molecules

Review
DNA Catalysis: Design, Function, and Optimization
Rebecca L. Stratton 1,† , Bishal Pokhrel 1,† , Bryce Smith 1 , Adeola Adeyemi 1 , Ananta Dhakal 1
and Hao Shen 1,2, *

1 Department of Chemistry and Biochemistry, Kent State University, Kent, OH 44242, USA;
[email protected] (R.L.S.); [email protected] (B.P.); [email protected] (B.S.); [email protected] (A.A.);
[email protected] (A.D.)
2 Advanced Materials and Liquid Crystal Institute, Kent State University, Kent, OH 44242, USA
* Correspondence: [email protected]
† These authors contributed equally to this work.

Abstract: Catalytic DNA has gained significant attention in recent decades as a highly efficient and
tunable catalyst, thanks to its flexible structures, exceptional specificity, and ease of optimization.
Despite being composed of just four monomers, DNA’s complex conformational intricacies enable
a wide range of nuanced functions, including scaffolding, electrocatalysis, enantioselectivity, and
mechano-electro spin coupling. DNA catalysts, ranging from traditional DNAzymes to innovative
DNAzyme hybrids, highlight the remarkable potential of DNA in catalysis. Recent advancements in
spectroscopic techniques have deepened our mechanistic understanding of catalytic DNA, paving
the way for rational structural optimization. This review will summarize the latest studies on
the performance and optimization of traditional DNAzymes and provide an in-depth analysis of
DNAzyme hybrid catalysts and their unique and promising properties.

Keywords: DNA catalysis; DNAzyme; DNA-nanoparticle hybrid; design; function; optimization

1. Introduction
Nucleic acid catalysts have long intrigued scientists, bridging the fields of kinetics and
supramolecular systems [1,2]. While protein enzymes were once considered the primary
Citation: Stratton, R.L.; Pokhrel, B.;
biological catalysts, Woese et al. proposed the idea of catalytically active RNA in the late
Smith, B.; Adeyemi, A.; Dhakal, A.;
1960s [3]. This theory led to the discovery of ribozymes [4], with the key studies conducted
Shen, H. DNA Catalysis: Design,
by Thomas R. Cech and Sidney Altman, who demonstrated that RNA sequences could
Function, and Optimization. Molecules
catalyze reactions without proteins [5]. These ribozymes are also referred to as “RNAzymes”
2024, 29, 5011. https://doi.org/
to highlight the catalytic role of RNA. These early RNAzymes, capable of splicing and
10.3390/molecules29215011
modifying phosphodiester bonds, paved the way for new research opportunities [6]. Over
Academic Editor: Ning Ma time, artificial selection and studies of native RNAs have expanded the number of known
Received: 30 September 2024
ribozymes [7–12]. Today, catalytic nucleic acids can be designed to selectively cleave RNA
Revised: 15 October 2024
molecules, offering promise for gene expression control, infection prevention, and related
Accepted: 17 October 2024 therapies [13–16].
Published: 23 October 2024 Since RNAzymes initially proved to be successful and modifiable nucleic acid catalysts,
their existence sparked intriguing theories about the potential for DNA-based catalysts [7].
Unlike RNA, which typically exists as short, single-stranded segments with accessible
active sites due to unpaired nucleotides folding into helical structures [17,18], DNA was
Copyright: © 2024 by the authors. initially considered less suitable for catalysis. This skepticism stemmed from DNA’s
Licensee MDPI, Basel, Switzerland.
structural characteristics: the absence of the 2′ -OH group, which limits the formation of
This article is an open access article
certain catalytically active conformations, and its common double-stranded form with
distributed under the terms and
fewer unpaired nucleotides [19]. Moreover, the lack of naturally occurring DNAzymes
conditions of the Creative Commons
presents further challenges in designing DNA catalysts. However, these very structural
Attribution (CC BY) license (https://
differences have since proven advantageous, enabling DNAzymes to compete impressively
creativecommons.org/licenses/by/
with their RNAzyme counterparts in versatility and effectiveness [20].
4.0/).

Molecules 2024, 29, 5011. https://doi.org/10.3390/molecules29215011 https://www.mdpi.com/journal/molecules

Molecules 2024, 29, 5011 2 of 20

Following the initial expansion of RNAzyme research, G. F. Joyce and R. R. Breaker lever-
aged the existing theoretical framework of ribozyme behavior to synthetically create the first
known RNA-cleaving DNAzyme [21]. This DNAzyme, now known as the 10–23 DNAzyme,
has since been applied in various contexts [22]. As anticipated, the list of known DNAzymes
has grown substantially, with the universal database “DNAmoreDB” now containing hun-
dreds of entries [23]. Currently, DNAzymes go beyond cleaving oligonucleotides at specific
sites [24–26]. They can facilitate reactions such as ligation [27–29], phosphorylation [30,31],
deglycosylation [32], and acylation [33]. They serve as effective mimics of many natu-
ral enzymes, including esterases [34,35], laccases [36], photolyases [37–39], phosphoserine
lyases [40], phosphatases [41], tyrosine kinases [42], chelatases [43–46], and particularly per-
oxidases [47,48]. In addition, DNAzymes have shown promise in modifying peptide chains
through side-chain modifications, linkages, and elimination reactions [49–51], offering signifi-
cant potential for targeted protein editing.
Beyond these functions, DNAzymes have been shown to catalyze a variety of chemical
reactions, including the cleavage of anilides and aromatic amides [35], thymine dimer
photoreversion [52], acylation of amines and lysines [53], oxidation of L-tyrosine and
amyloid β [54], reductive amination [55], enantioselective Diels–Alder reactions [56],
Friedel–Crafts alkylation [57], Michael addition [58], metal oxide degradation [59], aldol
reactions [60], and others [61]. Even more intriguing is the emerging practice of combin-
ing DNAzymes with other types of materials, successfully integrating both organic and
inorganic components [62].
The rapid expansion of available DNAzymes can be largely attributed to the develop-
ment of the in vitro selection method (also known as directed evolution or SELEX) method
in the 1990s [63–66]. This process typically involves multiple rounds of selection followed
by amplification, ensuring the precise identification of the most effective DNAzymes [67].
According to DNAzyme expert Silverman, the success of in vitro selection features a key
advantage of DNA catalysts over proteins: their sizes [68]. DNA consists of just four
chemically similar bases—guanine (G), thymine (T), adenine (A), and cytosine (C)—while
proteins are composed of a much larger and more diverse array of amino acids. This
vast diversity in proteins, while potentially offering numerous catalytic functions, makes
them practically impossible to evaluate and select through random methods like in vitro
selection [69–71].
Despite the utility of in vitro selection, it has limitations, particularly in the develop-
ment of large, complex DNAzymes. Some reactions may be too complicated for DNAzymes
selected through in vitro techniques [61,66,72,73]. Nevertheless, in vitro selection remains
a valuable method for discovering new DNAzymes, and more targeted approaches, such
as chemical evolution, have emerged to refine and improve existing DNAzymes rather
than restarting the selection process [24,74,75]. In recent years, research on catalytically
active DNA has become more advanced and specialized, making the design, function, and
optimization of DNAzymes essential for further development. One of the main challenges
in improving DNAzymes is the limited understanding of their sequence-specific activity,
conformational dynamics, and behavior within larger molecular complexes. Recent break-
throughs have begun to address these gaps, but few review articles focus on comparing the
attributes of DNAzymes, particularly in the context of supramolecular complexes.
The purpose of this review is to systematically organize catalytic DNA research,
starting with a focus on DNA-based catalysts that function primarily due to their inherent
DNA structure, without the influence of additional materials. This subgroup allows for the
exploration of sequential, environmental, conformational, and mechanical modifications
without the confounding effects of hybrid structures. Subsequent sections will delve into
the design, function, and optimization of DNAzyme hybrid structures. First, we will
explore the integration of DNA with inorganic materials, creating DNA–nanocore hybrids.
Later, we will examine hybrids that incorporate soft matter or biopolymers like DNA, RNA,
and proteins.
Molecules 2024, 29, x FOR PEER REVIEW 3 of 21

