Signal activated RNA interference

• US 20090234109A1
• Filed: 12/10/2008
• Published: 09/17/2009
• Est. Priority Date: 12/10/2007
• Status: Active Grant

• Current Assignee: California Institute of Technology
• Original Assignee: California Institute of Technology
• Inventors: Han, Si-Ping, Goddard, William A., Barish, Robert D.
• Granted Patent: US 9,029,524 B2
• Time in Patent Office: Days
• US Class Current: 536/24.500
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2310/3517 Marker; Tag
• CPC Class Codes: C12N 2310/3519 Fusion with another nucleic acid
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2320/10 in screening processes

Abstract

The invention provides compositions and methods for signal activated RNA interference (saRNAi), preferably in vivo. The invention provides polynucleotides that switches between an inactive form and an active form upon covalent or non-covalent interaction with one or more specific chemical signals, such as disease-specific mRNA, miRNA, or other cellular RNA products with sequences that characterize diseased states of the cell. The interaction between the subject polynucleotides and the signals is preferably mediated by hybridization, which exposes, facilitates the formation, and/or allows the formation of a substrate that can be processed by proteins of the RNAi pathway (such as Dicer). The input and output of multiple different polynucleotides of the invention can form an in vivo signaling network. In addition, the multiple input signals can be integrated to modulate the activity of the subject polynucleotides.

Claims

1. A signal-activated polynucleotide construct, comprising:

(1) a guide sequence and a sense sequence capable of forming a duplex region that can either become or be cleaved by Dicer to generate an siRNA or an miRNA comprising the guide sequence, wherein the guide sequence is substantially complementary to a transcript of a target gene; and

,

(2) a signal detecting sequence capable of hybridizing with either;

(a) one or more signal polynucleotides, or,

(b) one or more regions comprising part of the guide sequence and/or part of the sense sequence;

wherein,

(i) in the absence of the signal polynucleotides, the signal detecting sequence hybridizes with said one or more regions comprising part of the guide sequence and/or part of the sense sequence, wherein generation of the siRNA or miRNA is inhibited, and/or productive incorporation of said siRNA or miRNA into RISC is inhibited;

(ii) in the presence of the signal polynucleotides, the signal detecting sequence hybridizes with said signal polynucleotides, and allows the formation of said duplex region for the generation of said siRNA or said miRNA.

2-61. -61. (canceled)

62. A signal-activated polynucleotide construct, comprising:

(1) a guide sequence and a sense sequence capable of forming either an siRNA or an miRNA comprising the guide sequence, or a duplex region that can be cleaved by Dicer to generate said siRNA or miRNA, wherein the guide sequence is substantially complementary to a transcript of a target gene;

(2) an blocking sequence capable of hybridizing with one or more regions comprising part of the guide sequence and/or part of the sense sequence; and

,

(3) one or more signal detecting polynucleotides, each capable of hybridizing with one or more signal polynucleotides,

wherein,

(i) in the absence of the signal polynucleotides, the blocking sequence hybridizes with said one or more regions comprising part of the guide sequence and/or part of the sense sequence, wherein generation of the siRNA or miRNA is inhibited, and/or productive incorporation of said siRNA or miRNA into RISC is inhibited;

(ii) in the presence of the signal polynucleotides, the signal detecting polynucleotides hybridize with said signal polynucleotides, and allow the formation of said duplex region for the generation of said siRNA or said miRNA.

63-77. -77. (canceled)

78. A signal-activated polynucleotide construct, comprising:

(1) a hairpin structure comprising;

(a) a duplex region formed from a guide sequence and a sense sequence; and

,

(b) a single-stranded loop linking the guide sequence and the sense sequence;

wherein the duplex region can form a siRNA or an miRNA comprising the guide sequence, or be cleaved by Dicer to generate said siRNA or miRNA, wherein the guide sequence is substantially complementary to a transcript of a target gene;

(2) a single-stranded blocking sequence having a free end, and is linked to the guide sequence or the sense sequence, said blocking sequence comprising;

(c) a stretch of nuclease-resistant sequence at the free end that inhibits the degradation of said blocking sequence by exonuclease; and

,

(d) one or more signal detecting polynucleotides, each capable of hybridizing with one or more signal polynucleotides;

wherein,

(i) in the absence of the signal polynucleotides, the blocking sequence inhibits proper Dicer cleavage of the duplex region and/or proper loading of said guide sequence in a Dicer cleavage product into a RISC complex; and

,

(ii) in the presence of the signal polynucleotides, at least one of said signal detecting polynucleotides hybridize with said signal polynucleotides, resulting in cleavage of said single-stranded blocking sequence at one or more cleavage sites to create 5′

- or 3′

-OH groups, degradation of the blocking sequence, and proper loading of said guide sequence in the Dicer cleavage product into the RISC complex.

79-84. -84. (canceled)

85. A signal-activated polynucleotide construct, comprising:

(1) a duplex region that can be cleaved by Dicer to generate an siRNA or miRNA that inhibits the expression of a target gene via RNA interference mechanism; and

,

(2) a blocking sequence that hybridizes with a portion of said signal-activated polynucleotide, and creates steric hindrance that inhibits Dicer cleavage of said duplex region;

wherein the steric hindrance is relieved upon binding of a signal polynucleotide to the signal-activated polynucleotide, allowing said duplex region to be cleaved by Dicer to produce said siRNA or miRNA.

