable of directing sequencespecific inhibition of target mRNA as a RISC component. The first and second oligonucleotides of the DsiRNA agents of the instant 15 invention are not required to be completely complementary. In fact, in one embodiment, the 3'-terminus of the sense strand contains one or more mismatches. In one aspect, about two mismatches are incorporated at the 3' terminus of the sense strand. In another embodiment, the DsiRNA of the invention is a double stranded RNA molecule containing two RNA oligonucleotides each of which is an identical 20 number of nucleotides in the range of 27-35 nucleotides in length and, when annealed to each other, have blunt ends and a two nucleotide mismatch on the 3'-terminus of the sense strand (the 5'-terminus of the antisense strand). The use of mismatches or decreased thermodynamic stability (specifically at the 3'-sense / 5'-antisense position) has been proposed to facilitate or favor entry of the antisense strand into RISC 25 (Schwarz et al., 2003; Khvorova et al., 2003), presumably by affecting some ratelimiting unwinding steps that occur with entry of the siRNA into RISC. Thus, terminal base composition has been included in design algorithms for selecting active 21mer siRNA duplexes (Ui-Tei et al., 2004; Reynolds et al., 2004). With Dicer cleavage of the dsRNA region of this embodiment, the small end-terminal sequence 30 which contains the mismatches will either be left unpaired with the antisense strand (become part of a 3'-overhang) or be cleaved entirely off the final 21-mer siRNA. These specific forms of "mismatches", therefore, do not persist as mismatches in the final RNA component of RISC. The finding that base mismatches or destabilization of segments at the 3'-end of the sense strand of Dicer substrate improved the potency 74 Date Re9ue / Date Received 2022-03-14 of synthetic duplexes in RN Ai, presumably by facilitating processing by Dicer, was a surprising finding of past works describing the design and use of 25-30mer dsRNAs (also termed "DsiRNAs" herein; Rossi et al., U.S. Patent Application Nos. 2005 / 0277610, 2005 / 0244858 and 2007 / 0265220). It is now equally surprising that 5 DsiRNAs having base-paired deoxyribonucleotides at either passenger (sense) or guide (antisense) strand positions that are predicted to be 3' of the most 3' Dicer cleavage site of the respective passenger or guide strand are at least equally effective as RNA-RNA duplex-extended DsiRNA agents. Such agents may also harbor mismatches, with such mismatches being formed by the antisense strand either in 10 reference to (actual or projected hybridation with) the sequence of the sense strand of the DsiRNA agent, or in reference to the target RNA sequence. Exemplary mismatched or wobble base pairs of agents possessing mismatches are G:A, C:A, C:U, G:G, A:A, C:C, U:U, I:A, I:U and I:C. Base pair strength of such agents can also be lessened via modification of the nucleotides of such agents, including, e.g., 2- 15 amino- or 2,6-diamino modifications of guanine and adenine nucleotides. Exemplary Structures of DsiRNA Agent Compositions In one aspect, the present invention provides compositions for RNA interference (RNAi) that possess one or more base paired deoxyribonucleotides within 20 a region of a double stranded nucleic acid ( dsNA) that is positioned 3' of a projected sense strand Dicer cleavage site and correspondingly 5' of a projected anti sense strand Dicer cleavage site. The compositions of the invention comprise a dsNA which is a precursor molecule, i.e., the dsNA of the present invention is processed in vivo to produce an active small interfering nucleic acid (siRNA). The dsNA is processed by 25 Dicer to an active siRNA which is incorporated into RISC. In certain embodiments, the DsiRNA agents of the invention can have any of the following exemplary structures: In one such embodiment, the DsiRNA comprises: 5'-XXXXXXXXXXXXXXXXXXXXXXXXN*~DD-3' 30 3 '-YXXXXXXXXXXXXXXXXXXXXXXXXN*~XX-5' wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'- 75 Date Re9ue / Date Received 2022-03-14 O-methyl RNA monomers, "D"=DNA, and "N"=l to 50 or more, but is optionally 1- 15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2,'3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top 5 strand is the antisense strand. In a related embodiment, the DsiRNA comprises: 5' -XXXXXXXXXXXXXXXXXXXXXXXXN*Dt,DD-3' 3 ' - YXXXXXXXXXXXXXXXXXXXXXXXXN*Dt,DD-5' wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA 10 monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'O- methyl RNA monomers, "D"=DNA, and "N"=l to 50 or more, but is optionally 1- 15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the 15 antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. In another such embodiment, the DsiRNA comprises: 5 ' - XXXXXXXXXXXXXXXXXXXXXXXXN*Dt.DD-3 ' 3 ' - YXXXXXXXXXXXXXXXXXXXXXXXXN*DNZ Z-5' 20 wherein "X"=RNA, "X"=2'-O-methyl RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA, "Z"=DNA or RNA, and ''N"= 1 to 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 25 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-Omethyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic. 30 In another such embodiment, the DsiRNA comprises: 5' -XXXXXXXXXXXXXXXXXXXXXXXXN*Dt.DD-3' 3 ' - YXXXXXXXXXXXXXXXXXXXXXXXXN*Dt,Z Z-5' wherein "X"=RNA, "X"=2'-O-methyl RNA, "Y" is an optional overhang domain 76 Date Re9ue / Date Received 2022-03-14 comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of I -4 RNA monomers that are optionally 2'-O-methyl RNA monomers, "D"=DNA, "Z"=DNA or RNA, and "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 5 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2' -Omethyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic. 10 In another embodiment, the DsiRNA comprises: 5'-XXXXXXXXXXXXXXXXXXXXXXXXN*[Xl / Dl]NDD-3' 3 '-YXXXXXXXXXXXXXXXXXXXXXXXXN* [X2 / D2 J Nzz-5' wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, 15 "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'O- methyl RNA monomers, "D"=DNA, "Z"=DNA or RNA, and "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8, where at least one D 1 N is present in the top strand and is base paired with a corresponding D2N in the bottom strand. Optionally, DIN and DlN+J are base paired with corresponding D2N and D2N+1; DIN, DIN+1 and 20 D1N+2 are base paired with corresponding D2N, DlN+J and DIN+2, etc. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2' -O-methyl RNA monomers located at alternating residues along the top strand, rather than the 25 bottom strand presently depicted in the above schematic. In any of the above-depicted structures, the 5' end of either the sense strand or antisense strand optionally comprises a phosphate group. In another embodiment, the DNA:DNA-extended DsiRNA comprises strands having equal lengths possessing 1-3 mismatched residues that serve to orient Dicer 30 cleavage (specifically, one or more of positions 1, 2 or 3 on the first strand of the DsiRNA, when numbering from the 3 '-terminal residue, are mismatched with corresponding residues of the 5'-terminal region on the second strand when first and second strands are annealed to one another). An exemplary DNA:DNA-extended 77 Date Re9ue / Date Received 2022-03-14 DsiRNA agent with two terminal mismatched residues is shown: M-3' 5 ' -xxxxxxxxxxxxxxxxxxxxxxxxxxN*~M 3'-XXXXXXXXXXXXXXXXXXXXXXXXXXN*~ M-5' wherein "X"=RNA, "M"=Nucleic acid residues (RNA, DNA or non-natural or 5 modified nucleic acids) that do not base pair (hydrogen bond) with corresponding "M" residues of otherwise complementary strand when strands are annealed, "D"=DNA and "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. Any of the residues of such agents can optionally be 2'-O-methyl RNA monomers - alternating positioning 10 of2'-O-methyl RNA monomers that commences from the 3'-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above "blunt / fray" DsiRNA agent. In one embodiment, the top strand (first strand) is the sense strand, and the bottom strand (second strand) is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the 15 antisense strand. Modification and DNA:DNA extension patterns paralleling those shown above for asymmetric / overhang agents can also be incorporated into such "blunt / frayed" agents. In one embodiment, a length-extended DsiRNA agent is provided that comprises deoxyribonucleotides positioned at sites modeled to function via specific 20 direction of Dicer cleavage, yet which does not require the presence of a base-paired deoxyribonucleotide in the dsNA structure. An exemplary structure for such a molecule is shown: 5'-XXXXXXXXXXXXXXXXXXXDDXX-3' 3'-YXXXXXXXXXXXXXXXXXDDXXXX-5' 25 wherein "X"=RNA, "Y" is an optional overhang domain comprised of0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'O- methyl RNA monomers, and "D"=DNA. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom 30 strand is the sense strand and the top strand is the antisense strand. The above structure is modeled to force Dicer to cleave a minimum of a 21 mer duplex as its primary post-processing form. In embodiments where the bottom strand of the above structure is the antisense strand, the positioning of two deoxyribonucleotide residues 78 Date Re9ue / Date Received 2022-03-14 at the ultimate and penultimate residues of the 5' end of the antisense strand is likely to reduce off-target effects (as prior studies have shown a 2'-O-methyl modification of at least the penultimate position from the 5' terminus of the anti sense strand to reduce off-target effects; see, e.g., US 2007 / 0223427). 5 In one embodiment, the DsiRNA comprises: 5 ' - ~XXXXXXXXXXXXXXXXXXXXXXXXN• Y - 3 ' 3' -~XXXXXXXXXXXXXXXXXXXXXXXXN•-5' wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, 10 "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'O- methyl RNA monomers, "D"=DNA, and "N"=l to 50 or more, but is optionally 1- 15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top 15 strand is the antisense strand. In a related embodiment, the DsiRNA comprises: 5'-~XXXXXXXXXXXXXXXXXXXXXXXXN•DD-3' 3 '-~XXXXXXXXXXXXXXXXXXXXXXXXN*XX-5' wherein "X"=RNA, optionally a 2'-O-methyl RNA monomers "D"=DNA, "N"=l to 20 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. In another such embodiment, the DsiRNA comprises: 25 5 ' - ~XXXXXXXXXXXXXXXXXXXXXXXXN*DD-3 ' 3 ' - ~XXXXXXXXXXXXXXXXXXXXXXXXN• Z Z-5' wherein "X"=RNA, optionally a 2'-O-methyl RNA monomers "D"=DNA, ''N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8. ''N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. "Z"=DNA or RNA. In one embodiment, the top strand 30 is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2 '-Om ethyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic. 79 Date Re9ue / Date Received 2022-03-14 In another such embodiment, the DsiRNA comprises: 5'-~ZZXXXXXXXXXXXXXXXXXXXXXXXXN•DD-3' 3 ' - ~XXXXXXXXXXXXXXXXXXXXXXXXXXN*ZZ-5' wherein "X"=RNA, "X"=2'-O-methyl RNA, "D"=DNA, "Z"=DNA or RNA, and 5 "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2 '-O-methyl RNA monomers located at alternating residues along the top strand, rather than the 10 bottom strand presently depicted in the above schematic. In another such embodiment, the DsiRNA comprises: 5' -~ZZXXXXXXXXXXXXXXXXXXXXXXXXN•Y-3' 3 ' - ~xxxxxxxxxxxxxxxxxxxxxxxxxxN.-5' wherein "X"=RNA, "X"=2'-O-methyl RNA, "D"=DNA, "Z"=DNA or RNA, and 15 "N"= 1 to 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers. In one embodiment, the 20 top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic. In another embodiment, the DsiRNA comprises: 25 5 ' - [Xl / DlhXXXXXXXXXXXXXXXXXXXXXXXXN·DD-3' 3' - [X2 / D2 J NxxxxxxxxxxxxxxxxxxxxxxxxN.zz-5' wherein "X"=RNA, "D"=DNA, "Z"=DNA or RNA, and "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8, where at least one D 1 N is present in the top strand and is base paired with a corresponding D2N in the bottom strand. Optionally, DIN 30 and DlN+1 are base paired with corresponding D2N and D2N+1; DIN, DIN+I and DIN+2 are base paired with corresponding D2N, D 1 N+ 1 and D 1 N+2, etc. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom 80 Date Re9ue / Date Received 2022-03-14 strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic. In a related embodiment, the DsiRNA comprises: 5 5 ' - [ Xl / Dl hXXXXXXXXXXXXXXXXXXXXXXXXw Y - 3 ' 3' - [X2 / D2 hXXXXXXXXXXXXXXXXXXXXXXXXw-5' wherein "X"=RNA, "D"=DNA, "Y" is an optional overhang domain comprised of 0- 10 RNA monomers that are optionally 2'-O-methyl RNA monomers -in certain embodiments, "Y" is an overhang domain comprised of 1-4 RNA monomers that are 10 optionally 2'-O-methyl RNA monomers, and "N"=l to 50 or more, but is optionally 1- 15 or, optionally, 1-8, where at least one DIN is present in the top strand and is base paired with a corresponding D2N in the bottom strand. Optionally, DlN and DlN+I are base paired with corresponding D2N and D2N+1; DIN, DlN+l and DlN+z are base paired with corresponding D2N, DlN+l and DlN+2, etc. "N*"=0 to 15 or more, but is 15 optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand, with 2'-O-methyl RNA monomers located at alternating residues along the top strand, rather than the bottom strand presently depicted in the above schematic. 20 In any of the above-depicted structures, the 5' end of either the sense strand or antisense strand optionally comprises a phosphate group. In another embodiment, the DNA:DNA-extended DsiRNA comprises strands having equal lengths possessing 1-3 mismatched residues that serve to orient Dicer cleavage (specifically, one or more of positions 1, 2 or 3 on the first strand of the 25 DsiRNA, when numbering from the 3 '-terminal residue, are mismatched with corresponding residues of the 5 '-terminal region on the second strand when first and second strands are annealed to one another). An exemplary DNA:DNA-extended DsiRNA agent with two terminal mismatched residues is shown: M-3' 5 ' -Dr.XXXXXXXXXXXXXXXXXXXXXXXXXXN*M 30 3 ' - Dr.XXXXXXXXXXXXXXXXXXXXXXXXXXN*M M-5' wherein "X"=RNA, "M"=Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) that do not base pair (hydrogen bond) with corresponding 81 Date Re9ue / Date Received 2022-03-14 "M" residues of otherwise complementary strand when strands are annealed, "D"=DNA and "N"=l to 50 or more, but is optionally 1-15 or, optionally, 1-8. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. Any of the residues of such agents can optionally be 2'-O-methyl RNA monomers - alternating positioning 5 of2'-O-methyl RNA monomers that commences from the 3'-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above "blunt / fray" DsiRNA agent. In one embodiment, the top strand (first strand) is the sense strand, and the bottom strand (second strand) is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the 10 antisense strand. Modification and DNA:DNA extension patterns paralleling those shown above for asymmetric / overhang agents can also be incorporated into such "blunt / frayed" agents. In another embodiment, a length-extended DsiRNA agent is provided that comprises deoxyribonucleotides positioned at sites modeled to function via specific 15 direction of Dicer cleavage, yet which does not require the presence of a base-paired deoxyribonucleotide in the dsNA structure. Exemplary structures for such a molecule are shown: 5'-XXDDXXXXXXXXXXXXXXXXXXXXN*Y-3' 3'-DDXXXXXXXXXXXXXXXXXXXXXXN*-5' 20 or 5 ' - XDXDXXXXXXXXXXXXXXXXXXXXN* Y - 3 ' 3'-DXDXXXXXXXXXXXXXXXXXXXXXN*-5' wherein "X"=RNA, "Y" is an optional overhang domain comprised of 0-10 RNA monomers that are optionally 2'-O-methyl RNA monomers - in certain embodiments, 25 "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'O- methyl RNA monomers, and "D"=DNA. "N*"=0 to 15 or more, but is optionally 0, 1, 2, 3, 4, 5 or 6. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. The above structures are modeled to 30 force Dicer to cleave a minimum of a 2lmer duplex as its primary post-processing form. In any of the above embodiments where the bottom strand of the above structure is the antisense strand, the positioning of two deoxyribonucleotide residues at the ultimate and penultimate residues of the 5' end of the antisense strand is likely 82 Date Re9ue / Date Received 2022-03-14 to reduce off-target effects (as prior studies have shown a 2'-O-methyl modification of at least the penultimate position from the 5' terminus of the antisense strand to reduce off-target effects; see, e.g., US 2007 / 0223427). The extended DsiRNAs of the invention can carry a broad range of 5 modification patterns (e.g., 2'-O-methyl RNA patterns within extended DsiRNA agents). Certain preferred modification patterns of the second strand of the extended DsiRNAs of the invention are presented below - it is noted that while many of the below structures depict modification of non-extended DsiRNAs, the skilled artisan will recognize that the modification patterns shown are also readily applied to the full 10 range of extended DsiRN A structures described elsewhere herein. In one embodiment, the DsiRNA comprises: 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3 ' - YXXXXXXXXXXXXXXXXXXXXXXXXXp-5' wherein "X"=RNA, "p"=a phosphate group, "Y" is an overhang domain comprised of 15 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, and "D"=DNA. In one embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. In another embodiment, the DsiRNA comprises: 20 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3 ' - YXXXXXXXXXXXXXXXXXXXXXXXXXp-5' wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, underlined residues are 2 '-O-methyl RNA monomers, and 25 "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another embodiment, the DsiRNA comprises: 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3 ' - YXXXXXXXXXXXXXXXXXXXXXXXXXp-5' 30 wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, underlined residues are 2 '-O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense 83 Date Re9ue / Date Received 2022-03-14 strand. In further embodiments, the DsiRNA comprises: 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3 ' - YXXXXXXXXXXX~X~X~XXXXXXXXXp-5' 5 wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, underlined residues are 2' -O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In one embodiment, the DsiRNA comprises: 10 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3' -XXXXXXXXXXXXX~X~X~XXXXXXXXXp-5' wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In additional embodiments, the DsiRNA comprises: 15 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3 ' - Y~XXXXXXXXXX~X~X~XXXXXXXXXp-5' wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, underlined residues are 2'-O-methyl RNA monomers, and 20 "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In one embodiment, the DsiRNA comprises: 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3 ' -~X~XXXXXXXXXX~X~X~XXXXXXXXXp-5' wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, and "D"=DNA. 25 The top strand is the sense strand, and the bottom strand is the antisense strand. In other embodiments, the DsiRNA comprises: 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3 ' - Y~X~XXXXXXXX~X~X~XXXXXXXX~p-5' wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, "Y" is an 30 overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, underlined residues are 2'-O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense 84 Date Re9ue / Date Received 2022-03-14 strand. In one embodiment, the DsiRNA comprises: 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3 ' -XXXX~XXXXXXXX~X~X~XXXXXXXX~p-5' wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, and "D"=DNA. 5 The top strand is the sense strand, and the bottom strand is the antisense strand. In further embodiments, the DsiRNA comprises: 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3 ' - Y~X~X~XXXXXX~X~X~XXXXXXXXXp-5' wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, "Y" is an 10 overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, underlined residues are 2'-O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In one embodiment, the DsiRNA comprises: 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 15 3 '-XXXX~X~XXXXXX~X~X~XXXXXXXXXp-5' wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In additional embodiments, the DsiRNA comprises: 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 20 3 '-Y~X~X~XXXXXXX~X~XXXXXXXXXXp-5' wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, underlined residues are 2'-O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense 25 strand. In one embodiment, the DsiRNA comprises: 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3 ' - XXXX~X~XXXXXXX~X~XXXXXXXXXXp-5' wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. 30 In other embodiments, the DsiRNA comprises: 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3 '-Y~X~~XXXXXXXXXXXXXXXXXXXXp-5' 85 Date Re9ue / Date Received 2022-03-14 wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, underlined residues are 2 '-O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense 5 strand. In one embodiment, the DsiRNA comprises: 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3 '-XXXX!X!XXXXXXXXXXXXXXXXXXXXp-5' wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. 10 In certain additional embodiments, the DsiRNA comprises: 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3 ' - Y!X!X!X!XXXXXXXXXXXXXXXXXXp-5' wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl 15 RNA monomers, underlined residues are 2 '-O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In one embodiment, the DsiRNA comprises: 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3 ' -XXXX!X!X!XXXXXXXXXXXXXXXXXXp-5' 20 wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In additional embodiments, the DsiRNA comprises: 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3 ' - Y!X!XXXXXXXXXX!X!XXXXXXXX!p-5' 25 wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl RNA monomers, underlined residues are 2'-O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In one embodiment, the DsiRNA comprises: 86 Date Re9ue / Date Received 2022-03-14 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3 ' - XXXX~XXXXXXXXXX~X~XXXXXXXX~p-5' wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. 5 In further embodiments, the DsiRNA comprises: 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3 ' - Y~X~XXXXXXXXXX~X~XXXXXXXXXp-5' wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, "Y" is an overhang domain comprised of 1-4 RNA monomers that are optionally 2'-O-methyl 10 RNA monomers, underlined residues are 2 '-O-methyl RNA monomers, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In one embodiment, the DsiRNA comprises: 5'-pXXXXXXXXXXXXXXXXXXXXXXXDD-3' 3 '-XXXX~XXXXXXXXXX~X~XXXXXXXXXp-5' 15 wherein "X"=RNA, "p"=a phosphate group, "X"=2'-O-methyl RNA, and "D"=DNA. The top strand is the sense strand, and the bottom strand is the antisense strand. In another embodiment, the DsiRNA comprises strands having equal lengths possessing 1-3 mismatched residues that serve to orient Dicer cleavage (specifically, one or more of positions 1, 2 or 3 on the first strand of the DsiRNA, when numbering 20 from the 3 '-terminal residue, are mismatched with corresponding residues of the 5 ' - terminal region on the second strand when first and second strands are annealed to one another). An exemplary 27mer DsiRNA agent with two terminal mismatched residues is shown: 25 M-3' 5'-pxxxxxxxxxxxxxxxxxxxxxxxxxM 3'-XXXXXXXXXXXXXXXXXXXXXXXXXM Mp-5' wherein "X"=RNA, "p"=a phosphate group, "M"=Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) that do not base pair (hydrogen bond) with corresponding "M" residues of otherwise complementary strand when strands are annealed. Any of the residues of such agents can optionally be 2'-O-methyl RNA 30 monomers- alternating positioning of2'-O-methyl RNA monomers that commences from the 3'-terminal residue of the bottom (second) strand, as shown for above asymmetric agents, can also be used in the above "blunt / fray" DsiRNA agent. In one 87 Date Re9ue / Date Received 2022-03-14 embodiment, the top strand is the sense strand, and the bottom strand is the antisense strand. Alternatively, the bottom strand is the sense strand and the top strand is the antisense strand. As used herein "DsiRNAmm" refers to a DisRNA having a "mismatch 5 tolerant region" containing one, two, three or four mismatched base pairs of the duplex formed by the sense and antisense strands of the DsiRNA, where such mismatches are positioned within the DsiRNA at a location(s) lying between (and thus not including) the two terminal base pairs of either end of the DsiRNA. The mismatched base pairs are located within a "mismatch-tolerant region" which is 10 defined herein with respect to the location of the projected Ago2 cut site of the corresponding target nucleic acid. The mismatch tolerant region is located "upstream of' the projected Ago2 cut site of the target strand. "Upstream" in this context will be understood as the 5'-most portion of the DsiRNAmm duplex, where 5' refers to the orientation of the sense strand of the DsiRNA duplex. Therefore, the mismatch 15 tolerant region is upstream of the base on the sense (passenger) strand that corresponds to the projected Ago2 cut site of the target nucleic acid (see Fig 14); alternatively, when referring to the antisense (guide) strand of the DsiRNAmm, the mismatch tolerant region can also be described as positioned downstream of the base that is complementary to the projected Ago2 cut site of the target nucleic acid, that is, 20 the 3'-most portion of the antisense strand of the DsiRNAmm (where position I of the antisense strand is the 5' terminal nucleotide of the antisense strand, see Fig 20). In one embodiment, for example as depicted in Figure 33, the mismatch tolerant region is positioned between and including base pairs 3-9 when numbered from the nucleotide starting at the 5' end of the sense strand of the duplex. Therefore, 25 a DsiRNAmm of the invention possesses a single mismatched base pair at any one of positions 3, 4, 5, 6, 7, 8 or 9 of the sense strand of a right-hand extended DsiRNA (where position I is the 5' terminal nucleotide of the sense strand and position 9 is the nucleotide residue of the sense strand that is immediately 5' of the projected Ago2 cut site of the target RNA sequence corresponding to the sense strand sequence). In 30 certain embodiments, for a DsiRNAmm that possesses a mismatched base pair nucleotide at any of positions 3, 4, 5, 6, 7, 8 or 9 of the sense strand, the corresponding mismatched base pair nucleotide of the antisense strand not only forms a mismatched base pair with the DsiRNAmm sense strand sequence, but also forms a mismatched base pair with a DsiRNAmm target RNA sequence (thus, 88 Date Re9ue / Date Received 2022-03-14 complementarity between the antisense strand sequence and the sense strand sequence is disrupted at the mismatched base pair within the DsiRNAmm, and complementarity is similarly disrupted between the antisense strand sequence of the DsiRNAmm and the target RNA sequence). In alternative embodiments, the mismatch base pair 5 nucleotide of the antisense strand of a DsiRNAmm only form a mismatched base pair with a corresponding nucleotide of the sense strand sequence of the DsiRNAmm, yet base pairs with its corresponding target RNA sequence nucleotide (thus, complementarity between the antisense strand sequence and the sense strand sequence is disrupted at the mismatched base pair within the DsiRNAmm, yet complementarity 10 is maintained between the antisense strand sequence of the DsiRNAmm and the target RNA sequence). A DsiRNAmm of the invention that possesses a single mismatched base pair within the mismatch-tolerant region (mismatch region) as described above (e.g., a DsiRNAmm harboring a mismatched nucleotide residue at any one of positions 3, 4, 15 5, 6, 7, 8 or 9 of the sense strand) can further include one, two or even three additional mismatched base pairs. In preferred embodiments, these one, two or three additional mismatched base pairs of the DsiRNAmm occur at position(s) 3, 4, 5, 6, 7, 8 and / or 9 of the sense strand (and at corresponding residues of the antisense strand). In one embodiment where one additional mismatched base pair is present within a 20 DsiRNAmm, the two mismatched base pairs of the sense strand can occur, e.g., at nucleotides of both position 4 and position 6 of the sense strand (with mismatch also occurring at corresponding nucleotide residues of the antisense strand). In DsiRNAmm agents possessing two mismatched base pairs, mismatches can occur consecutively (e.g., at consecutive positions along the sense strand nucleotide 25 sequence). Alternatively, nucleotides of the sense strand that form mismatched base pairs with the antisense strand sequence can be interspersed by nucleotides that base pair with the antisense strand sequence (e.g., for a DsiRNAmm possessing mismatched nucleotides at positions 3 and 6, but not at positions 4 and 5, the mismatched residues of sense strand positions 3 and 6 are interspersed by two 30 nucleotides that form matched base pairs with corresponding residues of the anti sense strand). For example, two residues of the sense strand (located within the mismatchtolerant region of the sense strand) that form mismatched base pairs with the corresponding antisense strand sequence can occur with zero, one, two, three, four or five matched base pairs located between these mismatched base pairs. 