Molecules 2024, 29, 5011 3 of 20

Notably, metal nanoparticles such as gold (Au), platinum (Pt), and silver (Ag); metal
oxides like iron oxide (Fe2O3/Fe3O4), manganese oxide (Mn2O3), and titanium dioxide
(TiO2Notably, metal nanoparticles
); and carbon-based materials such
likeasgraphene
gold (Au), platinum
oxides, (Pt),nanotubes,
carbon and silver (Ag); metal
and fuller-
oxides like iron oxide (Fe2 O 3 /Fe 3 O4 ), manganese oxide (Mn 2 O 3 ), and
enes have been combined with DNA to generate hybrid catalysts [76–87]. These inorganictitanium dioxide
(TiO 2 ); and carbon-based
nanoparticles, materials like
known as nanozymes, graphene
often exhibitoxides, carbon
catalytic nanotubes,
activities on theirand
ownfullerenes
[88–91].
have been combined with DNA to generate hybrid catalysts [76–87]. These inorganic
When assembled with DNA, these nanozymes can achieve enhanced catalytic perfor-
nanoparticles, known as nanozymes, often exhibit catalytic activities on their own [88–91].
mance through complex and sometimes unexpected interactions. This synergy not only
When assembled with DNA, these nanozymes can achieve enhanced catalytic performance
complements the existing strengths of DNA catalysts but also mitigates undesirable be-
through complex and sometimes unexpected interactions. This synergy not only comple-
haviors, highlighting the importance and accessibility of modern DNAzyme applications.
ments the existing strengths of DNA catalysts but also mitigates undesirable behaviors,
highlighting the importance and accessibility of modern DNAzyme applications.
2. DNA-Dominant Catalysts
The complete
2. DNA-Dominant mechanism of activity for the 10–23 DNAzyme—
Catalysts
fromTheits unbound aptamer
complete mechanism stateofto activity
the pre-dissociation
for the 10–23activated complex—is
DNAzyme—from itsnow avail-
unbound
able as state
aptamer a theoretical framework activated
to the pre-dissociation for designing,
complex—isunderstanding,
now available and
as a optimizing
theoretical
DNAzymes. Like other catalysts, DNAzymes must perform multiple
framework for designing, understanding, and optimizing DNAzymes. Like other catalysts, tasks during each
catalytic cycle: substrate binding, catalysis, and product release
DNAzymes must perform multiple tasks during each catalytic cycle: substrate binding, [92]. Different regions of
the DNAzyme structure are typically responsible for each of these steps
catalysis, and product release [92]. Different regions of the DNAzyme structure are typically [93–95]. Most
DNAzymes for
responsible exhibit
eachtwo key features:
of these binding
steps [93–95]. armsDNAzymes
Most and a catalytic “motif”
exhibit two keyor active se-
features:
quence [1,96].
binding arms Figure 1a illustrates
and a catalytic the two-dimensional
“motif” or active sequence structure of a basic
[1,96]. Figure 1a RNA-cleaving
illustrates the
DNAzyme, with the
two-dimensional cleavage
structure of asite indicated
basic by an arrow.
RNA-cleaving The binding
DNAzyme, with the arms are repre-
cleavage site
sented by by
indicated thean 5′ arrow.
to 3′ strand, while the
The binding loop
arms areofrepresented
bases belowby the 5′ to 3′ strand,
structure
the shows the active
while the
catalytic sequence [21]. Binding arms are particularly adept at interacting
loop of bases below the structure shows the active catalytic sequence [21]. Binding arms with oligonu-
cleotide
are substrates,
particularly primarily
adept serving
at interacting to oligonucleotide
with “capture” and orient the substrate
substrates, primarilyinserving
the idealto
position for
“capture” and catalysis [1].substrate
orient the The catalytic
in themotifs, on the other
ideal position hand, are
for catalysis often
[1]. Theused as broader
catalytic motifs,
categories
on the other forhand,
comparing
are oftentheused
mechanisms
as broader of categories
different DNAzymes
for comparing [97].the
The binding arms
mechanisms of
surrounding
different each motif
DNAzymes can
[97]. Thebebinding
strategically
armsaltered to modify
surrounding eachthe
motifDNAzyme’s substrate
can be strategically
specificity
altered [32]. the DNAzyme’s substrate specificity [32].
to modify

Figure 1.
Figure 1. (a)
(a)The
Theinitial
initialstructure of of
structure ananRNA-cleaving
RNA-cleaving DNAzyme
DNAzyme [6]. [6].
(b) A(b)
more modern
A more render
modern of
ren-
the specific augments applied by Nguyen et al. in order to optimize 10–23 DNAzyme activity [98].
der of the specific augments applied by Nguyen et al. in order to optimize 10–23 DNAzyme
R-groups describe the chemical additions to the sugar backbone that create each respective
activity [98]. R-groups describe the chemical additions to the sugar backbone that create each re-
DNA/RNA/OMe/MOE monomer. (c) The innovative spatially and temporally resolved mechanism
spective DNA/RNA/OMe/MOE
of 10–23 DNAzyme activity proposedmonomer. (c) The
by Borggräfe et innovative spatially
al. [99]. (d) A andillustration
schematic temporallypublished
resolved
mechanism
by Borggräfe et al. Roman numerals show Sites I, II, and III [99]. Adapted from Ref. [6], Ref. illus-
of 10–23 DNAzyme activity proposed by Borggräfe et al. [99]. (d) A schematic [98],
tration
and Ref.published
[99] with by Borggräfe et al. Roman numerals show Sites I, II, and III [99]. Adapted from
permission.
Refs. [6,98,99] with permission.
Several variants of the RNA-cleaving 10–23 DNAzyme have been developed to im-
proveSeveral variants
catalytic of the RNA-cleaving
performance. 10–23 DNAzyme
Figure 1b illustrates have been developed
the two-dimensional structure of to im-
the
prove catalytic performance. Figure 1b illustrates the two-dimensional
most effective variant, Dz46. In this study, Nguyen et al. incorporated structure
2′-OMe, of the most
2′-MOE,
effective
LNA, andvariant, Dz46. In this
phosphorothioate study, Nguyen
modifications intoetDz46’s
al. incorporated 2′ -OMe,
catalytic core 2′ -MOE,
[98]. These LNA,
modifica-
and phosphorothioate modifications into Dz46’s catalytic core [98]. These modifications
tions enabled Dz46 to perform over sixty catalytic turnovers within 30 min in conditions
enabled Dz46 to perform over sixty catalytic turnovers within 30 min in conditions closely
resembling physiological environments. The authors further demonstrated that Dz46 is
Molecules 2024, 29, 5011 4 of 20

highly effective as an allele-specific gene-silencing agent, even against targets previously

