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Toehold-mediated DNA circuits are extensively employed to construct diverse DNA nanodevices and signal amplifiers. However, operations of these circuits are slow and highly susceptive to molecular noise such as the interference from bystander DNA strands. Herein, this work investigates the effects of a series of cationic copolymers on DNA catalytic hairpin assembly, a representative toehold-mediated DNA circuit. One copolymer, poly( L -lysine)-graft-dextran, significantly enhances the reaction rate by 30-fold due to its electrostatic interaction with DNA. Moreover, the copolymer considerably alleviates the circuit's dependency on the length and GC content of toehold, thereby enhancing the robustness of circuit operation against molecular noise. The general effectiveness of poly( L -lysine)-graft-dextran is demonstrated through kinetic characterization of a DNA AND logic circuit. Therefore, use of a cationic copolymer is a versatile and efficient approach to enhance the operation rate and robustness of toehold-mediated DNA circuits, paving the way for more flexible design and broader application., (© 2023 The Authors. Small published by Wiley-VCH GmbH.)

Nanosheets have thicknesses on the order of nanometers and planar dimensions in the micrometer range. Nanomaterials that are capable of converting reversibly between 2D nanosheets and 3D structures in response to specific triggers can enable construction of nanodevices. Supra-molecular lipid nanosheets and their triggered conversions to 3D structures including vesicles and cups are reported. They are produced from lipid vesicles upon addition of amphiphilic peptides and cationic copolymers that act as peptide chaperones. By regulation of the chaperoning activity of the copolymer, 2D to 3D conversions are reversibly triggered, allowing tuning of lipid bilayer structures and functionalities., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Control of protein conformation and function, induced by the binding of an effector, plays significant roles in modulating biochemical reaction. Although the DNAzymes catalytic activity is similar to protein-based enzymes, reports of allosterically controlled DNAzymes are still limited except for aptamer-DNAzymes hybrrids. Here, we report allosteric control of peroxidase-mimicking DNAzyme activity using cationic copolymers. The DNAzyme requires a structured G-quadruplex core and hemin for activity, and the DNAzyme with a parallel G-quadruplex core has higher DNAzyme activity than DNAzymes based on other types of structure. We previously reported that a cationic copolymer composed of a cationic backbone and hydrophilic dextran side chains selectively stabilizes parallel G-quadruplex structures. In this study, we investigated effects of the cationic copolymer on peroxidase-mimicking DNAzyme activity. The cationic copolymer enhanced the DNAzyme activity by more than 30-fold by stabilizing the parallel G-quadruplex structure. Furthermore, reversible allosteric control of DNAzyme activity was achieved by adding cationic and anionic polymers.

Photodriven DNA strand displacement by using a 2',6'-dimethylazobenzene-tethered strand and poly(l-lysine)-graft-dextran (PLL-g-Dex) as a chaperone is reported. Rapid strand displacement was reversibly induced by UV and visible-light irradiation without any toehold portion. To further improve the method, the concentration of PLL-g-Dex and the number of equivalents of the photoresponsive strand were optimised. Optimally, 64 % strand displacement was reversibly induced by alternating UV and visible-light irradiation., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

DNA sequences capable of forming triplexes induce DNA double-strand breaks that have attracted attention in genome editing technologies (e.g., CRISPR/Cas9 system, TALEN, and ZFN). Therefore, novel functional tools that stabilize triplex DNA structures must be further investigated to spark renewed interest. In this study, we investigated the unique character of cationic comb-type copolymers for the selective stabilization of triplex DNA. The melting temperature (T m ) of triplex DNA increased from 24.5 to 73.0 °C (ΔT m = 48.5 °C) by the addition of poly(allylamine)-graft-dextran (PAA-g-Dex) under physiological conditions (at pH 7.0), while PAA-g-Dex did not stabilize but rather destabilized the DNA duplex. On the other hand, poly(l-lysine)-graft-dextran (PLL-g-Dex) stabilized both the duplex and triplex structures at pH 7.0. Thermodynamic parameters evaluated by isothermal titration calorimetry (ITC) revealed that the binding constant (K a ) for the intermolecular triplex formation in the presence of PAA-g-Dex was 1.1 × 10 9 M -1 at 25 °C which is more than 10 times larger than that in the presence of PLL-g-Dex (8.6 × 10 7 M -1 ). Stabilizing activity and selectivity of cationic copolymers toward DNA assemblies were successfully controlled by selecting appropriate backbone structures of the copolymer. Various functional molecules that stabilize DNA duplexes have been developed and used in biological research. However, there are few cationic polymers that stabilize triplex DNA selectively. This study indicates that PAA-g-Dex has great potential to regulate the biological activities of triplex DNA.