89 Date Re9ue / Date Received 2022-03-14 For certain DsiRNAmm agents possessing three mismatched base pairs, mismatches can occur consecutively (e.g., in a triplet along the sense strand nucleotide sequence). Alternatively, nucleotides of the sense strand that form mismatched base pairs with the antisense strand sequence can be interspersed by 5 nucleotides that form matched base pairs with the antisense strand sequence (e.g., for a DsiRNAmm possessing mismatched nucleotides at positions 3, 4 and 8, but not at positions 5, 6 and 7, the mismatched residues of sense strand positions 3 and 4 are adjacent to one another, while the mismatched residues of sense strand positions 4 and 8 are interspersed by three nucleotides that form matched base pairs with 10 corresponding residues of the antisense strand). For example, three residues of the sense strand (located within the mismatch-tolerant region of the sense strand) that form mismatched base pairs with the corresponding antisense strand sequence can occur with zero, one, two, three or four matched base pairs located between any two of these mismatched base pairs. 15 For certain DsiRNAmm agents possessing four mismatched base pairs, mismatches can occur consecutively (e.g., in a quadruplet along the sense strand nucleotide sequence). Alternatively, nucleotides of the sense strand that form mismatched base pairs with the antisense strand sequence can be interspersed by nucleotides that form matched base pairs with the antisense strand sequence (e.g., for 20 a DsiRNAmm possessing mismatched nucleotides at positions 3, 5, 7 and 8, but not at positions 4 and 6, the mismatched residues of sense strand positions 7 and 8 are adjacent to one another, while the mismatched residues of sense strand positions 3 and 5 are interspersed by one nucleotide that forms a matched base pair with the corresponding residue of the antisense strand - similarly, the the mismatched residues 25 of sense strand positions 5 and 7 are also interspersed by one nucleotide that forms a matched base pair with the corresponding residue of the antisense strand). For example, four residues of the sense strand (located within the mismatch-tolerant region of the sense strand) that form mismatched base pairs with the corresponding antisense strand sequence can occur with zero, one, two or three matched base pairs 30 located between any two of these mismatched base pairs. In another embodiment, for example as depicted in Figure 39, a DsiRNAmm of the invention comprises a mismatch tolerant region which possesses a single mismatched base pair nucleotide at any one of positions 13, 14, 15, 16, 17, 18, 19, 20 or 21 of the antisense strand of a left-hand extended DsiRNA (where position I is the 90 Date Re9ue / Date Received 2022-03-14 5' terminal nucleotide of the antisense strand and position 13 is the nucleotide residue of the antisense strand that is immediately 3' (downstream) in the antisense strand of the projected Ago2 cut site of the target RNA sequence sufficiently complementary to the antisense strand sequence). In certain embodiments, for a DsiRNAmm that 5 possesses a mismatched base pair nucleotide at any of positions 13, 14, 15, 16, 17, 18, 19, 20 or 21 of the antisense strand with respect to the sense strand of the DsiRNAmm, the mismatched base pair nucleotide of the antisense strand not only forms a mismatched base pair with the DsiRNAmm sense strand sequence, but also forms a mismatched base pair with a DsiRNAmm target RNA sequence (thus, 10 complementarity between the antisense strand sequence and the sense strand sequence is disrupted at the mismatched base pair within the DsiRNAmm, and complementarity is similarly disrupted between the antisense strand sequence of the DsiRNAmm and the target RNA sequence). In alternative embodiments, the mismatch base pair nucleotide of the antisense strand of a DsiRNAmm only forms a mismatched base 15 pair with a corresponding nucleotide of the sense strand sequence of the DsiRNAmm, yet base pairs with its corresponding target RNA sequence nucleotide (thus, complementarity between the antisense strand sequence and the sense strand sequence is disrupted at the mismatched base pair within the DsiRNAmm, yet complementarity is maintained between the antisense strand sequence of the DsiRNAmm and the target 20 RNA sequence). A DsiRNAmm of the invention that possesses a single mismatched base pair within the mismatch-tolerant region as described above (e.g., a DsiRNAmm harboring a mismatched nucleotide residue at positions 13, 14, 15, 16, 17, 18, 19, 20 or 21 of the antisense strand) can further include one, two or even three additional mismatched 25 base pairs. In preferred embodiments, these one, two or three additional mismatched basepairsoftheDsiRNAmmoccuratposition(s) 13, 14, 15, 16, 17, 18, 19,20and / or 21 of the anti sense strand (and at corresponding residues of the sense strand). In one embodiment where one additional mismatched base pair is present within a DsiRNAmm, the two mismatched base pairs of the antisense strand can occur, e.g., at 30 nucleotides of both position 14 and position 18 of the antisense strand (with mismatch also occurring at corresponding nucleotide residues of the sense strand). In DsiRNAmm agents possessing two mismatched base pairs, mismatches can occur consecutively (e.g., at consecutive positions along the antisense strand nucleotide sequence). Alternatively, nucleotides of the antisense strand that form 91 Date Re9ue / Date Received 2022-03-14 mismatched base pairs with the sense strand sequence can be interspersed by nucleotides that base pair with the sense strand sequence (e.g., for a DsiRNAmm possessing mismatched nucleotides at positions 13 and 16, but not at positions 14 and 15, the mismatched residues of antisense strand positions 13 and 16 are interspersed 5 by two nucleotides that form matched base pairs with corresponding residues of the sense strand). For example, two residues of the antisense strand (located within the mismatch-tolerant region of the sense strand) that form mismatched base pairs with the corresponding sense strand sequence can occur with zero, one, two, three, four, five, six or seven matched base pairs located between these mismatched base pairs. For certain DsiRNAmm agents possessing three mismatched base pairs, mismatches can occur consecutively (e.g., in a triplet along the antisense strand nucleotide sequence). Alternatively, nucleotides of the antisense strand that form mismatched base pairs with the sense strand sequence can be interspersed by nucleotides that form matched base pairs with the sense strand sequence (e.g., for a 15 DsiRNAmm possessing mismatched nucleotides at positions 13, 14 and 18, but not at positions 15, 16 and 17, the mismatched residues of antisense strand positions 13 and 14 are adjacent to one another, while the mismatched residues of antisense strand positions 14 and 18 are interspersed by three nucleotides that form matched base pairs with corresponding residues of the sense strand). For example, three residues of the 20 anti sense strand (located within the mismatch-tolerant region of the anti sense strand) that form mismatched base pairs with the corresponding sense strand sequence can occur with zero, one, two, three, four, five or six matched base pairs located between any two of these mismatched base pairs. For certain DsiRNAmm agents possessing four mismatched base pairs, 25 mismatches can occur consecutively (e.g., in a quadruplet along the antisense strand nucleotide sequence). Alternatively, nucleotides of the antisense strand that form mismatched base pairs with the sense strand sequence can be interspersed by nucleotides that form matched base pairs with the sense strand sequence (e.g., for a DsiRNAmm possessing mismatched nucleotides at positions 13, 15, 17 and 18, but 30 not at positions 14 and 16, the mismatched residues of antisense strand positions 17 and 18 are adjacent to one another, while the mismatched residues of antisense strand positions 13 and 15 are interspersed by one nucleotide that forms a matched base pair with the corresponding residue of the sense strand- similarly, the the mismatched residues of antisense strand positions 15 and 17 are also interspersed by one 92 Date Re9ue / Date Received 2022-03-14 nucleotide that forms a matched base pair with the corresponding residue of the sense strand). For example, four residues of the antisense strand (located within the mismatch-tolerant region of the antisense strand) that form mismatched base pairs with the corresponding sense strand sequence can occur with zero, one, two, three, 5 four or five matched base pairs located between any two of these mismatched base pairs. In a further embodiment, for example as depicted in Figure 40, a DsiRNAmm of the invention possesses a single mismatched base pair nucleotide at any one of positions 11, 12, 13, 14, 15, 16, 17, 18or19oftheantisensestrandofaleft-hand 10 extended DsiRNA (where position 1 is the 5' terminal nucleotide of the antisense strand and position 11 is the nucleotide residue of the anti sense strand that is immediately 3' (downstream) in the antisense strand of the projected Ago2 cut site of the target RNA sequence sufficiently complementary to the antisense strand sequence). In certain embodiments, for a DsiRNAmm that possesses a mismatched 15 basepairnucleotideatanyofpositions 11, 12, 13, 14, 15, 16, 17, 18or 19ofthe anti sense strand with respect to the sense strand of the DsiRNAmm, the mismatched base pair nucleotide of the anti sense strand not only forms a mismatched base pair with the DsiRNAmm sense strand sequence, but also forms a mismatched base pair with a DsiRNAmm target RNA sequence (thus, complementarity between the 20 antisense strand sequence and the sense strand sequence is disrupted at the mismatched base pair within the DsiRNAmm, and complementarity is similarly disrupted between the antisense strand sequence of the DsiRNAmm and the target RNA sequence). In alternative embodiments, the mismatch base pair nucleotide of the antisense strand of a DsiRNAmm only forms a mismatched base pair with a 25 corresponding nucleotide of the sense strand sequence of the DsiRNAmm, yet this same antisense strand nucleotide base pairs with its corresponding target RNA sequence nucleotide (thus, complementarity between the antisense strand sequence and the sense strand sequence is disrupted at the mismatched base pair within the DsiRNAmm, yet complementarity is maintained between the antisense strand 30 sequence of the DsiRNAmm and the target RNA sequence). A DsiRNAmm of the invention that possesses a single mismatched base pair within the mismatch-tolerant region as described above (e.g., a DsiRNAmm harboring a mismatched nucleotide residue at positions 11, 12, 13, 14, 15, 16, 17, 18 or 19 of the antisense strand) can further include one, two or even three additional mismatched 93 Date Re9ue / Date Received 2022-03-14 base pairs. In preferred embodiments, these one, two or three additional mismatched base pairs of the DsiRNAmm occur at position(s) 11, 12, 13, 14, 15, 16, 17, 18 and / or 19 of the antisense strand (and at corresponding residues of the sense strand). In one embodiment where one additional mismatched base pair is present within a 5 DsiRNAmm, the two mismatched base pairs of the antisense strand can occur, e.g., at nucleotides of both position 14 and position 18 of the antisense strand (with mismatch also occurring at corresponding nucleotide residues of the sense strand). In DsiRNAmm agents possessing two mismatched base pairs, mismatches can occur consecutively (e.g., at consecutive positions along the antisense strand 10 nucleotide sequence). Alternatively, nucleotides of the antisense strand that form mismatched base pairs with the sense strand sequence can be interspersed by nucleotides that base pair with the sense strand sequence (e.g., for a DsiRNAmm possessing mismatched nucleotides at positions 12 and 15, but not at positions 13 and 14, the mismatched residues of antisense strand positions 12 and 15 are interspersed 15 by two nucleotides that form matched base pairs with corresponding residues of the sense strand). For example, two residues of the antisense strand (located within the mismatch-tolerant region of the sense strand) that form mismatched base pairs with the corresponding sense strand sequence can occur with zero, one, two, three, four, five, six or seven matched base pairs located between these mismatched base pairs. 