considered undruggable.
The 10–23 DNAzyme exemplifies the use of SELEX or chemical evolution in the
informed optimization of catalysts. Figure 1c depicts the stepwise model provided by
Borggräfe et al., with the DNA catalyst and RNA substrate shown in red and black,
respectively [99]. To validate the accuracy and significance of this structural model,
Borggräfe et al. employed a “rationally selected” single-atom replacement mutation, which
led to a six-fold increase in catalytic activity. This simple yet strategic modification signifi-
cantly enhanced the DNAzyme’s performance, highlighting the potential of well-informed
design adjustments.
Many DNAzyme species rely heavily on cofactors to adopt proper global folding
patterns necessary for their active conformations [100,101]. Common cofactors include
metals such as lead, magnesium, sodium, potassium, and lithium, or combinations of
these [22]. Modifications to a DNAzyme can also affect its cofactor preferences, potentially
shifting from a dependency on two metals to a reliance on just one. Larger molecules like
hemin [59], serotonin [52], histidine [36], and even lanthanides [102] have also been found
to enhance catalytic activity.
Despite their importance, cofactors are challenging to study due to their weak and
dynamic interactions with DNA catalysts. However, recent advancements in DNAzyme
imaging, such as those by Borggräfe et al., have shed light on these interactions [99]. Specif-
ically, the roles and locations of cofactors within the 10–23 DNAzyme system have been
identified with a fair degree of accuracy. NMR titration studies have revealed a structure,
shown in Figure 1d, that highlights three metal-binding sites within the DNAzyme. The
ion at Site I reduces repulsion between the phosphate backbones in the binding arm re-
gion. The ion at Site II triggers “conformational activation” on the 5′ side of the catalytic
loop, while the ion at Site III aligns the 3′ side of the catalytic loop with the scissile bond,
promoting cleavage.
Furthermore, various environmental factors, such as temperature, solvent, and pH, can
be adjusted to influence DNAzyme activity [103–108]. For instance, Li et al. demonstrated
that adding butanol enhances NaA43T DNAzyme activity [103]. Other studies have shown
that DNAzymes can function effectively across a range of temperatures, from high to
room temperature [104–106]. Additionally, pH plays a crucial role in regulating DNAzyme
function [107], with some DNAzymes operating in highly acidic conditions even in the
absence of cofactors [108]. These environmental adjustments work because the correct
positioning of DNA’s active sites is essential for catalytic activation. As illustrated in
Figure 1c,d, the three-dimensional structure of the catalytic motif must be properly oriented
for a DNAzyme to function.
An intriguing 2022 study by Li et al. used a magnetic bead to cyclically toggle a
DNAzyme’s activity manually and repeatedly [109]. In the experimental design shown
in Figure 2a, a magnetic bead was attached to the binding region of either a lead- or
magnesium-dependent DNAzyme. By applying an upward magnetic field, the DNAzyme
was activated or deactivated continuously or intermittently. The resulting fluorophore-
labeled product detection provided the data shown in Figure 2b,c. This study highlights
that distortion of a DNAzyme’s active sequence can hinder or completely block catalytic
reactions. When the key bases of a DNAzyme are stretched into an inaccessible position,
substrate molecules are either unable to bind or unable to react as they would with an
undistorted DNA sequence.
Molecules 2024,
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Figure 2.
Figure 2. (a)
(a) Magnetic
Magnetic bead
bead set-up
set-up used
used by
by Li
Li et
et al.
al. to
to stretch
stretch the
the active
active site
site of
of aa DNAzyme
DNAzyme into
into an
an
inactive conformation [109]. (b) A graph representing the effect of magnetic field (MF) application
inactive conformation [109]. (b) A graph representing the effect of magnetic field (MF) application on
on the activity of catalysts. Products were modified with a fluorescent probe to track their formation
the activity of catalysts. Products were modified with a fluorescent probe to track their formation via
via intensity. (c) Alternate representation of activity and MF relationship per “cycle” MF of activa-
intensity. (c) Alternate
tion. Adapted from Ref.representation of activity and MF relationship per “cycle” MF of activation.
[109] with permission.
Adapted from Ref. [109] with permission.
Apart from external modifications, internal changes to DNA structures can be
Apart from external modifications, internal changes to DNA structures can be achieved
achieved through chemical evolution. These changes may include sequence mutations or
through chemical evolution. These changes may include sequence mutations or chemical
chemical modifications, such as introducing functional groups to the bases [61,66,92,110–
modifications, such as introducing functional groups to the bases [61,66,92,110–122]. The
122]. The synthesis of modified nucleoside triphosphates (dNTPs) has gained considera-
synthesis of modified nucleoside triphosphates (dNTPs) has gained considerable interest
ble interest due to its numerous advantages. Hollenstein described this approach as “ele-
due to its numerous advantages. Hollenstein described this approach as “elegant” for
gant” for enhancing DNA-based enzyme-mimicking catalysts [117]. Modifications to
enhancing DNA-based enzyme-mimicking catalysts [117]. Modifications to dNTPs can
dNTPs can involve alterations to the sugar, base, or phosphate groups of nucleotides.
involve alterations to the sugar, base, or phosphate groups of nucleotides. These enhance-
These enhancements are often designed to improve the properties of DNA catalysts or to
ments are often designed to improve the properties of DNA catalysts or to introduce new
introduce new functionalities, such as incorporating non-standard bases or sugar analogs
functionalities, such as incorporating non-standard bases or sugar analogs [123]. For exam-
[123].
ple, For example, phosphorothioate
phosphorothioate bonds, which replace bonds, which replaceoxygen
a non-bridging a non-bridging
atom in the oxygen atom
phosphate
in the phosphate backbone with sulfur, have
backbone with sulfur, have been used to increase stability. been used to increase stability.
G-quadruplex DNAzymes
G-quadruplex DNAzymes primarilyprimarily function
function as as peroxidase
peroxidase mimics
mimics and and exemplify
exemplify
sequence-dependent catalysis [124]. In a 2016 study by Li et
sequence-dependent catalysis [124]. In a 2016 study by Li et al., the guanine-rich DNAzyme al., the guanine-rich
DNAzyme
Dz-00 was Dz-00
modified wastomodified
assess the to assess
impactthe of impact
adjacent of adenine
adjacent on adenine on its catalytic
its catalytic activity.
activity. Figure 3a shows that adding adenine ′ to the 3′ end, forming
Figure 3a shows that adding adenine to the 3 end, forming Dz-11, significantly enhanced Dz-11, significantly
enhanced
activity, activity,
while Dz-14, while
with Dz-14, with ancytosine,
an adjacent adjacentshowed
cytosine, showed
a slight a slightlikely
increase, increase,
duelikely
to its
due to its chemical similarity to adenine. Li noted that adenine’s effect
chemical similarity to adenine. Li noted that adenine’s effect is similar to distal histidine is similar to distal
in
histidine inenzymes
peroxidase peroxidase enzymes
(Figure (Figurea spacer
3b). When 3b). When chaina was
spacer chain was
introduced introduced
between be-
adenine
tween
and adenine
Dz-11’s activeand Dz-11’s
site, activityactive site, activity
decreased decreased
dramatically, dramatically,
indicating that even indicating
small sequence that
even small
changes cansequence changesturnover
greatly improve can greatly improve turnover rates.
rates.
Despite the
Despite thelack
lackofofa fully
a fully resolved
resolved 3D 3D structure,
structure, sequence
sequence modifications
modifications have re-
have revealed
vealed the key roles of specific bases in DNAzyme function. Li et
the key roles of specific bases in DNAzyme function. Li et al.’s work suggests the unpro- al.’s work suggests the
unprotonated form of adenine’s N1 likely optimizes Dz-00’s performance.
tonated form of adenine’s N1 likely optimizes Dz-00’s performance. Their modifications Their modifi-
cationsthat
imply imply that
Dz-00 andDz-00
Dz-11 and Dz-11via
operate operate via acid–base
a general a general acid–base
mechanism, mechanism, a theory
a theory supported
supported
by by other
other studies studies
[125]. Beyond[125]. Beyond
refining refining
known known mechanisms,
mechanisms, sequence modifi-
sequence modifications also
cations
raise thealso raise the
potential for potential
altering afor altering a function
DNAzyme’s DNAzyme’s and function
its activity and its activity
conditions condi-
[126].
tionsThis
[126].versatility highlights the complexity of DNAzymes. In the realm of RNA-
cleavingThisDNAzymes,
versatility highlights the complexity
the 8–17 motif has drawnofattention
DNAzymes. In thethe
alongside realm of RNA-cleav-
well-known 10–23
ing DNAzymes, the 8–17 motif has drawn attention alongside the well-known 10–23
DNAzyme [127]. The NaA43 DNAzyme, introduced in 2015 by Lu et al., likely cleaves sub-
strates
DNAzyme via a[127].
general Thebase mechanism
NaA43 DNAzyme, (Figure 3c). Remarkably,
introduced in 2015 by MaLu et al.
et discovered that a
al., likely cleaves
single-point
substrates via mutation
a general in NaA43 produced NaH1,
base mechanism (Figurea 3c).
species that operates
Remarkably, Mathrough a general
et al. discovered
acid
that amechanism
single-point under different
mutation pH conditions
in NaA43 produced [113].
NaH1,Ma aetspecies
al. conducted point mutations
that operates through a
on a four-base
general segment ofunder
acid mechanism NaH1different
(Figure 3d),pH revealing
conditionsthe importance
[113]. Ma et al. ofconducted
specific bases in
point
its catalytic activity. They also demonstrated NaH1’s ability to match
mutations on a four-base segment of NaH1 (Figure 3d), revealing the importance of spe- NaA43’s cleavage rate
but
cificatbases
a much in higher pH, with
its catalytic only one
activity. basealso
They change near the catalytic
demonstrated NaH1’s sequence
ability(Figure 3e).
to match
NaA43’s cleavage rate but at a much higher pH, with only one base change near the cata-
lytic sequence (Figure 3e).
Molecules 2024, 29, x FOR PEER REVIEW 6 of 21
Molecules 2024, 29, 5011 6 of 20

Figure 3. (a) Modifications to the 3′ and 5′ ends of Dz-00 and the activity variation for the mutated
Figure
species3.(Dz-11
(a) Modifications toDecrease
to Dz-18). (b) the 3′ andin5′activity
ends ofdue
Dz-00 and the in
to increase activity
spacervariation forAthe
length. (c) mutated
mechanism
species (Dz-11 to Dz-18). (b)
for DNAzyme-catalyzed Decrease
cleavage in activity due to
of oligonucleotide increase in(d)
monomers. spacer length.
Single point(c) A mechanism
mutations to the
for DNAzyme-catalyzed cleavage of oligonucleotide monomers. (d) Single point mutations to the
four main catalytic bases of NaH1 (left) and their respective rates. (e) A comparison of NaA43T to
four main catalytic bases of NaH1 (left) and their respective rates. (e) A comparison of NaA43T to
NaH1 at different pH values, including other mutations showing less significant results. Adapted
NaH1 at different pH values, including other mutations showing less significant results. Adapted
from Refs.
from Refs. [113,124]
[113,124] with
with permission.
permission.

