Catalytic Molecular Beacons

Abstract

We have constructed catalytic molecular beacons from a hammerhead-type deoxyribozyme by a modular design. The deoxyribozyme was engineered to contain a molecular beacon stem-loop module that, when closed, inhibits the deoxyribozyme module and is complementary to a target oligonucleotide. Binding of target oligonucleotides opens the beacon stem-loop and allosterically activates the deoxyribozyme module, which amplifies the recognition event through cleavage of a doubly labeled fluorescent substrate. The customized modular design of catalytic molecular beacons allows for any two single-stranded oligonucleotide sequences to be distinguished in homogenous solution in a single step. Our constructs demonstrate that antisense conformational triggers based on molecular beacons can be used to initiate catalytic events. The selectivity of the system is sufficient for analytical applications and has potential for the construction of deoxyribozyme-based drug delivery tools specifically activated in cells containing somatic mutations.

Molecular Beacons and Other Hairpin Probes

“…Catalytic MBs represent a next generation of molecular probes with the potential to amplify signals and thus to detect nucleic acid targets without PCR amplification. [15] These MBs are DNA constructs that combine the features of molecular beacons and hammerhead-type deoxyribozymes with RNase activity, which are located on two different modules. [15] In the absence of a target, the beacon hybridizes with the deoxyribozyme module.…”

“…[15] These MBs are DNA constructs that combine the features of molecular beacons and hammerhead-type deoxyribozymes with RNase activity, which are located on two different modules. [15] In the absence of a target, the beacon hybridizes with the deoxyribozyme module. When the target is present, the beacon changes its conformation and allows the substrate (a stemless fluorogenic oligonucleotide) to hybridize with the deoxyribozyme module.…”

“…This approach is just in the development stage, but in the proof-of-principle experiments, it was shown that it initiated catalytic events. [15] The combination of PNA-based technology with molecular beacons opened the possibility of targeting duplex DNA without prior denaturation. PNA openers are short PNA oligomers, which invade duplex DNA and locally expose a single-stranded region within duplex DNA forming a P-loop complex.…”

Homogeneous DNA-detection Based on the Non-enzymatic Reactions Promoted by Target DNA

“…56 Stojanovic et al synthesized the chimera DNA consisting of a DNAzyme and a MB to make the catalytic MB. 57 The catalytic activity of the DNAzyme moiety was restored by target binding to the MB moiety, making it possible to cleave fluorescent substrates in a catalytic manner. Sando et al reported catalytic signal amplification using self-cleaving DNAzyme 58 and a switchable cell-free translation system for luciferase synthesis.…”

Cis-regulatory hairpin-shaped mRNA encoding a reporter protein: catalytic sensing of nucleic acid sequence at single nucleotide resolution

“…1a) 3 utilize hybridizationinduced conformational change of the probe from the light-off hairpin structure to a light-on open form. Much recent attention has also been paid to catalytic gene sensing [4][5][6][7][8][9][10][11][12][13] through coupling to a signal-amplifying enzymatic [4][5][6][7][8] , chemical 9,10 , electrochemical 11 or magnetochemical 12 process. On the other hand, we reported a new strategy with MB-mRNA systems for selective genotyping 13 .…”