25 research outputs found
Enzyme-Free Amplified Detection of DNA by an Autonomous Ligation DNAzyme Machinery
The Zn<sup>2+</sup>-dependent ligation DNAzyme is implemented
as
a biocatalyst for the amplified detection of a target DNA by the autonomous
replication of a nucleic acid reporter unit that is generated
by the catalyzed ligation process. The reporter units enhance the
formation of active DNAzyme units, thus leading to the isothermal
autocatalytic formation of the reporter elements. The system was further
developed and applied for the amplified detection of Tay-Sachs genetic disorder
mutant, with a detection limit of 1.0 Ă 10<sup>â11</sup> M. Besides providing a versatile paradigm for the amplified detection
of DNA, the system reveals a new, enzyme-free, isothermal, autocatalytic
mechanism that introduces means for effective programmed synthesis
Zn<sup>2+</sup>-Ligation DNAzyme-Driven Enzymatic and Nonenzymatic Cascades for the Amplified Detection of DNA
A generic fluorescence sensing platform for analyzing
DNA by the
Zn<sup>2+</sup>-dependent ligation DNAzyme as amplifying biocatalyst
is presented. The platform is based on the target DNA induced ligation
of two substrate subunits and the subsequent opening of a beacon hairpin
probe by the ligated product. The strand displacement of the ligated
product by the beacon hairpin is, however, of limited efficiency.
Two strategies are implemented to overcome this limitation. By one
method, a âhelperâ nucleic acid sequence is introduced
into the system, and this hybridizes with the DNAzyme components and
releases the ligated product for opening of the hairpin. By the second
method, a nicking enzyme (Nt.BspQI) is added to the system, and this
nicks the duplex between the beacon and ligated product while recycling
the free ligation product. By combining the two coadded components
(âhelperâ sequence and nicking enzyme), the sensitive
detection of the analyte is demonstrated (detection limit, 20 pM).
The enzyme-free amplified fluorescence detection of the target DNA
is further presented by the Zn<sup>2+</sup>-dependent ligation DNAzyme-driven
activation of the Mg<sup>2+</sup>-dependent DNAzyme. According to
this method, the Mg<sup>2+</sup>-dependent DNAzyme subunits displace
the ligated product, and the resulting assembled DNAzyme cleaves a
fluorophore/quencher-modified substrate to yield fluorescence. The
method enabled the detection of the target DNA with a detection limit
corresponding to 10 pM. The different sensing platforms are implemented
to detect the TayâSachs genetic disorder mutant
pH-Programmable DNA Logic Arrays Powered by Modular DNAzyme Libraries
Nature performs complex information processing circuits,
such the
programmed transformations of versatile stem cells into targeted functional
cells. Man-made molecular circuits are, however, unable to mimic such
sophisticated biomachineries. To reach these goals, it is essential
to construct programmable modular components that can be triggered
by environmental stimuli to perform different logic circuits. We report
on the unprecedented design of artificial pH-programmable DNA logic
arrays, constructed by modular libraries of Mg<sup>2+</sup>- and UO<sub>2</sub><sup>2+</sup>-dependent DNAzyme subunits and their substrates.
By the appropriate modular design of the DNA computation units, pH-programmable
logic arrays of various complexities are realized, and the arrays
can be erased, reused, and/or reprogrammed. Such systems may be implemented
in the near future for nanomedical applications by pH-controlled regulation
of cellular functions or may be used to control biotransformations
stimulated by bacteria
Amplified Detection of DNA through the Enzyme-Free Autonomous Assembly of Hemin/G-Quadruplex DNAzyme Nanowires
An enzyme-free amplified detection platform is described
using
the horseradish peroxidase (HRP)-mimicking DNAzyme as an amplifying
label. Two hairpin structures that include three-fourths and one-fourth
of the HRP-mimicking DNAzyme in caged, inactive configurations are
used as functional elements for the amplified detection of the target
DNA. In the presence of the analyte DNA, one of the hairpins is opened,
and this triggers the autonomous cross-opening of the two hairpins
using the strand displacement principle. This leads to the formation
of nanowires consisting of the HRP-mimicking DNAzyme. The resulting
DNA nanowires act as catalytic labels for the colorimetric or chemiluminescent
readout of the sensing processes (the term âenzyme-freeâ
refers to a protein-free catalyst). The analytical platform allows
the sensing of the analyte DNA with a detection limit corresponding
to 1 Ă 10<sup>â13</sup> M. The optimized system acts as
a versatile sensing platform, and by coaddition of a âhelperâ
hairpin structure any DNA sequence may be analyzed by the system.