The introduction of functional groups to the active sites or adjacent bases of a DNAzyme
The introduction of functional groups to the active sites or adjacent bases of a
can significantly influence its catalytic behavior. In a recent study published by Zhang et al.,
DNAzyme can significantly influence its catalytic behavior. In a recent study published
researchers reported an impressive 700-fold increase in the efficiency of the 10–23 DNAzyme
by Zhang et al., researchers reported an impressive 700-fold increase in the efficiency of
after adding two small functional groups to its catalytic motif, generating the CaBn
the 10–23 DNAzyme after adding two small functional groups to its catalytic motif, gen-
species [128]. Carboxyl and benzyl groups were attached to positions 8 and 12 (Figure 4a),
erating the CaBn species [128]. Carboxyl and benzyl groups were attached to positions 8
selected based on insights from previous structural studies of the 10–23 DNAzyme. Figure 4b
and 12 (Figure
illustrates 4a), selected
the initial based onmade
modifications insights from
solely at previous
position 8,structural studies
chosen due of the
to the known10–
23 DNAzyme. Figure 4b illustrates the initial modifications made solely at position
tolerance of T8 to mutations [129,130]. Among the modifications, the carboxyl group (green 8, cho-
sen
linedue to the 4c)
in Figure known
had tolerance of T8 to mutations
the most substantial impact on [129,130].
DNAzyme Among the modifications,
activity. Interestingly,
the carboxyl group (green line in Figure 4c) had the most substantial
while the functional groups shown in Figure 4b exhibited significant individual impact on DNAzymeeffects,
activity.
Zhang’s Interestingly, while the
study demonstrated thatfunctional groups
the combined shown inofFigure
installation these 4b exhibited
groups signifi-
resulted in a
cant individual effects,
near-exponential Zhang’sofstudy
enhancement demonstrated
catalytic performance. that the combined installation of
theseBorggräfe
groups resulted in a near-exponential
et al. provided further insightsenhancement
suggesting of catalytic
that performance.
the CaBn species enhances
activity by increasing the number of magnesium ions available at the DNAzyme’s “Site II”,
thereby promoting more active conformations within the ensemble. Notably, this enhanced
ability to capture and interact with magnesium ions reduces the DNAzyme’s reliance on
high cofactor concentrations. Similar findings related to cofactor density have been reported
in other studies [131]. Zhang’s chemoenzymatic modification study parallels an earlier
optimization study by Nguyen et al., though Zhang’s work achieved a greater activity
boost through a simpler modification process. These findings reinforce the synergistic
nature of DNAzyme optimization and highlight the promising potential of this field.
Molecules
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Figure 4. (a) Red line shows the cleavage percentage of modified CaBn species compared to the
Figure 4. (a)DNAzyme
wild type Red line shows the cleavage
represented percentage
in black. of modified
Inset: specific CaBnand
locations species compared
structures to the wild
of modifications,
type DNAzyme represented in black. Inset: specific locations and structures of modifications, car-
carboxyl and benzyl groups. (b) Selection of two possible installations at position T8. (c) The
boxyl and benzyl groups. (b) Selection of two possible installations at position T8. (c) The compari-
comparison of each modification’s activity, with carboxyl (green) showing the best cleavage product
son of each modification’s activity, with carboxyl (green) showing the best cleavage product to time
to time relationship.
relationship. Adapted
Adapted from Ref. from
[128] Ref.
with[128] with permission.
permission.

Given the diverse reactions facilitated by known DNAzymes and the flexibility of
Borggräfe et al. provided further insights suggesting that the CaBn species enhances
single-stranded DNA aptamers, it may also be possible to expand DNAzyme applications
activity by increasing the number of magnesium ions available at the DNAzyme’s “Site
by substituting aptamers more suited to specific conditions. For instance, one challenge
II”, thereby promoting more active conformations within the ensemble. Notably, this en-
faced by many G-quadruplex catalysts is their inability to selectively recognize porphyrins,
hanced ability to capture and interact with magnesium ions reduces the DNAzyme’s reli-
along with their tendency to bind undesirable planar molecules [132–134]. Such behavior
ance on high cofactor concentrations. Similar findings related to cofactor density have
limits their usefulness in environments where non-reactive molecules could monopolize or
been reported in other studies [131]. Zhang’s chemoenzymatic modification study paral-
deactivate binding sites [135].
lels anInearlier
a 2024optimization
study, Gu et study by Nguyen
al. provided et al., though
an excellent exampleZhang’s
of aptamer work achieved aby
substitution
greater activity boost through a simpler modification process.
comparing two DNA catalysts: Hem1 and PS2.M. Hem1, which was developed using These findings reinforce the
synergistic
SELEX to nature of DNAzyme
specifically avoid formingoptimization and highlight
a G-quadruplex the promising
structure, potential
was confirmed of this
by multiple
field.
spectroscopic techniques to retain this property. As illustrated in Figure 5a, its activity
wasGiven the diverse
evaluated in the reactions
presence facilitated
of variousby known DNAzymes
cofactors. In contrast,and the flexibility
PS2.M, a classicalofG-
single-stranded DNA aptamers, it may also be possible to expand DNAzyme
quadruplex species, exhibits the common limitations of G-quadruplex catalysts, such as applications
byreduced
substituting aptamers
activity morecofactor
and distinct suited to specific conditions.
preferences, as shownFor instance,
in Figure 5b. one challenge
By substituting
faced by many G-quadruplex
the traditional G-quadruplexcatalysts
aptamer is theirwith
PS2.M inability to selectively
the rationally recognize
designed Hem1, porphy-
Gu et al.
rins, along with their tendency to bind undesirable planar molecules
successfully overcame the structural challenges of PS2.M and achieved a notable increase [132–134]. Such be-
havior limits their
in catalytic activity.usefulness in environments where non-reactive molecules could mo-
nopolizeTheor10–23
deactivate bindinghas
DNAzyme sites [135]. as a leading candidate in ushering the mod-
emerged
ernInera
a 2024 study, Gu
of rational et al. provided
optimization. an excellent
Other exampleare
DNA catalysts of following
aptamer substitution
closely behind,by
comparing
with studies twofocusing
DNA catalysts: Hem1 and
on the structural PS2.M. Hem1,
optimization of thewhich was developed
8–17 DNAzyme, using
peroxidase-
SELEX to specifically
mimicking DNAzymes, avoidandforming
various a G-quadruplex
others, yielding structure,
equallywas confirmed
impressive by multiple
results over the
spectroscopic techniquesWith
past decade [136–139]. to retain this property.
the availability As illustrated inthree-dimensional
of comprehensive Figure 5a, its activity was
structures
evaluated in the presence of various cofactors. In contrast, PS2.M, a classical
for more catalytic motifs, progress in this field is expected to accelerate significantly. G-quadruplex
species, exhibits such
Strategies the common
as in vitrolimitations
selection,of G-quadruplex
chemical evolution catalysts, such asenvironmental
technologies, reduced ac-
tivity and distinct
adjustments, cofactor
sequence preferences,functional
modifications, as showngroupin Figure 5b. By substituting
installations, nucleotide the tradi-
alterations,
tional G-quadruplex
mechanical aptamer PS2.M
manipulations, and aptamer with the rationally designed
substitutions have all Hem1, Gu et al.promising
demonstrated success-
fully overcame
results. the structural
However, challengestechniques
these optimization of PS2.M and areachieved
limited by a notable increase
the inherent in cat-
constraints
of DNA-dominant
alytic activity. catalysts. To address this, researchers have explored hybridizing DNA
with other materials, aiming for more substantial modifications and enhanced optimization.
Molecules
Molecules2024,
2024,29,
29,x5011
FOR PEER REVIEW 8 of 21
8 of 20

Figure
Figure5.5.(a)
(a)The
Theabsorbance
absorbanceandandtime
timerelationship
relationshipfor
forthe
theHem1
Hem1aptamer
aptamercomplex
complexwith
withsix
sixdifferent
different
cofactor
cofactor environments.
environments. TheThe sodium
sodiumandandmagnesium
magnesiumpairing
pairing vastly
vastly increases
increases activity
activity to roughly
to roughly dou-
double PS2.M’s
ble PS2.M’s highest
highest activity
activity withwith any cofactors.
any cofactors. (b) absorbance
(b) The The absorbance and relationship
and time time relationship for
for PS2.M
PS2.M
aptameraptamer complex
complex with
with six six different
different cofactor
cofactor environments.
environments. Potassium
Potassium shows
shows the most
the most prom-
promising
ising results but remains at roughly 0.3 a.u. Adapted from Ref. [135] with permission.
results but remains at roughly 0.3 a.u. Adapted from Ref. [135] with permission.