86-92. -92. (canceled)

93. A signal-activated polynucleotide construct, comprising:

(1) a guide sequence and a sense sequence forming a duplex region that can become an siRNA or an miRNA comprising the guide sequence, or be cleaved by Dicer to generate said siRNA or miRNA, wherein the guide sequence is substantially complementary to a transcript of a target gene; and

,

(2) a signal detecting sequence capable of hybridizing with either;

(a) one or more signal polynucleotides, or,

(b) one or more regions of the signal-activated polynucleotide other than the guide sequence and the sense sequence;

wherein,

(i) in the absence of the signal polynucleotides, the signal detecting sequence hybridizes with said one or more regions of the signal-activated polynucleotide, and creates steric hindrance to inhibit the generation of the siRNA or miRNA;

(ii) in the presence of the signal polynucleotides, the signal detecting sequence does not hybridizes with said regions of the signal-activated polynucleotide, and allows said duplex region to form said siRNA or said miRNA, or be cleaved by Dicer to generate said siRNA or miRNA.

94-104. -104. (canceled)

105. A signal-activated polynucleotide construct, comprising two or more tandem stem-loop structures, each comprising:

(1) a guide sequence and a sense sequence forming a duplex region that can become or be cleaved by Dicer to generate an siRNA or an miRNA comprising the guide sequence, wherein the guide sequence is substantially complementary to a transcript of a target gene; and

,

(2) a linker sequence linking two adjacent stem-loop structures;

wherein,

(i) in the absence of the signal polynucleotides, the loop regions of the two adjacent stem-loop structures hybridizes with each other to create steric hindrance and to inhibit the generation of said siRNA or miRNA;

(ii) in the presence of the signal polynucleotides, hybridization between the adjacent loop regions is disrupted to allows the adjacent duplex regions to generate said siRNA or said miRNA, or be cleaved by Dicer to generate said siRNA or miRNA.

106. (canceled)

107. A signal-activated polynucleotide construct, comprising two or more tandem stem-loop structures, each comprising:

(1) a guide sequence and a sense sequence forming a duplex region that can become an siRNA or an miRNA comprising the guide sequence, or be cleaved by Dicer to generate said siRNA or miRNA, wherein the guide sequence is substantially complementary to a transcript of a target gene; and

,

(2) a linker sequence linking two adjacent stem-loop structures;

wherein,

(i) in the absence of the signal polynucleotides, the loop regions of the two adjacent stem-loop structures hybridizes with each other to create steric hindrance and to inhibit the generation of said siRNA or miRNA;

(ii) in the presence of the signal polynucleotides, hybridization between the signal polynucleotides and the linker sequence results in the cleavage of the linker sequence and the relief of the steric hindrance, and allows the adjacent duplex regions to form said siRNA or said miRNA, or be cleaved by Dicer to generate said siRNA or miRNA.

108. A signal-activated polynucleotide construct, comprising:

(1) a guide sequence and a sense sequence capable of forming a duplex region that can become an siRNA or an miRNA comprising the guide sequence, or be cleaved by Dicer to generate said siRNA or miRNA, wherein the guide sequence is substantially complementary to a transcript of a target gene; and

,

(2) a single-stranded linker sequence linking said guide sequence and said sense sequence;

wherein,

(i) in the absence of one or more signal polynucleotides, the linker sequence hybridizes with a blocking polynucleotide to create steric hindrance and inhibit the formation of said duplex region and the generation of the siRNA or miRNA;

(ii) in the presence of the signal polynucleotides, said signal polynucleotides hybridize with said blocking sequence to remove the steric hindrance and allows the formation of said duplex region for the generation of said siRNA or said miRNA.

109. (canceled)

110. A signal-activated polynucleotide construct, comprising:

(1) a stem-loop structure comprising a double-stranded stem region and a single-stranded loop region, wherein one end of the stem region comprises a single-stranded overhang;

(2) a single-stranded second polynucleotide that hybridizes to the overhang to form an overhang duplex, wherein a single-stranded gap region links the overhang duplex to the stem region;

wherein,

(i) in the absence of the signal polynucleotides, the linker sequence hybridizes with a blocking polynucleotide to create steric hindrance and inhibit the formation of said duplex region and the generation of the siRNA or miRNA by Dicer;

(ii) in the presence of a signal polynucleotides, said signal polynucleotides hybridizes with the single-stranded gap region and becomes covalently linked to the overhang duplex and the stem region to create a continuous duplex region, wherein said continuous duplex region is capable of being cleaved by Dicer to generate an siRNA or an miRNA that mediates sequence-dependent gene silencing of a target gene.

111. A signal-activated polynucleotide construct, comprising a duplex region of less than 19 base pairs in length, wherein hybridization of the signal-activated polynucleotide with a signal sequence creates a duplex region of at least 19 base pairs in length, said duplex region capable of being cleaved by Dicer to generate an siRNA or miRNA that inhibits the expression of a target gene using an RNA interference mechanism.

112. A signal-activated polynucleotide construct comprising a duplex region having one or more mismatches that inhibits the duplex region from being cleaved by Dicer, wherein hybridization of the signal-activated polynucleotide with a signal sequence creates a duplex region, said duplex region capable of being cleaved by Dicer to generate an siRNA or miRNA that inhibits the expression of a target gene using an RNA interference mechanism.

113. A circular signal-activated polynucleotide construct, comprising:

(1) a duplex region of at least 19 nucleotides, and

(2) two single-stranded loop regions, one at each end of the duplex region, wherein one or both of the loop regions can hybridize with a signal polypeptide,

wherein,

(a) in the absence of the signal polypeptide, said duplex region is not a Dicer substrate,

(b) in the presence of the signal polypeptide, said one or both loop regions hybridize with said signal polypeptide and enables cleavage of the loop regions, wherein the duplex region becomes a Dicer substrate upon cleavage of said one or both loop regions, and wherein cleavage of said duplex region by Dicer produces an siRNA or miRNA that inhibits the expression of a target gene through an RNA interference mechanism.

114-122. -122. (canceled)