20 For certain DsiRNAmm agents possessing three mismatched base pairs, mismatches can occur consecutively (e.g., in a triplet along the antisense strand nucleotide sequence). Alternatively, nucleotides of the antisense strand that form mismatched base pairs with the sense strand sequence can be interspersed by nucleotides that form matched base pairs with the sense strand sequence (e.g., for a 25 DsiRNAmm possessing mismatched nucleotides at positions 13, 14 and 18, but not at positions 15, 16 and 17, the mismatched residues of antisense strand positions 13 and 14 are adjacent to one another, while the mismatched residues of antisense strand positions 14 and 18 are interspersed by three nucleotides that form matched base pairs with corresponding residues of the sense strand). For example, three residues of the 30 antisense strand (located within the mismatch-tolerant region of the antisense strand) that form mismatched base pairs with the corresponding sense strand sequence can occur with zero, one, two, three, four, five or six matched base pairs located between any two of these mismatched base pairs. 94 Date Re9ue / Date Received 2022-03-14 For certain DsiRNArnrn agents possessing four mismatched base pairs, mismatches can occur consecutively (e.g., in a quadruplet along the antisense strand nucleotide sequence). Alternatively, nucleotides of the antisense strand that form mismatched base pairs with the sense strand sequence can be interspersed by 5 nucleotides that form matched base pairs with the sense strand sequence (e.g., for a DsiRNAmm possessing mismatched nucleotides at positions 13, 15, 17 and 18, but not at positions 14 and 16, the mismatched residues of antisense strand positions 17 and 18 are adjacent to one another, while the mismatched residues of antisense strand positions 13 and 15 are interspersed by one nucleotide that forms a matched base pair 10 with the corresponding residue of the sense strand- similarly, the the mismatched residues of antisense strand positions 15 and 17 are also interspersed by one nucleotide that forms a matched base pair with the corresponding residue of the sense strand). For example, four residues of the antisense strand (located within the mismatch-tolerant region of the antisense strand) that form mismatched base pairs 15 with the corresponding sense strand sequence can occur with zero, one, two, three, four or five matched base pairs located between any two of these mismatched base palfS. In an additional embodiment, for example as depicted in Figure 41, a DsiRNAmm of the invention possesses a single mismatched base pair nucleotide at 20 any one of positions 15, 16, 17, 18, 19, 20, 21, 22 or 23 of the antisense strand of a left-hand extended DsiRNA (where position 1 is the 5' terminal nucleotide of the antisense strand and position 15 is the nucleotide residue of the anti sense strand that is immediately 3' (downstream) in the antisense strand of the projected Ago2 cut site of the target RNA sequence sufficiently complementary to the antisense strand 25 sequence). In certain embodiments, for a DsiRNAmm that possesses a mismatched base pair nucleotide at any of positions 15, 16, 17, 18, 19, 20, 21, 22 or 23 of the antisense strand with respect to the sense strand of the DsiRNAmm, the mismatched base pair nucleotide of the antisense strand not only forms a mismatched base pair with the DsiRNArnrn sense strand sequence, but also forms a mismatched base pair 30 with a DsiRNArnrn target RNA sequence (thus, complementarity between the antisense strand sequence and the sense strand sequence is disrupted at the mismatched base pair within the DsiRNArnrn, and complementarity is similarly disrupted between the antisense strand sequence of the DsiRNAmm and the target RNA sequence). In alternative embodiments, the mismatch base pair nucleotide of 95 Date Re9ue / Date Received 2022-03-14 the antisense strand of a DsiRNAmm only forms a mismatched base pair with a corresponding nucleotide of the sense strand sequence of the DsiRNAmm, yet this same antisense strand nucleotide base pairs with its corresponding target RNA sequence nucleotide (thus, complementarity between the antisense strand sequence 5 and the sense strand sequence is disrupted at the mismatched base pair within the DsiRNAmm, yet complementarity is maintained between the antisense strand sequence of the DsiRNAmm and the target RNA sequence). A DsiRNAmm of the invention that possesses a single mismatched base pair within the mismatch-tolerant region as described above (e.g., a DsiRNAmm harboring 10 a mismatched nucleotide residue at positions 15, 16, 17, 18, 19, 20, 21, 22 or 23 of the antisense strand) can further include one, two or even three additional mismatched base pairs. In preferred embodiments, these one, two or three additional mismatched base pairs of the DsiRNAmm occur at position(s) 15, 16, 17, 18, 19, 20, 21, 22 and / or 23 of the antisense strand (and at corresponding residues of the sense strand). In one 15 embodiment where one additional mismatched base pair is present within a DsiRNAmm, the two mismatched base pairs of the antisense strand can occur, e.g., at nucleotides of both position 16 and position 20 of the antisense strand (with mismatch also occurring at corresponding nucleotide residues of the sense strand). In DsiRNAmm agents possessing two mismatched base pairs, mismatches can 20 occur consecutively (e.g., at consecutive positions along the antisense strand nucleotide sequence). Alternatively, nucleotides of the antisense strand that form mismatched base pairs with the sense strand sequence can be interspersed by nucleotides that base pair with the sense strand sequence (e.g., for a DsiRNAmm possessing mismatched nucleotides at positions 16 and 20, but not at positions 17, 18 25 and 19, the mismatched residues of antisense strand positions 16 and 20 are interspersed by three nucleotides that form matched base pairs with corresponding residues of the sense strand). For example, two residues of the antisense strand (located within the mismatch-tolerant region of the sense strand) that form mismatched base pairs with the corresponding sense strand sequence can occur with 30 zero, one, two, three, four, five, six or seven matched base pairs located between these mismatched base pairs. For certain DsiRNAmm agents possessing three mismatched base pairs, mismatches can occur consecutively (e.g., in a triplet along the antisense strand nucleotide sequence). Alternatively, nucleotides of the antisense strand that form 96 Date Re9ue / Date Received 2022-03-14 mismatched base pairs with the sense strand sequence can be interspersed by nucleotides that form matched base pairs with the sense strand sequence (e.g., for a DsiRNAmm possessing mismatched nucleotides at positions 16, 17 and 21, but not at positions 18, 19 and 20, the mismatched residues of antisense strand positions 16 and 5 17 are adjacent to one another, while the mismatched residues of antisense strand positions 17 and 21 are interspersed by three nucleotides that form matched base pairs with corresponding residues of the sense strand). For example, three residues of the antisense strand (located within the mismatch-tolerant region of the antisense strand) that form mismatched base pairs with the corresponding sense strand sequence can 10 occur with zero, one, two, three, four, five or six matched base pairs located between any two of these mismatched base pairs. For certain DsiRNAmm agents possessing four mismatched base pairs, mismatches can occur consecutively (e.g., in a quadruplet along the antisense strand nucleotide sequence). Alternatively, nucleotides of the antisense strand that form 15 mismatched base pairs with the sense strand sequence can be interspersed by nucleotides that form matched base pairs with the sense strand sequence (e.g., for a DsiRNAmm possessing mismatched nucleotides at positions 17, 19, 21 and 22, but not at positions 18 and 20, the mismatched residues of antisense strand positions 21 and 22 are adjacent to one another, while the mismatched residues of antisense strand 20 positions 17 and 19 are interspersed by one nucleotide that forms a matched base pair with the corresponding residue of the sense strand- similarly, the the mismatched residues of antisense strand positions 19 and 21 are also interspersed by one nucleotide that forms a matched base pair with the corresponding residue of the sense strand). For example, four residues of the antisense strand (located within the 25 mismatch-tolerant region of the antisense strand) that form mismatched base pairs with the corresponding sense strand sequence can occur with zero, one, two, three, four or five matched base pairs located between any two of these mismatched base pairs. For reasons of clarity, the location(s) of mismatched nucleotide residues 30 within the above DsiRNAmm agents are numbered in reference to the 5' terminal residue of either sense or antisense strands of the DsiRNAmm. As noted for the different left-extended DsiRNAmm agents exemplified in Figures 20, 21 and 22, the numbering of positions located within the mismatch-tolerant region (mismatch region) of the antisense strand can shift with variations in the proximity of the 5' 97 Date Re9ue / Date Received 2022-03-14 terminus of the anti sense strand to the projected Ago2 cleavage site. Thus, the location(s) of preferred mismatch sites within either antisense strand or sense strand can also be identified as the permissible proximity of such mismatches to the projected Ago2 cut site. Accordingly, in one preferred embodiment, the position of a 5 mismatch nucleotide of the sense strand of a DsiRNAmm is the nucleotide residue of the sense strand that is located immediately 5' (upstream) of the projected Ago2 cleavage site of the corresponding target RNA sequence. In other preferred embodiments, a mismatch nucleotide of the sense strand of a DsiRNAmm is positioned at the nucleotide residue of the sense strand that is located two nucleotides 10 5' (upstream) of the projected Ago2 cleavage site, three nucleotides 5' (upstream) of the projected Ago2 cleavage site, four nucleotides 5' (upstream) of the projected Ago2 cleavage site, five nucleotides 5' (upstream) of the projected Ago2 cleavage site, six nucleotides 5' (upstream) of the projected Ago2 cleavage site, seven nucleotides 5' (upstream) of the projected Ago2 cleavage site, eight nucleotides 5' 15 (upstream) of the projected Ago2 cleavage site, or nine nucleotides 5' (upstream) of the projected Ago2 cleavage site. Exemplary single mismatch-containing 25 / 27mer DsiRNAs (DsiRNAmm) include the following structures (such mismatch-containing structures may also be 20 incorporated into other exemplary DsiRNA structures shown herein). s•-pxxMxxxxxxxxxxxxxxxxxxxxnD-3' 3 '-XXXXMXXXXXXXXXXXXXXXXXXXXXXp-5' 5'-pXxxMxxXXXXXXXXXXXXXXXXXDD-3' 25 3' -XXXXXMXXXXXXXXXXXXXXXXXXXXXp-5' 5'-pXXxxMxxxXXXXXXXXXXXXXXXDD-3' 3 ' - XXXXXXMXXXXXXXXXXXXXXXXXXXXp-5' 30 s•-pxxxxxMxxxxxxxxxxxxxxxxxDD-3' 3' -XXXXXXXMXXXXXXXXXXXXXXXXXXXp-5' 98 Date Re9ue / Date Received 2022-03-14 5'-pxxxxxxMxxxxxxxxxxxxxxxxDD-3' 3' -XXXXXXXXMXXXXXXXXXXXXXXXXXXp-5' 5'-pxxxxxxxMxxxxxxxxxxxxxxxnD-3' 5 3' -XXXXXXXXXMXXXXXXXXXXXXXXXXXp-5' 5'-pXXXXXXXXMXXXXXXXXXXXXXXDD-3' 3'-XXXXXXXXXXMXXXXXXXXXXXXXXXXp-5' 10 wherein "X"=RNA, "D"=DNA, "p"=a phosphate group, "M"=Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) that do not base pair (hydrogen bond) with corresponding "M" residues of otherwise complementary strand when strands are annealed. Any of the residues of such agents can optionally be 2'-Omethyl RNA monomers - alternating positioning of 2'-O-methyl RNA monomers that 15 commences from the 3'-terminal residue of the bottom (second) strand, as shown above, can also be used in the above DsiRNAmm agents. For the above mismatch structures, the top strand is the sense strand, and the bottom strand is the antisense strand. 20 In certain embodiments, a DsiRNA of the invention can contain mismatches that exist in reference to the target RNA sequence yet do not necessarily exist as mismatched base pairs within the two strands of the DsiRNA-thus, a DsiRNA can possess perfect complementarity between first and second strands of a DsiRNA, yet still possess mismatched residues in reference to a target RNA (which, in certain 25 embodiments, may be advantageous in promoting efficacy and / or potency and / or duration of effect). In certain embodiments, where mismatches occur between antisense strand and target RNA sequence, the position of a mismatch is located within the antisense strand at a position(s) that corresponds to a sequence of the sense strand located 5' of the projected Ago2 cut site of the target region - e.g., antisense 30 strand residue(s) positioned within the antisense strand to the 3' of the antisense residue which is complementary to the projected Ago2 cut site of the target sequence 99 Date Re9ue / Date Received 2022-03-14 (such region is indicated within, e.g., Figure 33 as a "mismatch region", which is distinct from the projected "seed region" of such DsiRNAs). Exemplary 25 / 27mer DsiRNAs that harbor a single mismatched residue in reference to target sequences include the following preferred structures. 5 Target RNA Sequence: .-3' 5'-.. AXXXXXXXXXXXXXXXXXXXX DsiRNAmm Sense Strand: 5'- pXXXXXXXXXXXXXXXXXXXXXXXDD-3' DsiRNAmm Antisense Strand: 3'- 10 EXXXXXXXXXXXXXXXXXXXXXXXXXXp-5' Target RNA Sequence: .-3' 5'-.. XAXXXXXXXXXXXXXXXXXXX . . DsiRNAmm Sense Strand: 5'- 15 pXXXXXXXXXXXXXXXXXXXXXXXDD-3' DsiRNAmm Antisense Strand: 3'XEXXXXXXXXXXXXXXXXXXXXXXXXXp- 5' Target RNA Sequence: 5'-... AXXXXXXXXXXXXXXXXXX . . 20 • -3 I 25 DsiRNAmm Sense Strand: 5'- pBXXXXXXXXXXXXXXXXXXXXXXDD-3' DsiRNAmm Antisense Strand: 3'XXEXXXXXXXXXXXXXXXXXXXXXXXXp- 5' Target RNA Sequence: • -3 I 5'-... XAXXXXXXXXXXXXXXXXX . . DsiRNAmm Sense Strand: 5'- pXBXXXXXXXXXXXXXXXXXXXXXDD-3' 30 DsiRNAmm Antisense Strand: 3'XXXEXXXXXXXXXXXXXXXXXXXXXXXp- 5' Target RNA Sequence: 5'-... XXAXXXXXXXXXXXXXXXX . . Date Re9ue / Date Received 2022-03-14 .-3' DsiRNAmm Sense Strand: 5'- pXXBXXXXXXXXXXXXXXXXXXXXDD-3' DsiRNAmm Antisense Strand: 3'- 5 XXXXEXXXXXXXXXXXXXXXXXXXXXXp-5' Target RNA Sequence: .