The 10–23 DNAzyme

3. DNA–Nanoparticle has emerged as a leading candidate in ushering the modern era
Hybrid
of rational optimization.
Nanozymes Other DNA
are nanoparticles withcatalysts are following
enzyme-like closely
properties, behind,
capable with studies
of converting vari-
focusing on the structural optimization of the 8–17 DNAzyme, peroxidase-mimicking
ous substrates into products. When combined with DNA, they form a new class of catalysts,
DNAzymes,
DNAzyme/nanozymeand various others,that
hybrids, yielding
provideequally
a highimpressive
surface area,results overcatalytic
multiple the past sites,
decadeen-
[136–139]. With the availability of comprehensive three-dimensional
hanced reaction specificity, and biocompatibility [91,140–143]. These hybrids often exhibit structures for more
catalytic
modifiedmotifs,
behavior progress
comparedin this
to field is expected
the parent to accelerate
nanozymes significantly.
or DNAzymes, as the nanoparticle
and Strategies
DNA componentssuch as in canvitro
serveselection, chemical during
distinct functions evolution technologies,
catalysis. environmental
This allows researchers
adjustments,
to independently sequence
optimizemodifications,
both components functional group installations,
for enhanced performance.nucleotide
Sometimes,altera-
a syn-
tions, mechanical manipulations, and aptamer substitutions have
ergistic effect occurs at the DNA–nanoparticle interfaces. DNA–nanoparticle hybrids canall demonstrated prom-
ising results. However,
be synthesized these optimization
through techniques techniqueslinkages
such as thiol-gold are limited
[144],by thechemistry
click inherent [145],
con-
straints of DNA-dominant
and noncovalent binding to catalysts. To address
the nitrogenous basesthis,
of researchers
DNA [62,146].have explored hybridiz-
ing DNA with other
A notable example materials, aiming for
of this approach more
was substantial by
demonstrated modifications
Shen and Mao, andwhoenhanced
linked
optimization.
DNA hairpins to gold nanospheres (AuNPs, Figure 6a) [62]. The DNA formed a corona-like
structure around the AuNP surface, leading them to name this catalyst the “coronazyme”.
3.Both
DNA–Nanoparticle
the original AuNPs Hybrid
and the resulting coronazyme function as peroxidase mimics,
catalyzing
Nanozymesthe oxidation of fluorogenic
are nanoparticles withamplex red in the
enzyme-like presencecapable
properties, of hydrogen peroxide.
of converting
However,
various the DNA-functionalized
substrates into products. When coronazyme
combined exhibited
with DNA,a five-fold increase
they form a new in catalytic
class of
efficiency compared to bare AuNPs (Figure 6b). Kinetic analyses
catalysts, DNAzyme/nanozyme hybrids, that provide a high surface area, multiple cata- and density functional
theory
lytic (DFT)
sites, calculations
enhanced reactionshowed that this
specificity, andimproved performance
biocompatibility stems from
[91,140–143]. These the strong
hybrids
interaction between the DNA and the substrate, enabling long-range
often exhibit modified behavior compared to the parent nanozymes or DNAzymes, as the catalysis exclusively
within the DNA
nanoparticle and corona. This interaction
DNA components transforms
can serve distinctindividual DNA bases
functions during into This
catalysis. reactive
al-
lows researchers to independently optimize both components for enhanced performance.is
sites, enhancing the coronazyme’s substrate selectivity. For instance, while resazurin
structurallyasimilar
Sometimes, to amplex
synergistic effect red,
occurs theatcoronazyme’s activity in interfaces.
the DNA–nanoparticle convertingDNA–nano-
resazurin is
significantly
particle hybridslower
can (Figure 6c), as the
be synthesized binding
through between such
techniques resazurin and the linkages
as thiol-gold coronazyme [144],is
weaker. This selective binding behavior allows the coronazyme
click chemistry [145], and noncovalent binding to the nitrogenous bases of DNA [62,146]. to mimic enzymes by
modulating its binding strength toward different substrates, depending on the surrounding
A notable example of this approach was demonstrated by Shen and Mao, who linked
DNA structure.
DNA hairpins to gold nanospheres (AuNPs, Figure 6a) [62]. The DNA formed a corona-
The authors further discovered that the catalytic performance of coronazymes is highly
like structure around the AuNP surface, leading them to name this catalyst the “corona-
dependent on the DNA sequence [147]. A regular DNA hairpin with randomized bases
zyme”. Both the original AuNPs and the resulting coronazyme function as peroxidase
showed higher activity compared to a GC-enriched hairpin (Figure 7a), as the latter tends
mimics, catalyzing the oxidation of fluorogenic amplex red in the presence of hydrogen
to immobilize charges within it. This finding suggests that DNA acts as a charge conduit,
peroxide. However, the DNA-functionalized coronazyme exhibited a five-fold increase in
and its charge-conducting capability directly influences the catalytic activity during redox
catalytic efficiency compared to bare AuNPs (Figure 6b). Kinetic analyses and density
reactions. Similar to DNA–hemin systems, internal charge transduction occurs within the
functional theory (DFT) calculations showed that this improved performance stems from
coronazyme, beginning with charge injection from the AuNP to the DNA hairpin. Since
the strong interaction between the DNA and the substrate, enabling long-range catalysis
the reaction substrates bind exclusively to the DNA bases, charges must transfer through
exclusively within the DNA corona. This interaction transforms individual DNA bases
the DNA strand to reach the bound substrate. Previous studies have shown that DNA
into reactive sites, enhancing the coronazyme’s substrate selectivity. For instance, while
ecules 2024, 29, x FOR PEER REVIEW 9 of 21

Molecules 2024, 29, 5011 9 of 20

resazurin is structurally similar to amplex red, the coronazyme’s activity in converting
resazurin is significantly lower (Figure 6c), as the binding between resazurin and the
coronazymecanis weaker.
transfer This selective
charges binding behavior
over distances allows
of tens of the coronazyme
nanometers, to mimic
but this charge conduction is
enzymes bysequence-dependent.
modulating its binding strength the
Therefore, toward different
activity of thesubstrates,
coronazyme depending
is closelyon
linked to the
the surrounding
DNADNA structure.
sequence.

Figure 6. (a) Figure

Schematic
6. (a)diagram of the
Schematic into coronazyme
diagram structure and
of the into coronazyme catalysis.
structure and (b) Activity
catalysis. (b)com-
Activity compari-
parison between bare 5 nm AuNPs and coronazymes for the oxidation of amplex red. Inset: Catalytic
son between bare 5 nm AuNPs and coronazymes for the oxidation of amplex red. Inset: Catalytic
efficiency of 5 nm AuNP compared to AuNP@DNA coronazyme. (c) Activity comparison between
efficiency of 5 nm AuNP compared to AuNP@DNA coronazyme. (c) Activity comparison between
bare 5 nm AuNPs and coronazymes for the reduction in resazurin. The coronazyme suppresses the
bare 5bynm
resazurin activity AuNPsits
blocking and coronazymes
access to AuNP. for the reduction
Adapted in [62]
from Ref. resazurin. The coronazyme suppresses the
with permission.
resazurin activity by blocking its access to AuNP. Adapted from Ref. [62] with permission.
The authors further discovered that the catalytic performance of coronazymes is
Additionally, because DNA is intrinsically chiral, electron spin is modulated as charges
highly dependent on the DNA sequence [147]. A regular DNA hairpin with randomized
pass through the DNA strand [148–150]. This recently discovered phenomenon is known as
bases showed higher activity compared to a GC-enriched hairpin (Figure 7a), as the latter
chiral-induced spin selectivity (CISS) [151,152]. The CISS effect suggests that DNA strands
tends to immobilize charges within it. This finding suggests that DNA acts as a charge
can act as both electron spin inducers and filters when attached to nanoparticles. The chiral
conduit, and its charge-conducting capability directly influences the catalytic activity dur-
structure of DNA, along with its preference for specific electron spins, can be leveraged
ing redox reactions. Similar to DNA–hemin systems, internal charge transduction occurs
to enhance the overall catalytic performance of DNA-based catalysts. This interpretation
within the coronazyme, beginning with charge injection from the AuNP to the DNA hair-
was supported by Shen and Mao’s findings, where they observed that DNA-wrapped
pin. Since the reaction substrates
coronazymes respondedbindtoexclusively to the DNA
external magnetic bases,
fields, charges spins
as electron must transfer
were aligned to the
through thefield
DNAdirection
strand toatreach the bound substrate. Previous studies
the Au-DNA interface (Figure 7b) [147]. Moreover, have shown that polarized
circularly
DNA can transfer charges over distances of tens of nanometers, but this charge conduction
light (CPL) can generate electron spin polarization at this interface. Notably, when exposed
is sequence-dependent.
to right-hand Therefore,
circularlythe activity of
polarized the(RHCP),
light coronazyme
whichis closely
matcheslinked to the of DNA,
the chirality
DNA sequence.the coronazyme exhibited consistently higher activity compared to left-hand circularly
polarized light (LHCP) (Figure 7c). Beyond utilizing magnetic fields and CPLs to modulate
coronazyme performance, the researchers also applied mechanical forces to stretch the DNA
hairpin during catalysis. The coronazyme’s activity responded to these force stimuli, as the
change in DNA conformation altered its chirality and, consequently, its charge-conduction
capabilities (Figure 7d). These discoveries demonstrate that DNA-based hybrid catalysts
can respond to various external stimuli, resulting in modulated catalytic performance.
The peroxidase-like activity of DNA-AuNP hybrids was also explored by Hizir et al.,
who capped AuNPs with single-stranded DNA (ssDNA) [78]. They found that the ssDNA-
AuNP system exhibited significantly enhanced TMB oxidation as the negatively charged
phosphate backbone of the ssDNA facilitated the electrostatic attraction of the substrate.
This highlights DNA’s ability to enhance catalysis by promoting substrate adsorption.
Similarly, Chen et al. synthesized ssDNA-encoded gold nanoparticle clusters (GNCs)
as programmable enzyme equivalents (PEEs) (Figure 8a) [153]. These ssDNA scaffolds
assemble into folded nanostructures with polyadenine (polyA) loops and double-stranded
stems acting as nucleation sites, leading to increased binding affinity for reaction substrates.
DNA’s intrinsic chirality has also been harnessed for substrate recognition. Recently,
Ouyang et al. modified DNA into a dopamine-binding aptamer (DBA) and conjugated
it to polyadenine-stabilized Au nanoparticles (pA-AuNPs) to create an aptananozyme
(Figure 8b) [154]. This aptananozyme catalyzed H2 O2 -mediated dopamine oxidation to
aminochrome through the aerobic oxidation of glucose. Compared to separate nanozyme/
aptamer units, the aptananozyme showed a 10-fold increase in dopamine oxidation by
H2 O2 and a 13-fold increase in the presence of glucose. This remarkable enhancement
 Molecules 2024, 29, 5011 10 of 20

Molecules 2024, 29, x FOR PEER REVIEW 10 of 21

was attributed to the concentration of dopamine at the catalytic interfaces, facilitated by
chiral-selective aptamer–dopamine binding.

