Ribozymes with product ejection by strand displacement

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.

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.