-3' 5'-... XXXAXXXXXXXXXXXXXXX . DsiRNAmm Sense Strand: 5'- 10 pXXXBXXXXXXXXXXXXXXXXXXXDD-3' DsiRNAmm Antisense Strand: 3'XXXXXEXXXXXXXXXXXXXXXXXXXXXp- 5' Target RNA Sequence: 5'-... XXXXAXXXXXXXXXXXXXX .. 15 , -3 I 20 DsiRNAmm Sense Strand: 5'- pXXXXBXXXXXXXXXXXXXXXXXXDD-3' DsiRNAmm Antisense Strand: 3'XXXXXXEXXXXXXXXXXXXXXXXXXXXp- 5' Target RNA Sequence: • -3 I 5'-... XXXXXAXXXXXXXXXXXXX . DsiRNAmm Sense Strand: 5'- pXXXXXBXXXXXXXXXXXXXXXXXDD-3' 25 DsiRNAmm Antisense Strand: 3'XXXXXXXEXXXXXXXXXXXXXXXXXXXp- 5' Target RNA Sequence: • -3 I 5'-... XXXXXXAXXXXXXXXXXXX . 30 DsiRNAmm Sense Strand: 5'- pXXXXXXBXXXXXXXXXXXXXXXXDD-3' DsiRNAmm Antisense Strand: 3'XXXXXXXXEXXXXXXXXXXXXXXXXXXp- 5' 101 Date Re9ue / Date Received 2022-03-14 Target RNA Sequence: . -3, 5'-. . XXXXXXXAXXXXXXXXXXX . DsiRNAmm Sense Strand: 5'- pXXXXXXXBXXXXXXXXXXXXXXXDD-3' 5 DsiRNAmm Antisense Strand: 3'XXXXXXXXXEXXXXXXXXXXXXXXXXXp- 5' Target RNA Sequence: • -3 I 5, - . . XXXXXXXXAXXXXXXXXXX . 10 DsiRNAmm Sense Strand: 5' - pXXXXXXXXBXXXXXXXXXXXXXXDD-3' DsiRNAmm Antisense Strand: 3'XXXXXXXXXXEXXXXXXXXXXXXXXXXp- 5' 15 wherein "X"=RNA, "D"=DNA, "p"=a phosphate group, "E"=Nucleic acid residues (RNA, DNA or non-natural or modified nucleic acids) that do not base pair (hydrogen bond) with corresponding "A" RNA residues of otherwise complementary (target) strand when strands are annealed, yet optionally do base pair with corresponding "B" residues ("B" residues are also RNA, DNA or non-natural or modified nucleic acids). 20 Any of the residues of such agents can optionally be 2'-O-methyl RNA monomers - e.g., alternating positioning of 2'-O-methyl RNA monomers that commences from the 3'-terminal residue of the bottom (second) strand, as shown above, or other patterns of 2'-O-methyl and / or other modifications as described herein can also be used in the above DsiRNA agents. 25 In addition to the above-exemplified structures, DsiRNAs of the invention can also possess one, two or three additional residues that form further mismatches with the target RNA sequence. Such mismatches can be consecutive, or can be interspersed by nucleotides that form matched base pairs with the target RNA sequence. Where interspersed by nucleotides that form matched base pairs, 30 mismatched residues can be spaced apart from each other within a single strand at an interval of one, two, three, four, five, six, seven or even eight base paired nucleotides between such mismatch-forming residues. As for the above-described DsiRNAmm agents, a preferred location within DsiRNAs for antisense strand nucleotides that form mismatched base pairs with target 102 Date Re9ue / Date Received 2022-03-14 RNA sequence (yet may or may not form mismatches with corresponding sense strand nucleotides) is within the anti sense strand region that is located 3' (downstream) of the antisense strand sequence which is complementary to the projected Ago2 cut site of the DsiRNA (e.g., in Figure 39, the region of the antisense 5 strand which is labeled as the "mismatch region" is preferred for mismatch-forming residues and happens to be located at positions 13-21 of the antisense strand for the agents shown in Figure 39). Thus, in one preferred embodiment, the position of a mismatch nucleotide (in relation to the target RNA sequence) of the antisense strand of a DsiRNAmm is the nucleotide residue of the antisense strand that is located 10 immediately 3' (downstream) within the antisense strand sequence of the projected Ago2 cleavage site of the corresponding target RNA sequence. In other preferred embodiments, a mismatch nucleotide of the antisense strand of a DsiRNAmm (in relation to the target RNA sequence) is positioned at the nucleotide residue of the antisense strand that is located two nucleotides 3' (downstream) of the corresponding 15 projected Ago2 cleavage site, three nucleotides 3' (downstream) of the corresponding projected Ago2 cleavage site, four nucleotides 3' ( downstream) of the corresponding projected Ago2 cleavage site, five nucleotides 3' (downstream) of the corresponding projected Ago2 cleavage site, six nucleotides 3' (downstream) of the projected Ago2 cleavage site, seven nucleotides 3' (downstream) of the projected Ago2 cleavage site, 20 eight nucleotides 3' ( downstream) of the projected Ago2 cleavage site, or nine nucleotides 3' (downstream) of the projected Ago2 cleavage site. In DsiRNA agents possessing two mismatch-forming nucleotides of the antisense strand (where mismatch-forming nucleotides are mismatch forming in relation to target RNA sequence), mismatches can occur consecutively (e.g., at 25 consecutive positions along the antisense strand nucleotide sequence). Alternatively, nucleotides of the anti sense strand that form mismatched base pairs with the target RNA sequence can be interspersed by nucleotides that base pair with the target RNA sequence (e.g., for a DsiRNA possessing mismatch-forming nucleotides at positions 13 and 16 (starting from the 5' terminus (position 1) of the antisense strand of the 30 structure shown in Figure 39), but not at positions 14 and 15, the mismatched residues of sense strand positions 13 and 16 are interspersed by two nucleotides that form matched base pairs with corresponding residues of the target RNA sequence). For example, two residues of the antisense strand (located within the mismatch-tolerant region of the anti sense strand) that form mismatched base pairs with the 103 Date Re9ue / Date Received 2022-03-14 corresponding target RNA sequence can occur with zero, one, two, three, four or five matched base pairs (with respect to target RNA sequence) located between these mismatch-forming base pairs. For certain DsiRNAs possessing three mismatch-forming base pairs 5 (mismatch-forming with respect to target RNA sequence), mismatch-forming nucleotides can occur consecutively (e.g., in a triplet along the antisense strand nucleotide sequence). Alternatively, nucleotides of the antisense strand that form mismatched base pairs with the target RNA sequence can be interspersed by nucleotides that form matched base pairs with the target RNA sequence (e.g., for a 10 DsiRNA possessing mismatched nucleotides at positions 13, 14 and 18, but not at positions 15, 16 and 17, the mismatch-forming residues of antisense strand positions 13 and 14 are adjacent to one another, while the mismatch-forming residues of antisense strand positions 14 and 18 are interspersed by three nucleotides that form matched base pairs with corresponding residues of the target RNA). For example, 15 three residues of the anti sense strand (located within the mismatch-tolerant region of the antisense strand) that form mismatched base pairs with the corresponding target RNA sequence can occur with zero, one, two, three or four matched base pairs located between any two of these mismatch-forming base pairs. For certain DsiRNAs possessing four mismatch-forming base pairs 20 (mismatch-forming with respect to target RNA sequence), mismatch-forming nucleotides can occur consecutively (e.g., in a quadruplet along the sense strand nucleotide sequence). Alternatively, nucleotides of the antisense strand that form mismatched base pairs with the target RNA sequence can be interspersed by nucleotides that form matched base pairs with the target RNA sequence (e.g., for a 25 DsiRNA possessing mismatch-forming nucleotides at positions 13, 15, 17 and 18, but not at positions 14 and 16, the mismatch-forming residues of antisense strand positions 17 and 18 are adjacent to one another, while the mismatch-forming residues of antisense strand positions 13 and 15 are interspersed by one nucleotide that forms a matched base pair with the corresponding residue of the target RNA sequence - 30 similarly, the mismatch-forming residues of antisense strand positions 15 and 17 are also interspersed by one nucleotide that forms a matched base pair with the corresponding residue of the target RNA sequence). For example, fourresidues of the antisense strand (located within the mismatch-tolerant region of the anti sense strand) that form mismatched base pairs with the corresponding target RNA sequence can 104 Date Re9ue / Date Received 2022-03-14 occur with zero, one, two or three matched base pairs located between any two of these mismatch-forming base pairs. The above DsiRNAmm and other DsiRNA structures are described in order to exemplify certain structures ofDsiRNAmm and DsiRNA agents. Design of the above 5 DsiRNAmm and DsiRNA structures can be adapted to generate, e.g., DsiRNAmm forms of a DNA-extended ("DNA handle") DsiRNA agent shown infra (including, e.g., design of mismatch-containing DsiRNAmm agents as shown in Figures 14-16 and 20-22). As exemplified above, DsiRNAs can also be designed that possess single mismatches (or two, three or four mismatches) between the antisense strand of the 10 DsiRNA and a target sequence, yet optionally can retain perfect complementarity between sense and antisense strand sequences of a DsiRNA. It is further noted that the DsiRNA agents exemplified infra can also possess insertion / deletion (in / del) structures within their double-stranded and / or target RNAaligned structures. Accordingly, the DsiRNAs of the invention can be designed to 15 possess in / del variations in, e.g., antisense strand sequence as compared to target RNA sequence and / or antisense strand sequence as compared to sense strand sequence, with preferred location(s) for placement of such in / del nucleotides corresponding to those locations described above for positioning of mismatched and / or mismatch-forming base pairs. 20 In certain embodiments, the "D" residues of any of the above structures include at least one PS-DNA or PS-RNA. Optionally, the "D" residues of any of the above structures include at least one modified nucleotide that inhibits Dicer cleavage. While the above-described "DNA-extended" DsiRNA agents can be categorized as either "left extended" or "right extended", DsiRNA agents comprising 25 both left- and right-extended DNA-containing sequences within a single agent (e.g., both flanks surrounding a core dsRNA structure are dsDNA extensions) can also be generated and used in similar manner to those described herein for "right-extended" and "left-extended" agents. In some embodiments, the DsiRNA of the instant invention further comprises 30 a linking moiety or domain that joins the sense and antisense strands of a DNA:DNAextended DsiRNA agent. Optionally, such a linking moiety domain joins the 3' end of the sense strand and the 5' end of the antisense strand. The linking moiety may be a chemical (non-nucleotide) linker, such as an oligomethylenediol linker, oligoethylene glycol linker, or other art-recognized linker moiety. Alternatively, the 105 Date Re9ue / Date Received 2022-03-14 linker can be a nucleotide linker, optionally including an extended loop and / or tetraloop. In one embodiment, the DsiRNA agent has an asymmetric structure, with the sense strand having a 27-base pair length, the antisense strand having a 29-nucleotide 5 length with a 2 base 3'-overhang (and, therefore, the DsiRNA agent possesses a blunt end at the 3' end of the sense strand / 5' end of the antisense strand), and with deoxyribonucleotides located at positions 24 and 25 of the sense strand (numbering from position 1 at the 5' of the sense strand) and each base paired with a cognate deoxyribonucleotide of the anti sense strand. In another embodiment, this DsiRNA 10 agent has an asymmetric structure further containing 2 deoxyribonucleotides at the 3' end of the sense strand. In another embodiment, the DsiRNA agent has an asymmetric structure, with the sense strand having a 30-nucleotide length, the antisense strand having a 28- nucleotide length, with a 2 nucleotide 3' overhang positioned at the 3' end of the 15 sense strand. The 3' end of the anti sense strand and 5' end of the sense strand of this DsiRNA agent form a blunt end, and starting from position 1 at the 5' terminus of the sense strand, positions 1-5 are deoxyribonucleotides that hybridize to form a duplex with cognate deoxyribonucleotides of the 3' end region of the antisense strand. Optionally, starting from position 1 at the 5' end of the antisense strand, positions 11- 20 21 of the antisense strand (in certain embodiments, positions 13-21) harbor one or more nucleotides that either form a mismatch base pairing with the corresponding nucleotide of the sense strand, or with the corresponding nucleotide of the target RNA sequence when the antisense strand and the target RNA sequence hybridize to form a duplex, or with both sense strand and target RNA sequence. Optionally, the ultimate 25 and penultimate nucleotides of the 5' terminus of the sense strand and the ultimate and penultimate nucleotides of the 3' end of the antisense strand comprise one or more phosphorothioates (optionally, the two antisense strand deoxyribonucleotides, the two sense strand deoxyribonucleotides, or all 4 deoxyribonucleotides constituting the ultimate and penultimate residues of both the 5' end of the sense strand and the 3' 30 end of the anti sense strand possess phosphorothioates). Modification of DsiRNAs One major factor that inhibits the effect of double stranded RNAs ("dsRNAs") is the degradation of dsRNAs (e.g., siRNAs and DsiRNAs) by nucleases. A 3'- 106 Date Re9ue / Date Received 2022-03-14 exonuclease is the primary nuclease activity present in serum and modification of the 3'-ends of antisense DNA oligonucleotides is crucial to prevent degradation (Eder et al., 1991). An RNase-T family nuclease has been identified called ERI-1 which has 3' to 5' exonuclease activity that is involved in regulation and degradation of siRNAs 5 (Kennedy et al., 2004; Hong et al., 2005). This gene is also known as Thexl (NM_02067) in mice or THEXl (NM_l53332) in humans and is involved in degradation of hi stone mRNA; it also mediates degradation of 3'-overhangs in siRNAs, but does not degrade duplex RNA (Yang et al., 2006). It is therefore reasonable to expect that 3'-end-stabilization of dsRNAs, including the DsiRNAs of 10 the instant invention, will improve stability. XRNl (NM 019001) is a 5' to 