Figure 7. (a) Comparison of the coronazyme activity between the randomized based pair sequence
Figure 7. (a) Comparison of the coronazyme activity between the randomized based pair sequence
and a GC-enriched sequence. (b) Magnetic field orientation-dependent coronazyme reactivity. (c) Top:
and a GC-enriched sequence. (b) Magnetic field orientation-dependent coronazyme reactivity. (c)
activity
Top: ratioratio
activity for individual coronazymes
for individual coronazymesunderunder
RHCPRHCP
and LHCP radiation.
and LHCP Bottom:
radiation. averaged
Bottom: activity
averaged
ratio (RHCP/LHCP) under various tensile force conditions. (d) Calculated activity polarization
activity ratio (RHCP/LHCP) under various tensile force conditions. (d) Calculated activity polariza- based
on the RHCP/LHCP ratio shown in (c). Top: activity polarization under 10.2 pN. Bottom:
tion based on the RHCP/LHCP ratio shown in (c). Top: activity polarization under 10.2 pN. Bottom: activity
activity ratio 0.6
ratio under under
pN.0.6 pN. Adapted
Adapted from
from Ref. Ref.with
[147] [147] with permission.
permission.

Similarly, Zhan
Additionally, et al. reported
because DNA isthe use of DNA-capped
intrinsically AuNPsspin
chiral, electron as chiral-selective
is modulatednano as
catalysts for glucose oxidase-mimicking reactions [155]. Uncapped
charges pass through the DNA strand [148–150]. This recently discovered phenomenon AuNPs showed is no
preference for glucose enantiomers, indicating that the chirality preference
known as chiral-induced spin selectivity (CISS) [151,152]. The CISS effect suggests that was introduced
exclusively
DNA strandsthrough
can act asthe addition
both of DNA.
electron The ssDNA-AuNPs
spin inducers and filters when displayed
attached higher activity
to nanopar-
for L-glucose
ticles. The chiral than D-glucose,
structure due to
of DNA, the stronger
along interaction for
with its preference with L-glucose,
specific which
electron was
spins,
likely driven by its stereo orientation preference. However, the
can be leveraged to enhance the overall catalytic performance of DNA-based catalysts.dsDNA-AuNPs, i-motif-
AuNPs,
This and G-quadruplex
interpretation AuNPs
was supported byshowed
Shen and more preference
Mao’s findings,forwhere
the D-glucose (Figure
they observed 8c).
that
Collectively, these studies underscore DNA’s potential in catalyst
DNA-wrapped coronazymes responded to external magnetic fields, as electron spins design, as it can effectively
adsorb
were reaction
aligned substrates,
to the enrich reactants
field direction at catalytic
at the Au-DNA surfaces,
interface and 7b)
(Figure introduce chirality for
[147]. Moreover,
selective reactions.
circularly polarized light (CPL) can generate electron spin polarization at this interface.
Notably,It iswhen
important to note
exposed that DNA–nanozyme
to right-hand hybrids are
circularly polarized lightnot(RHCP),
limited which
to AuNPs; other
matches
metals,
the suchofasDNA,
chirality copperthe(Cu), iron (Fe),exhibited
coronazyme and zincconsistently
(Zn), are also commonly
higher used.
activity For exam-
compared to
left-hand circularly polarized light (LHCP) (Figure 7c). Beyond utilizing magnetic fields
and CPLs to modulate coronazyme performance, the researchers also applied mechanical
Molecules 2024, 29, 5011 11 of 20

ple, Fu et al. reported platinum (Pt) nanozymes synthesized using G-C rich nucleation
centers [80], while Wei et al. developed DNA-based FeCuAg nanoclusters [156]. Both of
these DNA-nanoparticle hybrids demonstrated peroxidase-like activities. Additionally,
Liu et al. designed Cu-DNAzyme nanohybrids for delivering DNAzymes and Cu2+ into
cancer cells for combined catalytic therapy [157]. These nanohybrids exhibited enhanced
cell membrane permeability and excellent loading capacity (Figure 8d). The catalytic
10–23 DNAzyme in the nanohybrids cleaved human vascular endothelial growth factor-2
(VEGFR2) mRNA, leading to gene silencing. Simultaneously, the glutathione-induced
Molecules 2024, 29, x FOR PEER REVIEW 12 of 21
reduction of Cu2+ to Cu+ catalyzed the conversion of endogenous H2 O2 into cytotoxic
hydroxyl radicals, enabling dual-catalytic tumor therapy.

Figure 8.
Figure 8. (a)
(a)Schematics
Schematicsfor forssssDNA
DNA scaffold programmed
scaffold programmed Gold Nanoparticle
Gold NanoparticleClusters (GNCs)
Clusters for
(GNCs)
their peroxidase activity. (b) Schematics of the DNA aptamer-modified Au nanoparticles
for their peroxidase activity. (b) Schematics of the DNA aptamer-modified Au nanoparticles for for dual
catalytic activity of H2O2 mediated dopamine oxidation and aerobic oxidation of glucose for chemo-
dual catalytic activity of H2 O2 mediated dopamine oxidation and aerobic oxidation of glucose for
dynamic cancer treatment. (c) DNA concentration-dependent chiral selective catalysis of the AuNPs
chemodynamic cancer treatment. (c) DNA concentration-dependent chiral selective catalysis of the
with ssDNA (top left), dsDNA (top right), imotif (bottom left), and G-quadruplex (bottom right). (d)
AuNPs withfor
Schematics ssDNA
ss DNA(topscaffolds
left), dsDNA (top right),
programmed goldimotif (bottom clusters
nanoparticle left), and(GNCs)
G-quadruplex
for their(bottom
peroxi-
right). (d) Schematics
dase activity. Adaptedfor ss DNA
from Refs.scaffolds programmed
[153–155,157] gold nanoparticle clusters (GNCs) for their
with permission.
peroxidase activity. Adapted from Refs. [153–155,157] with permission.
It is also common to hybridize DNA with metal oxides, such as Fe3O4 and TiO2
It
[77,158].is also
The common
rationaleto hybridize
behind DNA with
DNA–metal metalhybrids
oxide oxides,issuch as Feto
similar 3 Othat
4 and
ofTiO 2 [77,158].
DNA–metal
The rationale behind DNA–metal oxide hybrids is similar to that of DNA–metal catalysts:
catalysts: the presence of DNA enhances substrate interaction, thereby improving the
the presence of DNA enhances substrate interaction, thereby improving the overall cat-
overall catalytic activity. For example, Zhang demonstrated the use of DNA with iron co-
alytic activity. For example, Zhang demonstrated the use of DNA with iron cobalt oxide
balt oxide nanosheets (FeCo-ONs) as a peroxidase mimic [159]. In a study by Liu et al.,
nanosheets (FeCo-ONs) as a peroxidase mimic [159]. In a study by Liu et al., DNA-
DNA-capped Fe3O4 nanoparticles showed a roughly 10-fold increase in activity compared
capped Fe3 O4 nanoparticles showed a roughly 10-fold increase in activity compared to
to bare nanoparticles (Figure 9a) [82]. The DNA ligands outperformed negatively charged
bare nanoparticles (Figure 9a) [82]. The DNA ligands outperformed negatively charged
polymers, such as polyacrylic acid (PAA) and polystyrene sulfonate (PSS), in nanoparticle
polymers, such as polyacrylic acid (PAA) and polystyrene sulfonate (PSS), in nanoparticle
modification, underscoring DNA’s superior substrate interactions. Beyond their nega-
modification, underscoring DNA’s superior substrate interactions. Beyond their negatively
tively charged backbones, DNA molecules feature additional hydrogen bonding capabil-
charged backbones, DNA molecules feature additional hydrogen bonding capabilities and
ities and π-π stacking interactions with substrates, significantly enhancing their binding
π-π stacking interactions with substrates, significantly enhancing their binding properties.
properties. In a study conducted by Zhang and his colleagues, it was found that the pe-
In a study conducted by Zhang and his colleagues, it was found that the peroxidase-like
roxidase-like
activity activity O
of ssDNA-Fe of ssDNA-Fe3O4 was doubled, dsDNA-Fe3O4 exhibited a 4.6-fold in-
3 4 was doubled, dsDNA-Fe3 O4 exhibited a 4.6-fold increase, hairpin
crease, hairpin DNA-NP
DNA-NP demonstrated an demonstrated an 8-fold
8-fold increase, and increase, and the hybridization
the hybridization chain reaction chain re-
(HCR)
action (HCR) H-DNA-Fe 3O4 revealed a 13-fold enhancement compared to bare Fe3O4 (Fig-
H-DNA-Fe3 O4 revealed a 13-fold enhancement compared to bare Fe3 O4 (Figure 9b) [77].
ure 9b) [77]. The variations in DNA binding to the nanoparticles, attributed to different
surface coverage, explain these differences in activity enhancement.
Molecules 2024, 29, 5011 12 of 20