This is exemplified with the detection of the BRCA1 oncogene with
a detection limit of 1 Ă 10<sup>â13</sup> M
Autonomous Replication of Nucleic Acids by Polymerization/Nicking Enzyme/DNAzyme Cascades for the Amplified Detection of DNA and the AptamerâCocaine Complex
The
progressive development of amplified DNA sensors and aptasensors using
replication/nicking enzymes/DNAzyme machineries is described. The
sensing platforms are based on the tailoring of a DNA template on
which the recognition of the target DNA or the formation of the aptamerâsubstrate
complex trigger on the autonomous isothermal replication/nicking processes
and the displacement of a Mg<sup>2+</sup>-dependent DNAzyme that catalyzes
the generation of a fluorophore-labeled nucleic acid acting as readout
signal for the analyses. Three different DNA sensing configurations
are described, where in the ultimate configuration the target sequence
is incorporated into a nucleic acid blocker structure associated with
the sensing template. The target-triggered isothermal autonomous replication/nicking
process on the modified template results in the formation of the Mg<sup>2+</sup>-dependent DNAzyme tethered to a free strand consisting of
the target sequence. This activates additional template units for
the nucleic acid self-replication process, resulting in the ultrasensitive
detection of the target DNA (detection limit 1 aM). Similarly, amplified
aptamer-based sensing platforms for cocaine are developed along these
concepts. The modification of the cocaine-detection template by the
addition of a nucleic acid sequence that enables the autonomous secondary
coupled activation of a polymerization/nicking machinery and DNAzyme
generation path leads to an improved analysis of cocaine (detection
limit 10 nM)
Multiplexed Analysis of Genes and of Metal Ions Using Enzyme/DNAzyme Amplification Machineries
The
progressive development of amplified DNA sensors using nucleic acid-based
machineries, involving the isothermal autonomous synthesis of the
Mg<sup>2+</sup>-dependent DNAzyme, is used for the amplified, multiplexed
analysis of genes (Smallpox, TP53) and metal ions (Ag<sup>+</sup>,
Hg<sup>2+</sup>). The DNA sensing machineries are based on the assembly
of two sensing modules consisting of two nucleic acid scaffolds that
include recognition sites for the two genes and replication tracks
that yield the nicking domains for Nt.BbvCI and two different Mg<sup>2+</sup>-dependent DNAzyme sequences. In the presence of any of the
genes or the genes together, their binding to the respective recognition
sequences triggers the nicking/polymerization machineries, leading
to the synthesis of two different Mg<sup>2+</sup>-dependent DNAzyme
sequences. The cleavage of two different fluorophore/quencher-modified
substrates by the respective DNAzymes leads to the fluorescence of
F<sub>1</sub> and/or F<sub>2</sub> as readout signals for the detection
of the genes. The detection limits for analyzing the Smallpox and
TP53 genes correspond to 0.1 nM. Similarly, two different nucleic
acid scaffolds that include Ag<sup>+</sup>-ions or Hg<sup>2+</sup>-ions recognition sequences and the replication tracks that yield
the Nt.BbvCI nicking domains and the respective Mg<sup>2+</sup>-dependent
DNAzyme sequences are implemented as nicking/replication machineries
for the amplified, multiplexed analysis of the two ions, with detection
limits corresponding to 1 nM. The ions sensing modules reveal selectivities
dominated by the respective recognition sequences associated with
the scaffolds
Smart Mesoporous SiO<sub>2</sub> Nanoparticles for the DNAzyme-Induced Multiplexed Release of Substrates
The fluorescent dyes methylene blue, MB<sup>+</sup>,
and thionine,
Th<sup>+</sup>, can be trapped in the pores of mesoporous silica,
MP-SiO<sub>2</sub>, by means of functional nanostructures consisting
of the Mg<sup>2+</sup>- or Zn<sup>2+</sup>-dependent DNAzyme sequences.
In the presence of Mg<sup>2+</sup> or Zn<sup>2+</sup> ions the respective
DNAzymes are activated, leading to the specific cleavage of the respective
caps, and the selective release of MB<sup>+</sup> or Th<sup>+</sup>. The enlargement of the conserved loop domains of the Mg<sup>2+</sup>- or Zn<sup>2+</sup>-dependent DNAzyme sequences with foreign nucleotides
prohibits the formation of active DNAzymes and eliminates the release
of the respective dyes. This is due to the flexibility of the loops
that lacks affinity for the association of the ions. The insertion
of aptamer sequences (e.g., the adenosine-5â˛-triphosphate (ATP)
aptamer) or ion-binding sequences (e.g., T-rich Hg<sup>2+</sup> ion-binding
domains) as foreign components to the loop regions allows the formation
of active Mg<sup>2+</sup>- or Zn<sup>2+</sup>-dependent DNAzyme structures
through the cooperative formation of aptamer-ATP complexes or T-Hg<sup>2+</sup>-T bridges. These aptamerâsubstrate complexes or T-Hg<sup>2+</sup>-T bridges allosterically stabilize and activate the DNAzymes,
thus allowing the selective release of the fluorescent substrates
MB<sup>+</sup> or Th<sup>+</sup>. The metal ion-driven DNAzyme release
of substrates from the pores of MP-SiO<sub>2</sub>, and particularly
the allosteric activation of the DNAzymes through cooperative aptamerâsubstrate
complexes or metal-ion bridges, has important future nanomedical implications
for targeted release of drugs. This is demonstrated with the triggered
release of the anticancer drug, doxorubicin, by the Mg<sup>2+</sup>-DNAzyme-locked pores or by the aptamer-ATP complex-triggered activation
of the Mg<sup>2+</sup>-dependent DNAzyme
Switching Photonic and Electrochemical Functions of a DNAzyme by DNA Machines
DNA nanostructures acting as DNA machines are described.