3' exonuclease that resides in P-bodies and has been implicated in degradation of mRNA targeted by miRNA (Rehwinkel et al., 2005) and may also be responsible for completing degradation initiated by internal cleavage as directed by a siRNA. XRN2 (NM _012255) is a distinct 5' to 3' 15 exonuclease that is involved in nuclear RNA processing. Although not currently implicated in degradation or processing of siRNAs and miRNAs, these both are known nucleases that can degrade RN As and may also be important to consider. RNase A is a major endonuclease activity in mammals that degrades RNAs. It is specific for ssRNA and cleaves at the 3'-end of pyrimidine bases. SiRNA 20 degradation products consistent with RNase A cleavage can be detected by mass spectrometry after incubation in serum (Turner et al., 2007). The 3'-overhangs enhance the susceptibility of siRNAs to RNase degradation. Depletion of RNase A from serum reduces degradation of siRNAs; this degradation does show some sequence preference and is worse for sequences having poly AIU sequence on the 25 ends (Haupenthal et al., 2006). This suggests the possibility that lower stability regions of the duplex may "breathe" and offer transient single-stranded species available for degradation by RNase A. RNase A inhibitors can be added to serum and improve siRNA longevity and potency (Haupenthal et al., 2007). In 21mers, phosphorothioate or boranophosphate modifications directly 30 stabilize the intemucleoside phosphate linkage. Boranophosphate modified RNAs are highly nuclease resistant, potent as silencing agents, and are relatively non-toxic. Boranophosphate modified RNAs cannot be manufactured using standard chemical synthesis methods and instead are made by in vitro transcription (IVT) (Hall et al., 2004 and Hall et al., 2006). Phosphorothioate (PS) modifications can be readily 107 Date Re9ue / Date Received 2022-03-14 placed in an RNA duplex at any desired position and can be made using standard chemical synthesis methods, though the ability to use such modifications within an RNA duplex that retains RNA silencing activity can be limited. As shown herein in Figure 2 (duplex #8), inclusion of a multiple PS-modified deoxyribonucleotide 5 residues in a tandem series configuration that base paired with a cognate tandem series of PS-modified deoxyribonucleotide residues abolished RNA silencing activity of an agent that was otherwise active with only unmodified deoxyribonucleotides present at these residues. Because PS moieties are likely to require greater spacing when included within an RNA duplex-containing agent in order to retain RNA 10 inhibitory acitivity, extended DsiRNAs such as those described herein can provide a means of including more PS modifications (either PS-DNA or PS-RNA) within a single DsiRNA agent than would otherwise be available were no such extension used. It is noted, however, that the PS modification shows dose-dependent toxicity, so most investigators have recommended limited incorporation in siRNAs, historically 15 favoring the 3 '-ends where protection from nucleases is most important (Harborth et al., 2003; Chiu and Rana, 2003; Braasch et al., 2003; Amarzguioui et al., 2003). More extensive PS modification can be compatible with potent RNAi activity; however, use of sugar modifications (such as 2'-O-methyl RNA) may be superior (Choung et al., 2006). 20 A variety of substitutions can be placed at the 2'-position of the ribose which generally increases duplex stability (Tm) and can greatly improve nuclease resistance. 2'-O-methyl RNA is a naturally occurring modification found in mammalian ribosomal RNAs and transfer RNAs. 2'-O-methyl modification in siRNAs is known, but the precise position of modified bases within the duplex is important to retain 25 potency and complete substitution of 2'-O-methyl RNA for RNA will inactivate the siRNA. For example, a pattern that employs alternating 2'-O-methyl bases can have potency equivalent to unmodified RNA and is quite stable in serum (Choung et al., 2006; Czauderna et al., 2003). The 2'-fluoro (2'-F) modification is also compatible with dsRNA (e.g., siRNA 30 and DsiRNA) function; it is most commonly placed at pyrimidine sites (due to reagent cost and availability) and can be combined with 2'-O-methyl modification at purine positions; 2'-F purines are available and can also be used. Heavily modified duplexes of this kind can be potent triggers of RNAi in vitro (Allerson et al., 2005; Prakash et al., 2005; Kraynack and Baker, 2006) and can improve performance and extend 108 Date Re9ue / Date Received 2022-03-14 duration of action when used in vivo (Morrissey et al., 2005a; Morrissey et al., 2005b ). A highly potent, nuclease stable, blunt l 9mer duplex containing alternative 2'-F and 2'-O-Me bases is taught by Allerson. In this design, alternating 2'-O-Me residues are positioned in an identical pattern to that employed by Czauderna, 5 however the remaining RNA residues are converted to 2'-F modified bases. A highly potent, nuclease resistant siRNA employed by Morrissey employed a highly potent, nuclease resistant siRNA in vivo. In addition to 2'-O-Me RNA and 2'-F RNA, this duplex includes DNA, RNA, inverted abasic residues, and a 3'-terminal PS internucleoside linkage. While extensive modification has certain benefits, more 10 limited modification of the duplex can also improve in vivo performance and is both simpler and less costly to manufacture. Soutschek et al. (2004) employed a duplex in vivo and was mostly RNA with two 2'-O-Me RNA bases and limited 3'-terminal PS internucleoside linkages. Locked nucleic acids (LNAs) are a different class of 2'-modification that can 15 be used to stabilize dsRNA (e.g., siRNA and DsiRNA). Patterns of LNA incorporation that retain potency are more restricted than 2'-O-methyl or 2'-F bases, so limited modification is preferred (Braasch et al., 2003; Grunweller et al., 2003; Elmen et al., 2005). Even with limited incorporation, the use of LNA modifications can improve dsRNA performance in vivo and may also alter or improve off target effect 20 profiles (Mook et al., 2007). Synthetic nucleic acids introduced into cells or live animals can be recognized as "foreign" and trigger an immune response. Immune stimulation constitutes a major class of off-target effects which can dramatically change experimental results and even lead to cell death. The innate immune system includes a collection of receptor 25 molecules that specifically interact with DNA and RNA that mediate these responses, some of which are located in the cytoplasm and some of which reside in endosomes (Marques and Williams, 2005; Schlee et al., 2006). Delivery of siRNAs by cationic lipids or liposomes exposes the siRNA to both cytoplasmic and endosomal compartments, maximizing the risk for triggering a type 1 interferon (IFN) response 30 both in vitro and in vivo (Morrissey et al., 2005b; Sioud and Sorensen, 2003; Sioud, 2005; Ma et al., 2005). RNAs transcribed within the cell are less immunogenic (Robbins et al., 2006) and synthetic RNAs that are immunogenic when delivered using lipid-based methods can evade immune stimulation when introduced unto cells by mechanical means, even in vivo (Heidel et al., 2004). However, lipid based 109 Date Re9ue / Date Received 2022-03-14 5 delivery methods are convenient, effective, and widely used. Some general strategy to prevent immune responses is needed, especially for in vivo application where all cell types are present and the risk of generating an immune response is highest. Use of chemically modified RN As may solve most or even all of these problems. Although certain sequence motifs are clearly more immunogenic than others~ it appears that the receptors of the innate immune system in general distinguish the presence or absence of certain base modifications which are more commonly found in mammalian RNAs than in prokaryotic RNAs. For example, pseudouridine, N6- methyl-A, and 2'-O-methyl modified bases are recognized as "self' and inclusion of 10 these residues in a synthetic RNA can help evade immune detection (Kariko et al., 2005). Extensive 2'-modification of a sequence that is strongly immunostimulatory as unmodified RNA can block an immune response when administered to mice ~ntravenously (Morrissey et al., 2005b ). However, extensive modification is not needed to escape immune detection and substitution of as few as two 2'-O-methyl 15 bases in a single strand of a siRNA duplex can be sufficient to block a type 1 IFN response both in vitro and in vivo; modified U and G bases are most effective (Judge et al., 2006). As an added benefit, selective incorporation of 2'-O-methyl bases can reduce the magnitude of off-target effects (Jackson et al., 2006). Use of 2'-O-methyl bases should therefore be considered for all dsRNAs intended for in vivo applications 20 as a means of blocking immune responses and has the added benefit of improving nuclease stability and reducing the likelihood of off-target effects. Although cell death can result from immune stimulation, assessing cell viability is not an adequate method to monitor induction of IFN responses. IFN responses can be present without cell death, and cell death can result from target 25 knockdown in the absence ofIFN triggering (for example, if the targeted gene is essential for cell viability). Relevant cytokines can be directly measured in culture medium and a variety of commercial kits exist which make performing such assays routine. While a large number of different immune effector molecules can be measured, testing levels of IFN-a, TNF-a, and IL-6 at 4 and 24 hours post 30 transfection is usually sufficient for screening purposes. It is important to include a "transfection reagent only control" as cationic lipids can trigger immune responses in certain cells in the absence of any nucleic acid cargo. Including controls for IFN pathway induction should be considered for cell culture work. It is essential to test for 110 Date Re9ue / Date Received 2022-03-14 immune stimulation whenever administering nucleic acids in vivo, where the risk of triggering IFN responses is highest. Modifications can be included in the DsiRNA agents of the present invention so long as the modification does not prevent the DsiRNA agent from serving as a 5 substrate for Dicer. Indeed, one surprising finding of the instant invention is that base paired deoxyribonucleotides can be attached to previously described DsiRNA molecules, resulting in enhanced RNAi efficacy and duration, provided that such extension is performed in a region of the extended molecule that does not interfere with Dicer processing (e.g., 3' of the Dicer cleavage site of the sense strand / 5' of the 10 Dicer cleavage site of the antisense strand). In one embodiment, one or more modifications are made that enhance Dicer processing of the DsiRNA agent. In a second embodiment, one or more modifications are made that result in more effective RNAi generation. In a third embodiment, one or more modifications are made that support a greater RN Ai effect. In a fourth embodiment, one or more modifications 15 are made that result in greater potency per each DsiRNA agent molecule to be delivered to the cell. Modifications can be incorporated in the 3 '-terminal region, the 5'-terminal region, in both the 3'-terminal and 5'-terminal region or in some instances in various positions within the sequence. With the restrictions noted above in mind, any number and combination of modifications can be incorporated into the DsiRNA 20 agent. Where multiple modifications are present, they may be the same or different. Modifications to bases, sugar moieties, the phosphate backbone, and their combinations are contemplated. Either 5'-terminus can be phosphorylated. Examples of modifications contemplated for the phosphate backbone include phosphonates, including methylphosphonate, phosphorothioate, and phosphotriester 25 modifications such as alkylphosphotriesters, and the like. Examples of modifications contemplated for the sugar moiety include 2'-alkyl pyrimidine, such as 2'-O-methyl, 2'-fluoro, amino, and deoxy modifications and the like (see, e.g., Amarzguioui et al., 2003). Examples of modifications contemplated for the base groups include abasic sugars, 2-O-alkyl modified pyrimidines, 4-thiouracil, 5-bromouracil, 5-iodouracil, and 30 5-(3-aminoallyl)-uracil and the like. Locked nucleic acids, or LNA's, could also be incorporated. Many other modifications are known and can be used so long as the above criteria are satisfied. Examples of modifications are also disclosed in U.S. Pat. Nos. 5,684,143, 5,858,988 and 6,291,438 and in U.S. published patent application No. 2004 / 0203145 Al. Other modifications are disclosed in Herdewijn (2000), Eckstein 111 Date Re9ue / Date Received 2022-03-14 (2000), Rusckowski et al. (2000), Stein et al. (2001); Vorobjev et al. (2001). One or more modifications contemplated can be incorporated into either strand. The placement of the modifications in the DsiRNA agent can greatly affect the characteristics of the DsiRNA agent, including conferring greater potency and 5 stability, reducing toxicity, enhance Dicer processing, and minimizing an immune response. In one embodiment, the antisense strand or the sense strand or both strands have one or more 2'-O-methyl modified nucleotides. In another embodiment, the antisense strand contains 2'-O-methyl modified nucleotides. In another embodiment, the antisense stand contains a 3' overhang that is comprised of 2'-O-methyl modified 10 nucleotides. The antisense strand could also include additional 2'-O-methyl modified nucleotides. In certain embodiments of the present invention, the DsiRNA agent has one or more properties which enhance its processing by Dicer. According to these embodiments, the DsiRNA agent has a length sufficient such that it is processed by 15 Dicer to produce an active siRNA and at least one of the following properties: (i) the DsiRNA agent is asymmetric, e.g., has a 3' overhang on the antisense strand and (ii) the DsiRNA agent has a modified 3' end on the