Target-dependent nucleic acid adapter
In accordance with the present invention, there is provided a nucleic acid target-dependent adapter linked to a nucleic acid sequence. The adapter comprises linked together a biosensor having a specific sequence complementary to a target sequence of a substrate, the biosensor improving the specificity of the nucleic acid sequence for the substrate, and a blocker stem sequence complementary to a portion of the nucleic acid sequence. In absence of the target sequence of the substrate, the blocker stem sequence forms an intramolecular stem with the nucleic acid sequence linked thereto, preventing exposition of the sequence of the nucleic acid sequence, thus locking the nucleic acid sequence so linked to the adapter in an inactive conformation, and in presence of the target sequence, the blocker stem sequence dissociating from the nucleic acid sequence, thus exposing the nucleic acid sequence linked to the adapter in an active conformation.
1. A method for turning on or off an enzymatic activity of a nucleic acid molecule having a catalytic core providing an enzymatic activity adapted to be matched to a substrate comprising a target sequence, said method comprising the steps of attaching to said nucleic acid molecule a nucleic acid target dependent switch adapter having a nucleic acid sequence comprising:
i) a blocker stem sequence complementary to a portion of the catalytic core of said nucleic acid molecule; and
ii) a biosensor sequence having a sequence complementary to said target sequence, being spaced from the catalytic core by at least three nucleotides and having an affinity level for the target sequence greater than the affinity level of the blocker stem sequence for the catalytic center,
wherein in absence of the target sequence of said substrate, said blocker stem sequence forms an intramolecular stem with the catalytic core of said nucleic acid molecule, preventing folding of the catalytic core of the nucleic acid molecule, thus locking said nucleic acid molecule in an inactive conformation, turning off the enzymatic activity and, in presence of said target sequence of said substrate, said biosensor sequence forming conventional Watson-Crick base pairs with said target sequence and said blocker stem sequence dissociating from the intramolecular stem of the catalytic core, thus permitting folding of the catalytic core of the nucleic acid molecule exposing said nucleic acid molecule in an active conformation, turning on the enzymatic activity.
2. The method of claim 1 , further comprising a nucleic acid sequence forming a stabilizing stem, whereby the 3′ end of the nucleic acid sequence of the switch is paired up with said nucleic acid sequence forming an intramolecular stem at the 3′ end of said nucleic acid sequence of the switch, thus preventing or reducing degradation of said nucleic acid sequence switch.
3. The method of claim 2 , wherein the stabilizing stem has two complementary strands, a first strand of which is linked to the 5′-end of the biosensor, and a second strand of which is complementary to the first strand and is adapted to be linked at its 5′-end to the 3′-end of the nucleic acid sequence of the switch, thus preventing exposure of a single stranded 3′-end sequence susceptible to degradation by cellular nuclease.
4. The method of claim 3 , wherein the first strand of the stabilizing stem has a sequence as set forth from residue 4 to 11 of SEQ ID NO:1 and the second strand of the stabilizing stem has a sequence as set forth from residue 96 to 103 of SEQ ID NO:1.
5. The method of claim 1 , wherein the blocker has a sequence specific for a ribozyme.
6. The method of claim 5 , wherein the ribozyme is ribozyme delta.
7. The method of claim 6 , wherein the biosensor has a sequence as set forth from residue 15 to 29 of SEQ ID NO:1.
8. The method of claim 1 , wherein the blocker stem sequence has a sequence as set forth from residue 30 to 33 of SEQ ID NO:1.
9. The method of claim 1 , wherein the switch adapter has the sequence as set forth in SEQ ID NO:1.
10. The method of claim 1 , wherein the blocker stem sequence is linked to the biosensor.
11. The method of claim 6 , wherein the nucleic acid molecule attached to said adapter is ribozyme delta.