Ribozymes with product ejection by strand displacement

Claim 1

A synthetic ribozyme polynucleotide having a first end and a second end, comprising from said first end to said second end: a catalytic region having endonuclease activity specific for a target polynucleotide sequence of a substrate; a first substrate binding region linked to said catalytic region, said first substrate binding region comprising a first competitive binding nucleotide sequence capable of forming a first hybrid with said substrate; and a first displaceable antisense arm linked to said first substrate binding region, wherein said first displaceable antisense arm comprises a first stabilization region and a second stabilization region, said first stabilization region being capable of hybridizing with said second stabilization region, and a first displacement region capable of forming a second hybrid with said competitive binding nucleotide sequence.

Claim 22

A method for producing synthetic ribozyme polynucleotides, comprising the steps of: constructing at least one set of synthetic ribozyme oligonucleotides comprising variable length substrate binding regions and displaceable antisense arm regions, and further comprising an inactivated catalytic site; constructing a desired substrate; contacting said set of synthetic oligonucleotides with said substrate at a temperature which is less than a preselected temperature; capturing complexes of said substrate and said oligonucleotides; subjecting said complexes to said preselected temperature; capturing and amplifying any oligonucleotides released at said preselected temperature; repeating the steps of constructing synthetic ribozyme oligonucleotides, contacting with substrate, capturing complexes, subjecting complexes to said preselected temperature, and capturing and amplifying released complexes, until a constant binding and release is found; cloning, isolating, and sequencing any oligonucleotides released after said constant binding and release is achieved; and activating the catalytic sites of any oligonucleotides released after said constant binding and release is achieved.

Claim 23

A method for producing synthetic ribozyme polynucleotides, comprising the steps of: constructing at least one set of synthetic ribozyme oligonucleotides comprising variable length substrate binding regions and displaceable antisense arm regions, and further comprising an inactivated catalytic site; constructing a desired substrate; contacting said set of synthetic oligonucleotides with said substrate at a temperature which is less than a preselected temperature; capturing complexes of said substrate and said oligonucleotides; subjecting said complexes to said preselected temperature; capturing and amplifying any oligonucleotides released at said preselected temperature; repeating the steps of constructing synthetic ribozyme oligonucleotides, contacting with substrate, capturing complexes, subjecting complexes to said preselected temperature, and capturing and amplifying released complexes, until a constant binding and release is found; cloning, isolating, and sequencing any oligonucleotides released after said constant binding and release is achieved; and activating the catalytic sites of any oligonucleotides released after said constant binding and release is achieved.

Claim 24

A method of cleaving a target nucleotide sequence of a substrate, comprising the steps of: providing said substrate; providing a synthetic ribozyme polynucleotide having a first end and a second end, comprising from said first end to said second end a catalytic region having endonuclease activity specific for said target polynucleotide sequence and a first substrate binding region linked to said catalytic region, said substrate binding region further comprising a first competitive binding nucleotide sequence capable of forming a first hybrid with said substrate, and a first displaceable antisense arm linked to said substrate binding region, said first displaceable antisense arm further comprising a first stabilization region, a second stabilization region, and a first displacement region capable of forming a second hybrid with said first competitive binding nucleotide sequence; and contacting said synthetic ribozyme polynucleotide and said substrate to allow said catalytic region to cleave said substrate at said target nucleotide sequence.

Claim 25

A method of cleaving a target nucleotide sequence of a substrate, comprising the steps of: providing said substrate; providing a synthetic ribozyme polynucleotide having a first end and a second end, comprising from said first end to said second end a catalytic region having endonuclease activity specific for said target polynucleotide sequence and a first substrate binding region linked to said catalytic region, said substrate binding region further comprising a first competitive binding nucleotide sequence capable of forming a first hybrid with said substrate, and a first displaceable antisense arm linked to said substrate binding region, said first displaceable antisense arm further comprising a first stabilization region, a second stabilization region, and a first displacement region capable of forming a second hybrid with said first competitive binding nucleotide sequence; and contacting said synthetic ribozyme polynucleotide and said substrate to allow said catalytic region to cleave said substrate at said target nucleotide sequence.