sense strand to direct orientation of Dicer binding and processing of the dsRNA region to an active siRNA. In certain such embodiments, the presence of one or more base paired deoxyribonucleotides in a 20 region of the sense strand that is 3' to the projected site of Dicer enzyme cleavage and corresponding region of the antisense strand that is 5' of the projected site of Dicer enzyme cleavage can also serve to orient such a molecule for appropriate directionality of Dicer enzyme cleavage. In certain embodiments, the length of the dsDNA region (or length of the 25 region comprising DNA:DNA base pairs) is 1-50 base pairs, optionally 2-30 base pairs, preferably 2-20 base pairs, and more preferably 2-15 base pairs. Thus, a DNA:DNA-extended DsiRNA of the instant invention may possess a dsDNA region that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47, 30 48, 49, 50 or more (e.g., 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more) base pairs in length. In some embodiments, the longest strand in the dsNA comprises 29-43 nucleotides. In one embodiment, the DsiRNA agent is asymmetric such that the 3' end of the sense strand and 5' end of the anti sense strand form a blunt end, and the 3' 112 Date Re9ue / Date Received 2022-03-14 5 end of the antisense strand overhangs the 5' end of the sense strand. In certain embodiments, the 3' overhang of the antisense strand is 1-10 nucleotides, and optionally is 1-4 nucleotides, for example 2 nucleotides. Both the sense and the antisense strand may also have a 5' phosphate. In certain embodiments, the sense strand of a DsiRNA of the invention that comprises base paired deoxyribonucleotide residues has a total length of between 26 nucleotides and 39 or more nucleotides (e.g., the sense strand possesses a length of 26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48, 49, 50 or more (e.g., 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more) nucleotides). In 10 certain embodiments, the length of the sense strand is between 26 nucleotides and 39 nucleotides, optionally between 27 and 35 nucleotides, or, optionally, between 27 and 33 nucleotides in length. In related embodiments, the antisense strand has a length of between 27 and 43 or more nucleotides ( e.g., the sense strand possesses a length of 26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48, 15 49, 50 or more (e.g., 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more) nucleotides). In certain such embodiments, the antisense strand has a length of between 27 and 43 nucleotides in length, or between 27 and 39 nucleotides in length, or between 27 and 35 nucleotides in length, or between 28 and 37 nucleotides in length, or, optionally, between 29 and 35 nucleotides in length. 20 In certain embodiments, the presence of one or more base paired deoxyribonucleotides in a region of the sense strand that is 3' of the projected site of Dicer enzyme cleavage and corresponding region of the antisense strand that is 5' of the projected site of Dicer enzyme cleavage can serve to direct Dicer enzyme cleavage of such a molecule. While certain exemplified agents of the invention possess a sense 25 strand deoxyribonucleotide that is located at position 24 or more 3' when counting from position 1 at the 5' end of the sense strand, and having this position 24 or more 3' deoxyribonucleotide of the sense strand base pairing with a cognate deoxyribonucleotide of the anti sense strand, in some embodiments, it is also possible to direct Dicer to cleave a shorter product, e.g., a 19mer or a 20mer via inclusion of 30 deoxyribonucleotide residues at, e.g., position 20 of the sense strand. Such a position 20 deoxyribonucleotide base pairs with a corresponding deoxyribonucleotide of the antisense strand, thereby directing Dicer-mediated excision of a l 9mer as the most prevalent Dicer product (it is noted that the antisense strand can also comprise one or two deoxyribonucleotide residues immediately 3' of the antisense residue that base 113 Date Re9ue / Date Received 2022-03-14 pairs with the position 20 deoxyribonucleotide residue of the sense strand in such embodiments, to further direct Dicer cleavage of the anti sense strand). In such embodiments, the double-stranded DNA region (which is inclusive of modified nucleic acids that block Dicer cleavage) will generally possess a length of greater than 5 1 or 2 base pairs (e.g., 3 to 5 base pairs or more), in order to direct Dicer cleavage to generate what is normally a non-preferred length of Dicer cleavage product. A parallel approach can also be taken to direct Dicer excision of 20mer siRNAs, with the positioning of the first deoxyribonucleotide residue of the sense strand (when surveying the sense strand from position 1 at the 5' terminus of the sense strand) 10 occurring at position 21. In certain embodiments, the sense strand of the DsiRNA agent is modified for Dicer processing by suitable modifiers located at the 3' end of the sense strand, i.e., the DsiRNA agent is designed to direct orientation of Dicer binding and processing via sense strand modification. Suitable modifiers include nucleotides such as 15 deoxyribonucleotides, dideoxyribonucleotides, acyclonucleotides and the like and sterically hindered molecules, such as fluorescent molecules and the like. Acyclonucleotides substitute a 2-hydroxyethoxymethyl group for the 2'deoxyribofuranosyl sugar normally present in dNMPs. Other nucleotide modifiers could include 3'-deoxyadenosine (cordycepin), 3'-azido-3'-deoxythymidine (AZT), 20 2',3'-dideoxyinosine (ddI), 2',3'-dideoxy-3'-thiacytidine (3TC), 2',3'-didehydro-2',3'dideoxythymidine (d4T) and the monophosphate nucleotides of 3'-azido-3'deoxythymidine (AZT), 2',3'-dideoxy-3'-thiacytidine (3TC) and 2',3'-didehydro-2',3'dideoxythymidine ( d4T). In one embodiment, deoxyribonucleotides are used as the modifiers. When nucleotide modifiers are utilized, 1-3 nucleotide modifiers, or 2 25 nucleotide modifiers are substituted for the ribonucleotides on the 3' end of the sense strand. When sterically hindered molecules are utilized, they are attached to the ribonucleotide at the 3' end of the antisense strand. Thus, the length of the strand does not change with the incorporation of the modifiers. In another embodiment, the invention contemplates substituting two DNA bases in the DsiRNA agent to direct the 30 orientation of Dicer processing of the anti sense strand. In a further embodiment of the present invention, two terminal DNA bases are substituted for two ribonucleotides on the 3'-end of the sense strand forming a blunt end of the duplex on the 3' end of the sense strand and the 5' end of the antisense strand, and a two-nucleotide RNA overhang is located on the 3'-end of the antisense strand. This is an asymmetric 114 Date Re9ue / Date Received 2022-03-14 composition with DNA on the blunt end and RNA bases on the overhanging end. In certain embodiments of the instant invention, the modified nucleotides (e.g., deoxyribonucleotides) of the penultimate and ultimate positions of the 3' terminus of the sense strand base pair with corresponding modified nucleotides (e.g., 5 deoxyribonucleotides) of the antisense strand ( optionally, the penultimate and ultimate residues of the 5' end of the antisense strand in those DsiRNA agents of the instant invention possessing a blunt end at the 3' terminus of the sense strand / 5' terminus of the antisense strand). The sense and antisense strands of a DsiRNA agent of the instant invention 10 anneal under biological conditions, such as the conditions found in the cytoplasm of a cell. In addition, a region of one of the sequences, particularly of the antisense strand, of the DsiRNA agent has a sequence length of at least 19 nucleotides, wherein these nucleotides are in the 21-nucleotide region adjacent to the 3' end of the antisense strand and are sufficiently complementary to a nucleotide sequence of the RNA 15 produced from the target gene to anneal with and / or decrease levels of such a target RNA. The DsiRNA agent of the instant invention may possess one or more deoxyribonucleotide base pairs located at any positions of sense and antisense strands that are located 3' of the projected Dicer cleavage site of the sense strand and 5' of the 20 projected Dicer cleavage site of the antisense strand. In certain embodiments, one, two, three or all four of positions 24-27 of the sense strand (starting from position 1 at the 5' terminus of the sense strand) are deoxyribonucleotides, each deoxyribonucleotide of which base pairs with a corresponding deoxyribonucleotide of the antisense strand. In certain embodiments, the deoxyribonucleotides of the 5' 25 region of the antisense strand (e.g., the region of the antisense strand located 5' of the projected Dicer cleavage site for a given DsiRNA molecule) are not complementary to the target RNA to which the DsiRNA agent is directed. In related embodiments, the entire region of the anti sense strand located 5' of the projected Dicer cleavage site of a DsiRNA agent is not complementary to the target RNA to which the DsiRNA 30 agent is directed. In certain embodiments, the deoxyribonucleotides of the antisense strand or the entire region of the antisense strand that is located 5' of the projected Dicer cleavage site of the DsiRNA agent is not sufficiently complementary to the target RNA to enhance annealing of the antisense strand of the DsiRNA to the target RNA when the antisense strand is annealed to the target RNA under conditions 115 Date Re9ue / Date Received 2022-03-14 sufficient to allow for annealing between the antisense strand and the target RNA (e.g., a "core" antisense strand sequence lacking the DNA-extended region anneals equally well to the target RNA as the same "core" antisense strand sequence also extended with sequence of the DNA-extended region). 5 The DsiRNA agent may also have one or more of the following additional properties: ( a) the antisense strand has a right or left shift from the typical 21 mer, (b) the strands may not be completely complementary, i.e., the strands may contain simple mismatch pairings and (c) base modifications such as locked nucleic acid(s) maybe included in the 5' end of the sense strand. A "typical" 21mer siRNA is 10 designed using conventional techniques. In one technique, a variety of sites are commonly tested in parallel or pools containing several distinct siRNA duplexes specific to the same target with the hope that one of the reagents will be effective (Ji et al., 2003). Other techniques use design rules and algorithms to increase the likelihood of obtaining active RNAi effector molecules (Schwarz et al., 2003; 15 Khvorova et al., 2003; Ui-Tei et al., 2004; Reynolds et al., 2004; Krol et al., 2004; Yuan et al., 2004; Boese et al., 2005). High throughput selection of siRNA has also been developed (U.S. published patent application No. 2005 / 0042641 Al). Potential target sites can also be analyzed by secondary structure predictions (Heale et al., 2005). This 21mer is then used to design a right shift to include 3-9 additional 20 nucleotides on the 5' end of the 21 mer. The sequence of these additional nucleotides may have any sequence. In one embodiment, the added ribonucleotides are based on the sequence of the target gene. Even in this embodiment, full complementarity between the target sequence and the antisense siRNA is not required. The first and second oligonucleotides of a DsiRNA agent of the instant 25 invention are not required to be completely complementary. They only need to be substantially complementary to anneal under biological conditions and to provide a substrate for Dicer that produces a siRNA sufficiently complementary to the target sequence. Locked nucleic acids, or LNA's, are well known to a skilled artisan (Elman et al., 2005; Kurreck et al., 2002; Crinelli et al., 2002; Braasch and Corey, 2001; 30 Bondensgaard et al., 2000; Wahlestedt et al., 2000). In one embodiment, an LNA is incorporated at the 5' terminus of the sense strand. In another embodiment, an LNA is incorporated at the 5' terminus of the sense strand in duplexes designed to include a 3' overhang on the antisense strand. In certain embodiments, the DsiRNA agent of the instant invention has an 116 Date Re9ue / Date Received 2022-03-14 asymmetric structure, with the sense strand having a 27-base pair length, and the antisense strand having a 29-base pair length with a 2 base 3'-overhang. Such agents optionally may possess between one and four deoxyribonucleotides of the 3' terminal region (specifically, the region 3' of the projected Dicer cleavage site) of the sense 5 strand, at least one of which base pairs with a cognate deoxyribonucleotide of the 5' terminal region (specifically, the region 5' of the projected Dicer cleavage site) of the antisense strand. In other embodiments, the sense strand has a 28-base pair length, and the antisense strand has a 30-base pair length with a 2 base 3'-overhang. Such agents optionally may possess between one and five deoxyribonucleotides of the 3' 10 terminal region (specifically, the region 3' of the projected Dicer cleavage site) of the sense strand, at least one of which base pairs with a cognate deoxyribonucleotide of the 5' terminal region (specifically, the region 5' of the projected Dicer cleavage site) of the antisense strand. In additional embodiments, the sense strand has a 29-base pair length, and the anti sense strand has a 31-base pair length with a 2 base 3 '- 15 overhang. Such agents optionally possess between one and six deoxyribonucleotides of the 3' terminal region (specifically, the region 3' of the projected Dicer cleavage site) of the sense strand, at least one of which base pairs with a cognate deoxyribonucleotide of the 5' terminal region (specifically, the region 5' of the projected Dicer cleavage site) of the antisense strand. In further embodiments, the 20 sense strand has a 30-base pair length, and the antisense strand has a 32-base pair length with a 2 base 3'-overhang. Such agents optionally possess between one and seven deoxyribonucleotides ...