Molecules 2024, 29, x FOR PEER REVIEW 13 of 21

The variations in DNA binding to the nanoparticles, attributed to different surface coverage,
explain these differences in activity enhancement.

Figure 9. (a)
(a)Activity
Activitycomparison
comparisonofofbare
bareFeFe
3O34O
, with Fe3Fe
4 , with O43 O
modified withwith
4 modified DNA, polystyrene
DNA, sul-
polystyrene
fonate (PSS), and polyacrylic acid (PAA). (b) Relative catalytic rate for TMB oxidation
sulfonate (PSS), and polyacrylic acid (PAA). (b) Relative catalytic rate for TMB oxidation for for
Fe 3O4/dsDNA, Fe3O4/ssDNA, Fe3O4/H-DNA, and Fe3O4/HCR. Adapted from Refs. [77,82] with per-
Fe3 O4 /dsDNA, Fe3 O4 /ssDNA, Fe3 O4 /H-DNA, and Fe3 O4 /HCR. Adapted from Refs. [77,82]
mission.
with permission.

4. DNA–Carbon Hybrids
DNA can also be hybridized with various carbon materials to enhance their catalytic
properties. Carbon-based
Carbon-based materials,
materials, such
such as carbon dots [160] and graphene [161], have have
previously been explored for their high efficiency, efficiency, large
large surface
surface area,
area, and
and versatility
versatility in
different
different forms,
forms, making
making themthem ideal
ideal candidates
candidates for for hybridization
hybridization with with DNA
DNA [162,163].
[162,163].
In a study by Qu et al., a DNA-modified graphene/Pd nanoparticle
DNA-modified graphene/Pd nanoparticle hybrid hybrid (DNA-G-
(DNA-G-
Pd) was wasassembled
assembled for formic
for formic acid electro-oxidation
acid electro-oxidation and the
and the Suzuki Suzuki
reaction reaction
(Figure 10a)
(Figure 10a) [164].
[164]. These hybrids These hybrids demonstrated
demonstrated higher
higher catalytic catalytic
activity, activity, lifespan,
extended extendedand lifespan,
easy
and easy recyclability.
recyclability. Cyclic voltammetry
Cyclic voltammetry (CV) analysis
(CV) analysis revealed revealed
that thethat the mass-normalized
mass-normalized peak
peak current for DNA-G-Pd was 2.5 times higher than PVP-G-Pd
current for DNA-G-Pd was 2.5 times higher than PVP-G-Pd and approximately 3.5 times and approximately
3.5 times
better thanbetter
Pd/C,than Pd/C, highlighting
highlighting the crucial role theof
crucial
DNA role of DNA in electrochemical
in electrochemical applications (Fig- ap-
plications (Figure 10b). The DNA lattice not only distributed active
ure 10b). The DNA lattice not only distributed active sites evenly but also depleted oxygensites evenly but also
depleted oxygenpreventing
in the solution, in the solution, preventing
Pd from formingPd from forming
passive PdO and passive PdO and
supporting supporting
formic acid in-
formic acid intermediates
termediates to promote
to promote catalysis. catalysis.study
In another In another
by Das study
and byhisDas and his coworkers,
coworkers, a G-quad-
aruplex/hemin
G-quadruplex/hemin network crosslinked
network crosslinked by carbonby carbon quantum
quantum dots showeddots increased
showed increased
catalytic
catalytic
activity and enhanced stability (Figure 10c) [165]. The carbon dots (CDs)(CDs)
activity and enhanced stability (Figure 10c) [165]. The carbon dots interacted
interacted non-
non-covalently
covalently withwith the hemin/GQ
the hemin/GQ network,
network, facilitating
facilitating electron
electron transfer
transfer through
through keto keto
car-
carbonyl functional
bonyl functional groups
groups andand creatinga aconfined
creating confinedyetyetbeneficial
beneficialmicroenvironment
microenvironment for for the
the
ABTS
ABTS oxidation
oxidation reaction,
reaction, leading
leading toto aa faster
faster catalytic
catalytic rate.
rate.
Li
Li et
et al.
al. developed
developed aa nanocomposite
nanocomposite composedcomposed of of platinum,
platinum, ssDNA,
ssDNA, and and reduced
reduced
graphene oxide (ssDNA-RGO/cf-Pt), which exhibited 2.5 times greater catalytic activity
graphene oxide (ssDNA-RGO/cf-Pt), which exhibited 2.5 times greater catalytic activity
than RGO-Pt and 3.8 times higher activity than regular Pt nanoparticles for methanol
than RGO-Pt and 3.8 times higher activity than regular Pt nanoparticles for methanol ox-
oxidation [166]. In methanol oxidation, CO adsorption typically poisons the catalytic
idation [166]. In methanol oxidation, CO adsorption typically poisons the catalytic effi-
efficiency, but the additional oxygen groups in the ssDNA and residual oxygen species
ciency, but the additional oxygen groups in the ssDNA and residual oxygen species in the
in the RGO enhanced CO oxidation, providing resistance to CO poisoning. The anti-
RGO enhanced CO oxidation, providing resistance to CO poisoning. The anti-poisoning
poisoning ratio was 1.75 times higher than that of RGO/Pt and Pt nanoparticles. These
ratio was 1.75 times higher than that of RGO/Pt and Pt nanoparticles. These studies high-
studies highlight the diverse role of DNA in generating superior catalysts and enhancing
light the diverse role of DNA in generating superior catalysts and enhancing catalytic per-
catalytic performance.
formance.
Molecules 2024, 29, x FOR PEER REVIEW 14 of 21