Electrostatic interactions play a major role in protein-DNA interactions. As a model system of a cationic protein, herein we focused on a comb-type copolymer of a polycation backbone and dextran side chains, poly(L-lysine)-graft-dextran (PLL-g-Dex), which has been reported to form soluble interpolyelectrolyte complexes with DNA strands. We investigated the effects of PLL-g-Dex on the conformation and thermodynamics of DNA oligonucleotides forming various secondary structures. Thermodynamic analysis of the DNA structures showed that the parallel conformations involved in both DNA duplexes and triplexes were significantly and specifically stabilized by PLL-g-Dex. On the basis of thermodynamic parameters, it was further possible to design DNA switches that undergo structural transition responding to PLL-g-Dex from an antiparallel duplex to a parallel triplex even with mismatches in the third strand hybridization. These results suggest that polycationic molecules are able to induce structural polymorphism of DNA oligonucleotides, because of the conformation-selective stabilization effects., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the presence of poly(L-lysine)-graft-dextran, an in-stem molecular beacon involving three perylene-anthraquinone pairs in the stem region had a signal/background ratio of as high as 570. Response speed was also remarkable; equilibrium was attained within 5 minutes after addition of substrate DNA at 20 °C., (This journal is © The Royal Society of Chemistry 2012)

The biological importance of left-handed Z-DNA was supported by the identification of Z-DNA binding proteins. We previously reported that poly(l-lysine)-graft-dextran copolymers composed of a poly(l-lysine) main chain and dextran graft chains induced the B-Z transition under low salt conditions. Not only the cationic main chain but also the dextran grafts play an important role in induction of the B-Z transition. In this study, we prepared a series of poly(l-lysine) graft copolymers with different graft chains (dextran, poly(ethylene glycol) (PEG), or both) and graft contents to further elucidate the roles of copolymer structures and properties. The copolymers with higher graft contents more effectively stabilized the Z form of DNA than those having lower graft contents. An increase in the B-Z transition rate was observed as graft content increased. We propose that the side chains of the copolymers change microenvironmental factors, such as water activity and dielectric constant, around the DNA and stabilize the Z conformer. Interestingly, hetero graft copolymers with both dextran and PEG side chains stabilized Z-DNA more effectively than the homo graft copolymers with either dextran or PEG side chains.

Biological roles of transition from B form to Z form of DNA (B-Z transition) have recently received attention. The B-Z transition was also employed as driving machinery of a nano-mechanical DNA device. However, there are little reports of effective inducer of the B-Z transition. We previously reported that poly((L)-Lysine)-graft-dextran induces B-Z transition and grafted dextran plays an important role for the B-Z transition. In this report, we designed cationic graft copolymer as a potential inducer of B-Z transition. Series of the copolymers consisting of poly((L)-Lysine) backbone and abundant of dextran (Dex) or poly(ethylene glycol) (PEG) side chains were prepared. The B-Z transition of poly(dG-dC)/Poly(dG-dC) were observed in the presence of these copolymers. The copolymers having higher content of Dex or PEG effectively induced the B-Z transition compared to that having lower content. The result indicated that not only electrostatic interaction between DNA and the poly((L)-Lysine) backbone but also hydrophilic graft chains play a role for the B-Z transition. We speculated that Dex- or PEG-enriched environment is favorable for Z form.

The intermolecular DNA quadruplex is attractive materials for design of nanomolecular constructs and machines. This folding kinetics is, however, slow, hampering its application to these materials. We have reported that the cationic comb-type copolymer accelerated duplex and triplex formation. In this study, the effect of the copolymer on the intermolecular quadruplex folding was investigated. The copolymer was found to accelerate the association of intermolecular quadruplex by three-orders. Furthermore, the copolymer induced the strand exchange reaction between intermolecular quadruplex and its single-stranded counterparts. We suggested that the copolymer acts as a nucleic acid chaperone for the intermolecular quadruplex.

Poly( L-lysine)- graft-dextran (PLL- g-dex), graft copolymers with dextran side chains grafted onto a poly( L-lysine) backbone, previously shown to be effective as stabilizers of DNA triple helices and as carriers of functional genes to target cells or tissues, were employed in this work to prevent nonspecific adsorption of proteins, as determined by means of optical waveguide lightmode spectroscopy. PLL- g-dex copolymers readily adsorb from aqueous solution onto negatively charged oxide surfaces and significantly reduce nonspecific protein adsorption onto bare silica-titania surfaces. While effective and equivalent surface adsorption and antifouling properties were observed for PLL- g-dex copolymers in a variety of architectures, nanotribological analysis by atomic force microscopy was able to distinguish between the different brush densities produced.