Claims

168 What is claimed is:

1. A double stranded nucleic acid molecule comprising: a sense strand of 21-50 nucleotides in length; and an antisense strand of 15-22 nucleotides in length, where said sense and antisense strands form a duplex; wherein the sense and antisense strands are separate strands; wherein the sense strand comprises a tetraloop adjacent to said duplex; wherein said tetraloop has a nucleic acid sequence selected from the group consisting of UNCG, GNRA, CUUG, d(GNNA), d(CNNG), and d(TNCG); and wherein said antisense strand is sufficiently complementary to a target mRNA along at least 15 nucleotides of said antisense strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammal or a mammalian cell.

2. The double stranded nucleic acid molecule of claim 1, wherein the tetraloop has a nucleic acid sequence selected from the group consisting of UUCG, GAAA, d(GTTA), and d(TTCG).

3. The double stranded nucleic acid molecule of claim 1 or 2, wherein the tetraloop is flanked at the 5' end by a nucleic acid sequence selected from the group consisting of C, CC, G, and GG.

4. The double stranded nucleic acid molecule of claim 1 or 2, wherein the tetraloop is flanked at the 5' end by a nucleic acid sequence that duplexes with a nucleic sequence selected from the group consisting of C, CC, G, and GG.

5. The double stranded nucleic acid molecule of any one of claims 1-4, wherein the tetraloop is flanked at the 3' end by a nucleic acid sequence selected from the group consisting of C, CC, G, and GG.

6. The double stranded nucleic acid molecule any one of claims 1-4, wherein the tetraloop is flanked at the 3' end by a nucleic acid sequence that duplexes with a nucleic acid sequence selected from the group consisting of C, CC, G, and GG. CA 3151965 Date reçue / Received date 2024-12-10 169 7. The double stranded nucleic acid molecule of any one of claims 1-6, wherein the tetraloop has a nucleic acid sequence of GAAA.

8. The double stranded nucleic acid molecule of any one of claims 1-7, wherein the double stranded nucleic acid molecule is formulated in a pharmaceutical composition comprising a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is suitable for human administration.

9. The double stranded nucleic acid molecule any one of claims 1-8, wherein the double stranded nucleic acid molecule is conjugated to a non-nucleic acid moiety.

10. The double stranded nucleic acid molecule of claim 9, wherein the non-nucleic acid moiety is a peptide or an organic compound.

11. The double stranded nucleic acid molecule of claim 10, wherein the organic compound is a dye.

12. The double stranded nucleic acid molecule of claim 10, wherein the organic compound is cholesterol.

13. The double stranded nucleic acid molecule any one of claims 1-12, wherein the double stranded nucleic acid molecule comprises at least one deoxyribonucleotide.

14. The double stranded nucleic acid molecule of any one of claims 1-13, wherein the double stranded nucleic acid molecule comprises at least one modified nucleotide.

15. The double stranded nucleic acid molecule of claim 14, wherein the at least one modified nucleotides comprises a modified base and / or a modified sugar moiety.

16. The double stranded nucleic acid molecule of claim 14, wherein the at least one modified nucleotide is selected from the group consisting of: dideoxyribonucleotides, acyclonucleotides 3'-deoxyadenosine (cordycepin), 3'-azido-3'-deoxythymidine (AZT), CA 3151965 Date reçue / Received date 2024-12-10 170 2’,3'-dideoxyinosine (ddI), 2',3'-dideoxy-3'-thiacytidine (3TC), 2',3'-didehydro-2',3'-dideoxythymidine (d4T), monophosphate nucleotides of 3'-azido-3'-deoxythymidine (AZT), monophosphate nucleotides of 2',3'-dideoxy-3'-thiacytidine (3TC), and monophosphate nucleotides of 2',3'-didehydro-2',3'-dideoxythymidine (d4T).

17. The double stranded nucleic acid molecule of any one of claims 1-16, further comprising at least one internucleoside linkage modification selected from the group consisting of: phosphonate, phosphorothioate, and phosphotriester modifications.

18. The double stranded nucleic acid molecule of claim 14, wherein the at least one modified nucleotide comprises a locked nucleic acid modification.

19. The double stranded nucleic acid molecule of claim 14, wherein the at least one modified nucleotide comprises a 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-0-[2- (methylamino)-2-oxoethyl], 4-thio, 4'-CH2-O-2'-bridge, 4'-(CH2)2-O-2-bridge, 2-locked nucleic acid, 2'-amino or 2'-O-(N-methylcarbamate) modification.

20. The double stranded nucleic acid molecule of any one of claims 1-14, wherein said antisense strand comprises a 3' overhang.

21. The double stranded nucleic acid molecule of claim 20, wherein the 3' overhang comprises at least one modified nucleotide.

22. The double stranded nucleic acid molecule of claim 21, wherein the modified nucleotide of said 3' overhang is a 2'-O-methyl ribonucleotide.

23. The double stranded nucleic acid molecule of claim 21, wherein all nucleotides of said 3' overhang are modified nucleotides. CA 3151965 Date reçue / Received date 2024-12-10 171 24. The double stranded nucleic acid molecule of any one of claims 1-23, wherein the sense strand has a nucleotide sequence that is at least 80%, 90%, 95% or 100% complementary to the antisense strand nucleotide sequence.

25. The double stranded nucleic acid molecule of any one of claims 18-24, further comprising a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester.

26. The double stranded nucleic acid molecule of any one of claims 1-25, wherein the double stranded nucleic acid molecule reduces target gene expression in a mammalian cell by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50%, at least 80%, and at least 90%.

27. The double stranded nucleic acid molecule of any one of claims 1-26, wherein the double stranded nucleic acid molecule, when introduced into a mammalian cell, reduces target gene expression by at least 70% when transfected into said cell at a concentration between 1 nM and 10 PM.

28. The double stranded nucleic acid molecule of any one of claims 1-27, wherein at least 50% of the nucleotides of said double stranded nucleic acid molecule are modified nucleotides.

29. The double stranded nucleic acid molecule of any one of claims 1-28, wherein every nucleotide of said double stranded nucleic acid molecule is a modified nucleotide.

30. The double stranded nucleic acid molecule of claim any one of claims 1-29, wherein the sense strand is of a length selected from the group consisting of: 25 to 30 nucleotides, 33 to 49 nucleotides, 35 to 49 nucleotides and 37 to 49 nucleotides.

31. The double stranded nucleic acid molecule of any one of claims 1-30, wherein the antisense strand possesses a 3' overhang of 1-4, 1-3 or 1-2 nucleotides in length.

32. Use of an effective amount of the double stranded nucleic acid molecule of any one of claims 1-31 for reducing expression of the target gene in said cell or in said animal, in comparison to a reference double stranded nucleic acid molecule. CA 3151965 Date reçue / Received date 2024-12-10 172 33. A pharmaceutical composition for reducing expression of the target gene in a cell of a subject comprising the double stranded nucleic acid molecule of any one of claims 1-31, in an amount effective to reduce expression of the target gene in the cell in comparison to a reference double stranded nucleic acid molecule, and a pharmaceutically acceptable carrier. CA 3151965 Date reçue / Received date 2024-12-10