Multicomponent nucleic acid enzymes and methods for their use
A multi-component nuclease and nucleic acid technology, applied in the multi-component nuclease and its application field, can solve problems such as noise
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Embodiment 1
[0450] Example 1: Application of MNAzyme to directly detect target nucleic acid (human RPLPO sequence)
[0451] 1.1 Partzyme Oligonucleotide
[0452] Four designs for MNAzyme based on 8:17 DNAse were tested (Figures 8-10). Those skilled in the art should understand that the sensor arm (target binding) sequence represented by "N" can be replaced with a target-specific sequence of any known nucleic acid target (Figures 8-10). The substrate arm sequence that binds to the reporter substrate can be universal and can be used for many targets. Those skilled in the art should understand that the substrate sequence represented by "N" in Figures 8-10 can be replaced with DNA, RNA or DNA / RNA chimeric sequences, and those represented by "r" can be replaced by other and / or different numbers. Nucleotide sequence substitution.
[0453] In the experiment used to measure the catalytic activity of the RPLPO MNAzyme described in Figures 8-10, the A and B oligonucleotide partzymes for the target exon...
Embodiment 2
[0487] Example 2: MNAzyme for detecting miR-20 or a short DNA sequence homologous to miR-20
[0488] 2.1. Partzyme Oligonucleotides
[0489] Detection using MNAzyme can also be used to analyze miR. In this example, MNAzymes are only formed when the correct miR sequence is present. Such MNAzymes can distinguish related miR sequences such as hsa-miR-20 and hsa-miR-93.
[0490] In experiments performed to measure the catalytic activity of the MNAzyme described in Figure 11, A and B partzyme oligonucleotides were designed to target hsa-miR-20. The sequences of partzyme A and B oligonucleotides from 5'to 3'are shown below. In the following sequence, Underline The bases formed in the assembled MNAzyme form part of the catalytic core, the bases in bold hybridize with the target, and the bases in italics hybridize with the substrate.
[0491] SEQ ID NO: 10: Partzyme A2:
[0492] miR20A2 / 1: CGGTCGAA
[0493] SEQ ID NO: 11: Partzyme B3:
[0494] miR20B3 / 1: CCGAGC
[0495] 2.2. Repo...
Embodiment 3
[0523] Example 3: MNAzyme for direct detection of nucleic acid targets (designs 5 and 6)
[0524] 3.1. Partzyme Oligonucleotides
[0525] The catalytic activity of designs 5 and 6 of MNAzyme based on 10:23 DNase was tested (Figure 13). Those skilled in the art should understand that the sensor arm (target binding) sequence represented by "N" can be replaced with a target-specific sequence of any known nucleic acid target. The substrate arm sequence that binds to the reporter substrate can be universal and can be used for many targets. Those skilled in the art should understand that the substrate sequence represented by "N" in Figure 13 can be replaced by DNA, RNA or DNA / RNA chimeric sequences.
[0526] In conducting experiments to measure the catalytic activity of the RPLPO MNAzyme described in Figure 13, A and B oligonucleotide partzymes targeting exon 5 of the RPLPO gene were designed. The sequences of partzymes A and B from 5’ to 3’ are shown below, where Underline The bases f...
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