We have reported that poly(L-lysine)-graft-dextran (PLL-g-Dex) accelerates DNA hybridization and increases stability of a double stranded DNA (dsDNA). Furthermore, PLL-g-Dex was found to stimulate the DNA strand exchange reaction between dsDNA and its complementary single stranded DNA (ssDNA). In order to better understand these phenomena, we evaluate PLL-g-Dex/DNA interaction by the single molecular observation and intermolecular force measurement at single molecular level. We have been able to observe PLL-g-Dex/DNA complex at single molecular level. PLL-g-Dex was found to increases the dissociation force of a dsDNA.

We have previously reported that poly(l-lysine)-graft-dextran cationic comb-type copolymers accelerate strand exchange reaction between duplex DNA and its complementary single strand by >4 orders of magnitude, while stabilizing duplex. However, the stabilization of the duplex is considered principally unfavourable for the accelerating activity since the strand exchange reaction requires, at least, partial melting of the initial duplex. Here we report the effects of different cationic moieties of cationic comb-type copolymers on the accelerating activity. The copolymer having guanidino groups exhibited markedly higher accelerating effect on strand exchange reactions than that having primary amino groups. The high accelerating effect of the former is considered to be due to its lower stabilizing effect on duplex DNA, resulting from its increased affinity to single-stranded DNA. The difference in affinity was clearly demonstrated by a fluorescence correlation spectroscopy study; the interaction of the former with single-stranded DNA still remained high even at 1 M NaCl, while that of the latter completely disappeared. These results suggest that some modes of interactions, such as hydrogen bonding, other than electrostatic interactions between the copolymers having guanidino groups and DNAs may be involved in strand exchange activation.

DNA sequence 5'-TGGGGT-3'; TG(4)T takes an intermolecular quadruplex structure which composed of four TG(4)T strands associated together by four layers of G-quartets with parallel strand orientation. Interestingly, its association rate is considerably slow, hampering its application of DNA nanobiotechnology. We have reported that the cationic comb-type copolymer seemingly promoted the nucleation step of DNA hybridization to accelerate duplex and triplex formation. Then, we presumed that the copolymer also influenced kinetics of the quadruplex formation. In this study, the effect of the copolymer on the intermolecular quadruplex folding was investigated. The copolymer was found to accelerate both the association and dissociation of tetramolecular quadruplex. We concluded that PLL-g-Dex acts as a nucleic acid chaperone for tetramolecular quadruplex formation.

The structural transition of a double helical DNA from B-DNA to Z-DNA, B-Z transition, has been received growing attention because of its potential roles in biological systems and its applicability to nanobiotechnology. It was known that the B-Z transition is induced by cationic molecules. However, there are only a few reports describing cationic polymers as potential B-Z inducers. In this study, we demonstrated that cationic comb-type copolymer, poly(L-lysine)-graft-dextran, induced the B-Z transition in which distinct intermediate formation was involved. Furthermore, we suggested that the grafted-dextran plays an important role in the B-Z transition.

Delivery systems of small interfering RNA (siRNA) are the key to siRNA therapeutic application. In this study, we prepared and evaluated a series of cationic comb-type copolymers (CCCs) possessing a polycationic backbone (less than 30 weight (wt) %) and abundant water-soluble side chains (more than 70 wt.%) as a siRNA carrier with prolonged blood circulation time. Markedly, the CCC with the higher side chain content (10 wt.% PLL and 90 wt.% PEG) showed stronger interaction with siRNA than that with the lower content (30 wt.% PLL and 70 wt.% PEG), suggesting that highly dense PEG brush reinforces interpolyelectrolyte complex between the PLL backbone and siRNA. The siRNA complexed with the CCC was resistant to nucleases in 90% plasma for 24 h in vitro. The CCC having the higher side chain content increased circulation time of siRNA in mouse bloodstream by 100-fold. Surprisingly, even when the CCC and siRNA were separately injected into mouse at 20 min interval, blood circulation of post-injected siRNA was significantly increased. These results imply that the CCC has higher selectivity in its ionic interaction with siRNA than other anionic substances in blood stream. To our knowledge, this is the first example of a polyplex carrier that prolongs blood circulation time of unmodified siRNA without resource-consuming preparation process.