Molecules 2024, 29, x FOR PEER REVIEW 14 of 21

Molecules 2024, 29, 5011 13 of 20

Figure 10. (a) Schematic of DNA-modified Graphene/Pd NPs (DNA-G-Pd) synthesis. (b) Cyclic volt-
ammogram for oxidation of formic acid (c) Mechanism for CD–G quadruplex–hemin nanonetwork
interaction for DNAzyme activity enhancement. Adapted from Refs. [164,165] with permission.
Figure 10.
Figure (a)Schematic
10.(a) Schematicofof DNA-modified
DNA-modified Graphene/Pd
Graphene/Pd NPsNPs (DNA-G-Pd)
(DNA-G-Pd) synthesis.
synthesis. (b) Cyclic
(b) Cyclic volt-
5. DNA–Soft
ammogram for Matter Catalysts
oxidation of formic acid (c) Mechanism for CD–G quadruplex–hemin nanonetwork
voltammogram for oxidation of formic acid (c) Mechanism for CD–G quadruplex–hemin nanonetwork
interaction
interaction for
for DNAzyme
DNAzyme
It is common activity
activity
practice enhancement.
enhancement.
to hybridize Adapted
Adapted from
DNAzymes withRefs.
from Refs. [164,165]
[164,165]
natural with
with permission.
permission.
biopolymers like pep-
tides and synthetic
5. DNA–Soft
DNA–Soft Matterpolymers
Catalyststo create hybrid catalysts [167,168]. For instance, Ding and
5. Matter Catalysts
his colleagues synthesized a DNA/peptide nanoparticle that exhibited enhanced peroxi-
It is
It is common
common practice
practiceto hybridize
hybridizeDNAzymes with natural biopolymers likelike
peptides
dase-like activity through atosynergistic DNAzymes
mechanism with
[169]. natural biopolymers
The incorporation pep-
of histidine
and
tides synthetic
andfrom polymers
synthetic to
polymers create hybrid catalysts
to createhydrogen
hybrid catalysts [167,168]. For
[167,168]. instance,
Forthe Ding
instance, and his
residues the peptides facilitates bonding, mimicking role ofDingdistalandar-
colleagues
his colleagues synthesized a DNA/peptide nanoparticle that exhibited enhanced peroxidase-
ginine residuessynthesized
found in naturala DNA/peptide
peroxidases. nanoparticle that exhibited
This interaction stabilizesenhanced
the heminperoxi-aggre-
like activity
dase-like through
activity a synergistic
through mechanism [169]. The
a synergistic incorporation of histidine residues
gates on the guanine quartet of the DNAmechanism
framework,[169]. The the
enabling incorporation of histidine
hybrid to demonstrate
from the peptides facilitates hydrogen bonding, mimicking the role of distal arginine
residues
superior from the peptidesproperties.
peroxidase-like facilitates hydrogen
The observed bonding, mimicking
synergistic the role
catalytic of distal
behavior stemsar-
residues found in natural peroxidases. This interaction stabilizes the hemin aggregates on
ginine
from residues found
the complementary in natural peroxidases. This interaction stabilizes the hemin aggre-
the guanine quartet of thechemical and structural
DNA framework, features
enabling of the peptides
the hybrid and DNA
to demonstrate com-
superior
gates on the guanine quartet of the DNA framework, enabling the hybrid to demonstrate
ponents.
peroxidase-like properties. The observed synergistic catalytic behavior stems from the
superior peroxidase-like
Similarly, Xiang andproperties.
his structuralThe observed
team reported synergistic
thatofcationic peptide catalytic behavior stems
complementary chemical and features the peptides andconjugates covalently
DNA components.
from
linked the complementary
to DNAzymes chemical
resulted and structural
in areported
catalytically features of
active DNA-peptide the peptides
conjugateand DNA
(Figure com-
11a)
Similarly, Xiang and his team that cationic peptide conjugates covalently linked
ponents.
[170]. This conjugate exhibited increased peroxidase and oxidase activities, by up to four-
to DNAzymes resulted in a catalytically active DNA-peptide conjugate (Figure 11a) [170].
fold Similarly,
This and
conjugate
Xiang
threefold,
exhibited
and his team
respectively.
increasedThe
reported that cationic
enhancement
peroxidase
peptide conjugates
was attributed
and oxidase activities,tobythe
covalently
up electrostatic
to fourfold and in-
linked
teractionto DNAzymes
between the resulted
peptides inand
a catalytically
DNA active DNA-peptide
phosphates, as well as the conjugate
π–π stacking (Figure
between11a)
threefold, respectively. The enhancement was attributed to the electrostatic interaction between
[170]. Thisand
histidine conjugate
DNA exhibited increased
nucleobases. These peroxidase stabilized
interactions and oxidase theactivities,
parallel by upG-quad-
DNA to four-
the peptides and DNA phosphates, as well as the π–π stacking between histidine and DNA
fold and
ruplex threefold,
structures
nucleobases. Theseand respectively.
promoted hemin
interactions
The enhancement
binding.
stabilized
was
Wang further
the parallel
attributed to the electrostatic
demonstratedstructures
DNA G-quadruplex the engineer- in-
and
teraction between the
ing of a peroxidase-mimicking
promoted peptides
hemin binding. Wang and DNA phosphates,
nanoparticle, as
utilizing hemin
further demonstrated well as the π–π
the engineering stacking
encapsulatedofbetween between
DNA
a peroxidase-
histidine and
G-quadruplexes DNA and nucleobases.
lysine-rich These
peptidesinteractions
(Figure stabilized
11b) [171]. the
This
mimicking nanoparticle, utilizing hemin encapsulated between DNA G-quadruplexes and parallel
scaffolded DNA G-quad-
architecture
ruplex
resulted structures
lysine-rich in peptidesand(Figure
enhanced promoted 11b)hemin
peroxidase-like binding.
[171].activity Wang
due
This scaffolded further
to the demonstrated
increased
architecture resultedthe
substrate in engineer-
binding
enhanced ca-
ing of
pacity. a peroxidase-mimicking nanoparticle, utilizing hemin
peroxidase-like activity due to the increased substrate binding capacity. encapsulated between DNA
G-quadruplexes and lysine-rich peptides (Figure 11b) [171]. This scaffolded architecture
resulted in enhanced peroxidase-like activity due to the increased substrate binding ca-
pacity.

Figure 11. (a)

Figure 11. (a) Schematics
Schematics of
of the
the DNA-peptide conjugate hybrid
DNA-peptide conjugate hybrid formation.
formation. (b)
(b) Activity
Activity comparison
comparison
among peptide–DNA/hemin hybrid, G–DNA/hemin hybrid and peptide/hemin
among peptide–DNA/hemin hybrid, G–DNA/hemin hybrid and peptide/hemin hybrid for TMB
hybrid ox-
for TMB
idation. Adapted from Refs. [170,171] with permission.
oxidation. Adapted from Refs. [170,171] with permission.
Figure 11. (a) Schematics of the DNA-peptide conjugate hybrid formation. (b) Activity comparison
6. Conclusions
among peptide–DNA/hemin hybrid, G–DNA/hemin hybrid and peptide/hemin hybrid for TMB ox-
idation. Adapted from
Significant Refs. [170,171]
progress has beenwith permission.
made in DNA-based catalysts over the years. Initially
a theoretical concept in the 1960s, catalytic nucleic acids have evolved into a versatile
Molecules 2024, 29, 5011 14 of 20

class of multifunctional catalysts. Recent advancements have generated various DNA-

based catalysts, incorporating structural, conformational, and chemical modifications,
aptamer engineering, and hybridization with metals, polymers, and biopolymers. These
improvements have resulted in catalysts with higher stability, enhanced performance,
better efficiency, chiral selectivity, and catalytic modulation via light, magnetic fields,
or mechanical force. This progress offers enormous potential for DNA-based catalysts
to address the limitations associated with natural enzymes. Further research into the
dynamics and functional mechanisms of non-hybridized DNAzymes will help unlock the
full potential of DNA-dominant catalysts and strengthen the foundation for optimizing
DNA hybrid catalysts.
The selection of new DNAzymes via in vitro selection and chemical evolution is well-
documented and has been enriched by advances in three-dimensional structure-resolution
technologies. Understanding DNA’s activity across pH, temperature, solvent, cofactor, and
substrate variations has been extensively reviewed. Mutation studies have identified key
bases that must be conserved for catalytic activity. These insights, combined with spatially
resolved, catalytically active DNAzyme structures, have revealed unexpected mechanisms.
Simple functional modifications have improved DNAzyme activity up to 700-fold.
DNA’s biocompatibility makes it ideal for hybridization with other catalysts, com-
bining catalytic traits to create superior hybrid catalysts. DNAzyme-hybrid catalysts,
particularly those with metallic nanoparticles, represent a significant advancement due
to their enhanced properties and versatility. Gold nanoparticles, for instance, conjugated
with DNAzymes, can serve as sensitive biosensors or effective catalysts in redox reactions.
These hybrids leverage the selective nature of DNAzymes and the catalytic and optical
properties of nanoparticles. Incorporating other materials can further improve stability
and catalytic activity, making DNAzyme hybrids suitable for challenging chemical environ-
ments. The precise control over nanoparticle size, shape, and composition offers another
layer of optimization for DNA hybrid catalysts, enhancing performance in fields like envi-
ronmental sensing, diagnostics, and drug delivery. The synergy between DNAzymes and
nanoparticles is expected to drive future advancements in these areas.
Furthermore, hybrid systems like DNA@AuNPs provide insights into fundamental
concepts like chirality and charge transfer, offering new opportunities to understand DNA
catalysis. DNA’s substrate-binding affinity and charge transfer are crucial to its catalytic
performance, and these factors are influenced by DNA sequence and conformational
flexibility. Increasing active sites or modulating DNA flexibility could optimize binding
and catalysis, and altering DNA length, base sequence, or conformational dynamics can
enhance efficiency.
Despite groundbreaking discoveries, some aspects of DNA catalysis remain poorly
understood, and hybridization introduces additional complexity. Emerging single-molecule
techniques, such as single-particle force spectroscopy and single-molecule fluorescence
microscopy, offer unprecedented insights at high resolution. Computational approaches,
integrated with artificial intelligence and machine learning, may also play a key role in
decoding catalytic mechanisms. Future research will focus on the application of DNAzymes
in complex biological environments and industrial processes. Improved screening and
selection methods will enable the discovery and refinement of these catalytic tools for
practical use. DNA’s structural flexibility, sequence programmability, and capacity for
hybridization ensure that these catalysts will continue to drive innovation across fields,
from diagnostics to therapeutic interventions.

Funding: This research was funded by Kent State University’s Farris Family Innovation Award and
National Science Foundation (NSF) (No. CHE2247709).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Molecules 2024, 29, 5011 15 of 20

Acknowledgments: The authors thank Kent State University’s Farris Family Innovation Award for
financial support. H.S. acknowledges the National Science Foundation (NSF) (No. CHE2247709) for
grant support.
Conflicts of Interest: The authors declare no conflict of interest.

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