dsRNA endoribonucleases
The invention relates to a new double-stranded RNA endoribonuclease, its derivative and/or variant, which has a loop locating in and interacting with the major groove of the double-stranded RNA, exhibiting sequence specific properties in the double-stranded RNA cleavage.
1. A method of sequence specific cleavage of dsRNA substrate by a dsRNA endoribonuclease, comprising the steps of
a) combining the dsRNA endoribonuclease with dsRNA substrate in a mixture, wherein said dsRNA endoribonuclease comprises amino acid sequence of SEQ ID NO:1 with a D94R mutation; and has the loop that is locating in and interacting with a major groove of dsRNA, which corresponds to the loop locating in and interacting with a major groove of dsRNA in the model of structure of endoribonuclease Mini III in complex with dsRNA; and wherein said dsRNA endoribonuclease exhibits the dsRNA sequence specific activity within the consensus sequence (SEQ ID NOS: 111 and 121)
5′ DACCUHD 3′
3′ HUGGADH 5′
where H=A or C or U; D=A or G or U; preferably said dsRNA endoribonuclease exhibits the dsRNA sequence specific activity within the consensus sequence SEQ ID NOS: 112 and 122)
5′ YGACCUCGNNG 3′
3′ RCUGGAGCNNC 5′
where Y=C or U; R=A or G; N=G or A or U or C; and wherein the specific sequence in dsRNA substrate recognized by said dsRNA endoribonuclease is the consensus sequence (SEQ ID NOS: 111 and 121)
5′ DACCUHD 3′
3′ HUGGADH 5′
where H=A or C or U; D=A or G or U; preferably is the consensus sequence (SEQ ID NOS: 112 and 122)
5′ YGACCUCGNNG 3′
3′ RCUGGAGCNNC 5′
where Y=C or U; R=A or G; N=G or A or U or C,
b) cleaving the said dsRNA substrate within said recognition sequence by said dsRNA endoribonuclease.
2. The method of sequence specific cleavage of dsRNA substrate according to claim 1 , wherein the cleaving of ds RNA is performed in temperature from 35° C. till 45° C.; and/or in sodium chloride concentration from 5 to 60 mM; and preferably in Mg 2+ concentration of 1 to 2.5 mM.
3. An dsRNA endoribonuclease, wherein dsRNA endoribonuclease comprises amino acid sequence of SEQ ID NO:1 with D94R mutation; and has the loop that is locating in and interacting with a major groove of dsRNA, which corresponds to the loop locating in and interacting with a major groove of dsRNA in the model of structure of endoribonuclease Mini III in complex with dsRNA; and wherein the specific sequence in dsRNA substrate recognized by said dsRNA endoribonuclease is the consensus sequence (SEQ ID NOS: 111 and 121)
5′ DACCUHD 3′
3′ HUGGADH 5′
where H=A or C or U; D=A or G or U; preferably is the consensus sequence (SEQ ID NOS: 112 and 122)
5′ YGACCUCGNNG 3′
3′ RCUGGAGCNNC 5′
where Y=C or U; R=A or G; N=G or A or U or C; and wherein said dsRNA endoribonuclease exhibits the dsRNA sequence specific activity within said consensus sequence.
4. A method for producing dsRNA endoribonuclease, wherein the method comprises the step of expressing of dsRNA endoribonuclease as defined in claim 3 .
5. A genetic construct, characterized in that it comprises the nucleotide sequence encoding the dsRNA endoribonuclease as defined in claim 3 .
6. A host cell comprising the genetic construct as defined in claim 5 .
7. The kit, wherein it comprises the dsRNA endoribonuclease as defined in claim 3 .
8. A method for sequence specific cleavage of a dsRNA substrate with a dsRNA endoribonuclease comprising an amino acid sequence of SEQ ID NO:1 with a D94R mutation; and has the loop that is locating in and interacting with a major groove of dsRNA, which corresponds to the loop locating in and interacting with a major groove of dsRNA in the model of structure of endoribonuclease Mini III in complex with dsRNA; and wherein said dsRNA endoribonuclease exhibits the dsRNA sequence specific activity within the consensus sequence (SEQ ID NOS: 111 and 121)
5′ DACCUHD 3′
3′ HUGGADH 5′
where H=A or C or U; D=A or G or U; preferably said dsRNA endoribonuclease exhibits the dsRNA sequence specific activity within the consensus sequence (SEQ ID NOS: 112 and 122)
5′ YGACCUCGNNG 3′
3′ RCUGGAGCNNC 5′
where Y=C or U; R=A or G; N=G or A or U or C, and wherein the specific sequence in dsRNA substrate recognized by said dsRNA endoribonuclease is the consensus sequence (SEQ ID NOS: 111 and 121)
5′ DACCUHD 3′
3′ HUGGADH 5′
where H=A or C or U; D=A or G or U; preferably is the consensus sequence (SEQ ID NOS: 112 and 122)
5′ YGACCUCGNNG 3′
3′ RCUGGAGCNNC 5′
where Y=C or U; R=A or G; N=G or A or U or C, and wherein said dsRNA substrate comprises and is cleaved within said recognition sequence by said dsRNA endoribonuclease.