We have demonstrated that cationic comb-type copolymers consisting of a polycation backbone and abundant grafts of water-soluble polymers stabilize DNA hybrids. Furthermore, the copolymers were found to accelerate strand exchange reaction between a double-stranded DNA and its complementary single-stranded DNA. In this article, we investigated the effects of PLL-g-Dex on base pairs of a self-complementary DNA octamer, d(GGAATTCC). The soluble interpolyelectrolyte complex (IPEC) between the DNA and copolymer allowed us to characterize the complex by using spectroscopic methods under physiological ionic condition. Chemical shifts of nucleobase proton signals were not changed by PLL-g-Dex. Furthermore, the copolymer slightly changed the von't Hoff DeltaH accompanying the helix-coil transition of the octamer. These results indicated that the base pairs of the duplex DNA in the IPEC were not perturbed by the polycationic copolymer. It was obviously shown by temperature dependencies of proton and phosphorus NMR spectra that DNA/copolymer interaction was considerably enhanced in response to ds DNA formation. An increase in the density and total number of DNA negative charges upon hybrid formation likely caused the higher affinity of the copolymer with the ds form over that of the copolymer with the ss form. The IPEC formation of CCCs with DNA, however, seems highly sensitive to the coil-helix transition of the DNA.

We have focused on the DNA strand exchange reaction (SER) as a format of a single-base mismatch detection. We employed a partially double-stranded (PDS) DNA probe having a single-stranded (ss) portion. We could rapidly recognize a single-base mismatch by the SER rates between the PDS probes and target ss DNAs. In the presence of cationic comb-type copolymers (PLL-g-Dex) and betaine, the PDS probes could clearly discriminate between A:T matched and A:G mismatched targets. Furthermore, the PDS probes could discriminate among homo- and hetero-type alleles.

For the better applications and developments of DNA nanomachines, their responding kinetics, output, and sequence-selectivity need to be improved. Furthermore, the DNA nanomachines currently have several limitations in operating conditions. Here we show that a simple addition of a cationic comb-type copolymer, poly(l-lysine)-graft-dextran, produces the robust and quick responses of DNA nanomachines under moderate conditions including physiologically relevant conditions even at very low strand concentrations (nanomoles per liter range) through hybrid stabilization and DNA strand exchange acceleration.

Cationic comb-type copolymer (CCC) consisting of a polycationic backbone and abundant graft water-soluble chains exhibited considerable stabilization effect on DNA hybrids, such as double- and triple-stranded DNAs. Here, we describe the effect of CCC on antiparallel G-quadruplex folding of human telomeric DNA, d(GGGTTA)(n) in the presence of sodium ions. CCC did not significantly alter the circular dichroism (CD) spectra of d((GGGTTA)(3)GGG) and d((GGGTTA)(7)GGG) indicating that the CCC did not influence the antiparallel folding of the telomeric repeats. Hence, the ionic interaction of CCC with the DNA sequence did not interfere with specific interaction of the DNA with sodium ions to form G-quartets. Interestingly, CCC did not change the melting temperature of the d((GGGTTA)(3)GGG) suggesting negligible stabilizing effect of CCC on the antiparallel quadruplex structure.

We have focused on design of biomaterials that are capable of engineering nucleic acids folding. In this study, we explored the effect of poly(L-lysine)-g-dextran, PLL-g-Dex, copolymer on hybridization kinetics of DNA. The hybridization kinetics was studied by using a fluorescence resonance energy transfer (FRET) assay in the absence and presence of the copolymer. The copolymer at nano molar concentration significantly accelerated the hybridization rate over 200-fold under physiologically relevant ionic conditions.

In order to evaluate the effect of cationic copolymer structures on their nucleic acid-chaperoning activity, we prepared various copolymers having different cationic residues or backbone molecular weight. It was revealed that nucleic acid-chaperoning activity increases with increasing molecular weight of the copolymer backbone and that the copolymer having the guanidino groups is effective for increasing nucleic acid-chaperoning activity. Compared with PLL-g-Dex, GPLL-g-Dex has weak activity to stabilize ds DNA. This weak stabilization effect of GPLL-g-Dex may contribute to the higher accelerating effect.