Specifically,
DNA âwalkersâ assembled on nucleic acid scaffolds and
triggered by fuel/antifuel strands are activated in solution or on
surfaces, for example, electrodes or semiconductor CdSe/ZnS quantum
dots (QDs). The DNA machines led to the switchable formation or dissociation
of the hemin/G-quadruplex DNAzyme on the DNA scaffolds. This enabled
the chemiluminescence, chemiluminescence resonance energy transfer
(CRET), electrochemical, or photoelectrochemical transduction of the
switchable states of the different DNA machines
Evaluation of DNA Methyltransferase Activity and Inhibition via Isothermal Enzyme-Free Concatenated Hybridization Chain Reaction
Methyltransferase
(MTase)-catalyzed DNA methylation plays a vital
role in the biological epigenetic processes of key diseases and has
attracted increasing attention, making the amplified detection of
MTase activity of great significance in clinical disease diagnosis
and treatment. Herein, we developed an isothermal, enzyme-free, and
autonomous strategy for analyzing MTase activity based on concatenated
hybridization chain reaction (C-HCR)-mediated FoĚrster resonance
energy transfer (FRET). In a typical C-HCR procedure without MTase
(Dam), Y-shaped initiator DNA activates upstream HCR-1 to assemble
a double-stranded DNA (dsDNA) copolymeric nanowire consisting of multiple
tandem DNA trigger units that motivate downstream HCR-2 to successively
bring a fluorophore donor/acceptor (FAM/TAMRA) pair into close proximity,
leading to the generation of an amplified FRET readout signal. The
target Dam MTase and auxiliary DpnI endonuclease can sequentially
and specifically recognize/methylate and cleave the Y-shaped initiator
oligonucleotide, respectively, and thus prohibit the C-HCR process
and FRET signal generation, resulting in the construction of a signal-on
sensing platform for MTase assay. Our proposed isothermal enzyme-free
C-HCR amplification approach was further utilized for screening MTase
inhibitors. Furthermore, the proposed C-HCR approach can be easily
adapted for probing other different MTases and for screening the corresponding
inhibitors just by changing the recognition sequence of Y-shaped initiator
DNA through a âplug-and-playâ format. It provides a
versatile and robust tool for highly sensitive detection of various
biotransformations and thus holds great promise in clinical assessment
and diagnosis
Programmed DNAzyme-Triggered Dissolution of DNA-Based Hydrogels: Means for Controlled Release of Biocatalysts and for the Activation of Enzyme Cascades
Acrylamide/acrylamide-modified nucleic
acid copolymer chains provide building units for the construction
of acrylamideâDNA hydrogels. Three different hydrogels are
prepared by the cross-linking of the acrylamideâDNA chains
with metal ion-dependent DNAzyme sequences and their substrates. The
metal ion-dependent DNAzyme sequences used in the study include the
Cu<sup>2+</sup>-, Mg<sup>2+</sup>-, and Zn<sup>2+</sup>-dependent
DNAzymes. In the presence of the respective metal ions, the substrates
of the respective DNAzymes are cleaved, leading to the separation
of the cross-linking units and to the dissolution of the hydrogel.
The different hydrogels were loaded with a fluorophore-modified dextran
or with a fluorophore-functionalized glucose oxidase. Treatment of
the different hydrogels with the respective ions led to the release
of the loaded dextran or the enzyme, and the rates of releasing of
the loaded macromolecules followed the order of Cu<sup>2+</sup> >
Mg<sup>2+</sup> > Zn<sup>2+</sup>. Also, the different hydrogels
were loaded with the enzymes β-galactosidase (β-Gal),
glucose oxidase (GOx), or horseradish peroxidase (HRP). In the presence
of the appropriate metal ions, the respective hydrogels were dissolved,
resulting in the activation of the β-Gal/GOx or GOx/HRP bienzyme
cascades and of the β-Gal/GOx/HRP trienzyme cascade