A series of comb-type copolymers comprised of various polycation backbones and dextran (Dex) side chains were prepared to study the DNA/copolymer interaction. While the cationic copolymers with a lower degree of dextran grafts maintained an ability to condense DNA molecules into a globule form those with a higher degree of dextran grafting interacted with DNA without inducing DNA condensation. The structural differences in cationic backbones diversely influenced DNA hybridization as evaluated by circular dichroism (CD) spectrometry and UV-melting analyses. The copolymer having a polyallylamine (PAA) backbone induced B-->A-type transformation of DNA duplex, whereas the copolymers having either alpha-poly(l-lysine) (alpha PLL) or epsilon-poly(l-lysine) (epsilon PLL) backbone induced B-->C-type transformation. The PAA copolymer is the first example of the artificial polymer that induces B-->A-type transformation under physiologically relevant condition. UV-melting analyses of DNA strands indicated that the alpha PLL copolymers showed the highest stabilization efficacy toward poly(dA).poly(dT) duplex and poly(dA).2poly(dT) triplex without affecting reversibility of inter DNA association. Melting temperatures (T(m)) of the triplex increased from 38 degrees Celsius to 99 degrees Celsius by the addition of the alpha PLL copolymer with an appropriate grafting degree. While the PAA copolymers had higher density of cationic groups along the backbone than alpha PLL copolymers, these copolymers moderately increased T(m) of the DNA triplex. The PAA copolymer caused considerable hysteresis in thermal melting/reassociation processes. Note that the PLL copolymers increased T(m) of the DNA triplex and not the duplex, suggesting their potential as a triplex selective stabilizer. Chemical structures of the cationic backbones of the copolymers were characteristically affected on the copolymer/DNA interaction even if their backbones were surrounded by abundant side chains (> wt%) of dextran. The study suggested that tailor-made design of "functional polycounterion" is a strategy to engineer molecular assembling of DNA.

We have shown that the cationic comb-type copolymers (CCCs) accelerate DNA hybridization and increase stability of DNA duplexes and triplexes. The CCCs were considered to act as "nucleic acid chaperones," promoting the formation of the most stable hybrids. In this study, CCCs with primary amino or guanidino groups were prepared to evaluate the effects of different cationic moieties upon the nucleic acid chaperone activity. CCCs having guanidino groups have higher accelerating effect on DNA strand exchange reactions than that having primary amino groups. It was suggested that some modes of interactions, such as hydrogen bonding, other than ionic interactions between the copolymers and DNAs may be involved upon the strand exchange activation.

Human telomeric DNA, d(GGGTTA)n, tends to adopt complex intramolecular G-quadruplex structure in solution. The sequence folds into G-quadruplex with the antiparallel orientation in the presence of sodium ions, while antiparallel and parallel conformations were coexisted in solution containing relatively high concentration of potassium ions. Here, we describe that cationic comb-type copolymer, poly(l-lysine)-graft-dextran (PLL-g-Dex), triggers selective folding of d[GGG(TTAGGG)3] into G-quadruplex with the parallel orientation. The parallel G-quadruplex formed in the presence of the copolymer has the melting temperature > 90 degrees C, including strong stabilization effect of the copolymer.

We have demonstrated that cationic comb-type copolymers (CCCs) accelerate DNA hybridization and increase stability of DNA duplexes and triplexes. The CCCs were considered to act as nucleic acid chaperones that catalyze structural transitions of nucleic acid hybrids. In this study, CCCs with primary amino or guanidino groups were prepared to evaluate the effects of different cationic moieties upon the nucleic acid chaperoning activity. CCCs having guanidino groups have higher accelerating effect on DNA strand exchange reactions (SFRs) than that having primary amino groups. It was suggested that some modes of interactions, such as hydrogen bonding, other than ionic interactions between the copolymers and DNAs may be involved upon the strand exchange activation.

We examined the effect of the combination of the two triplex-stabilizing factors, poly(L-lysine)-graft-dextran (PLL-g-Dex) copolymer and 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) backbone modification of triplex-forming oligonucleotide (TFO), on the pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The combination of both stabilizing factors that was the triplex involving the 2',4'-BNA TFO in the presence of the copolymer synergistically increased the thermal stability of the pyrimidine motif triplex at neutral pH. The present results certainly support the idea that the combination of the stabilizing factors can be a key method for triplex stabilization and may lead to progress in therapeutic applications of the antigene strategy in vivo.

We have reported previously that the cationic comb-type copolymer, PLL-g-Dex, accelerated DNA strand exchange reaction while stabilizing DNA duplexes. In this research, we studied the effects of PLL-g-Dex on a DNA-fueled nanomachine. The copolymer enabled rapid and secure operations of the DNA nanomachine which utilized the structural transition between intramolecular G-quadruplex and intermolecular double helix.