Introduction

The correct folding of biopolymers is essential to their functions as catalysts, information carriers, and substance transporters. Molecular chaperones are proteins that facilitate the correct folding and assembly of other biomacromolecules and that prevent misfolding into nonfunctional structures. For example, the assembly of DNA and histones into nucleosomes is assisted by nucleoplasmin, the first identified molecular chaperone. Mixing of DNA and histones without this chaperone protein results in the formation of heterogeneous aggregates [1]. Artificial chaperones having resource-effectiveness and durability are demanded in nanotechnological fields employing biopolymers.

Effective folding of nucleic acids is crucial for their biological functions in living cells and for their bionanotechnological applications. Nucleic acids frequently fold into metastable structures (Fig. 1). These kinetically trapped structures do not rearrange into the thermodynamically most stable structure because of the high energy barrier for the dissociation of misfolded base-paired structures. In experiments with purified RNA or DNA, the metastable structures are disrupted, and samples are induced to form the thermodynamically most stable structure by heating followed by slow cooling. At physiological temperature, molecular chaperones, such as retroviral nucleocapsid protein NCp7 [2], host factor Hfq [3], histone-like protein StpA [4], and cold shock proteins CspA [5] and CspE [6], can mediate folding into thermodynamically stable structures. Nucleic acid chaperone protein activities generally destabilize nucleic acid base pairing, which means that these protein mediators are not suitable for nanotechnological applications, where both base-pairing accuracy and duplex stability are required. Moreover, insufficient stability and availability limit the utility of chaperone proteins in many applications.

Fig. 1
figure 1

Schematic of nucleic acid folding

We have pioneered the use of synthetic polymers as nucleic acid chaperones. These polymers form soluble interpolyelectrolyte complexes (IPECs) with negatively charged biopolymers. By grafting hydrophilic polysaccharides onto the polycationic main chain, the comb-type copolymer shown in Fig. 2 was designed. This copolymer increases the solubility of IPECs, and the structural deformation of nucleic acids due to IPEC formation is suppressed [7, 8]. A soluble IPEC without DNA condensation is obtained when the cationic comb-type copolymer is composed of >80 wt.% grafted chains (dextran) and <20 wt.% polycationic main chain (polylysine) [8,9,10]. Based on single-molecule observations, the addition of the copolymer does not induce the collapse of flow-stretched DNA into globule structures, whereas the polylysine homopolymer or a copolymer with a lower grafting degree do [9]. Moreover, the Brownian motion of DNA segments is maintained in the presence of the copolymer [9]. Circular dichroism (CD) and NMR measurements also confirmed that the copolymer does not alter the DNA secondary structure [8, 10]. The chaperone activity of the cationic comb-type copolymer and its applications in DNA nanotechnology are described in this review. Moreover, peptide folding chaperoned by the copolymer is also discussed.

Fig. 2
figure 2

Chemical structure and schematic of the cationic comb-type copolymer with a polylysine backbone and dextran graft chains (PLL-g-Dex)

Nucleic acid chaperone activity of cationic comb-type copolymers

Enhancement of thermal stability of nucleic acids

DNA folding chaperoned by PLL-g-Dex has been characterized spectroscopically. UV melting analysis indicated that the presence of PLL-g-Dex with 90 wt.% grafted dextran markedly increases the thermal stability of DNA duplexes [8] and triplexes [7]. PLL-g-Dex increased the melting temperature (Tm) of poly(dA)·poly(dT) duplexes and poly(dT)·poly(dA)·poly(dT) triplexes by 20 and 50 °C, respectively. The melting profile of a DNA triplex is typically biphasic, as the transition of a triplex to a duplex is followed by a duplex to single-stranded DNA transition at a higher melting temperature. In contrast, a DNA triplex stabilized by PLL-g-Dex has a single transition at a higher temperature than either of the transitions observed in the absence of the copolymer (Fig. 3). A reversible transition occurs upon cooling, indicating that the interaction with PLL-g-Dex does not alter duplex or triplex rehybridization. At equivalent positive-to-negative charge ratios, PLL-g-Dex has a stronger stabilizing effect on the DNA structure than spermine, a polyamine commonly used as a DNA duplex stabilizer. DNA duplex and triplex structures were unchanged by the addition of PLL-g-Dex with a high degree of grafting, as shown by CD spectroscopy [7, 8, 10]. CD signals were similar to those in the absence of the polymer even when an excess amount of copolymer was added. The 1H NMR spectra confirmed that the polymer increased Tm without affecting the local base-paired structures of a DNA duplex [11].

Fig. 3
figure 3

Triplex thermal stability was enhanced in the presence of PLL-g-Dex. UV absorbance was used to monitor the melting transition of the triplex in the absence (solid line) and presence (dashed line) of PLL-g-Dex at polymer positive charge to nucleotide negative charge ratio (N/P) of 2. Tm1 and Tm2 indicate the transition of triplex to duplex and duplex to single strand, respectively. Tm indicates a single transition of triplex to single strand in the presence of the copolymer. Adapted with permission from ref. [7]. Copyright 1997 American Chemical Society

The PLL backbone stabilizes DNA hybridization by reducing electrostatic repulsion between the negative charges on the polynucleotide backbone, and the Dex chains prevent DNA dehydration and compaction. Strong but locally dynamic interactions with PLL-g-Dex preserve the native structure and self-assembly properties of DNA when Watson–Crick or Hoogsteen hydrogen bonds are involved in the secondary structure. In contrast, polycationic homopolymers cause alterations in the secondary structure of DNA and irreversibly form insoluble IPECs with DNA.

Thermodynamic and kinetic effects on DNA structure formation

Thermodynamic and kinetic studies have revealed the unique mechanism by which the copolymer stabilizes nucleic acid structures. Thermodynamic parameters showed similar enthalpies (ΔH°) of structure formation in the presence and absence of PLL-g-Dex. There is no difference in enthalpy because the polymer does not disrupt hydrogen bonding or base stacking. Reduced entropy (ΔS°) was observed in the presence of PLL-g-Dex [11]. This observation suggests that the counterion condensation effect on DNA is reduced by the association of PLL-g-Dex with DNA, leading to the stabilization of the DNA assemblies. The effect on counterion condensation is discussed more fully in section 2.3. Kinetic analysis demonstrated that the addition of PLL-g-Dex accelerates the hybridization rate of a DNA duplex by approximately 200-fold under physiological ionic conditions (Fig. 4) [12] and increases the binding constant (i.e., the association rate constant divided by the dissociation rate constant) of a DNA triplex by ~100-fold [13]. Certain chemical modifications of DNA and conjugation with some DNA-binding molecules stabilize nucleic acid structures by decreasing the duplex dissociation rate. In contrast, PLL-g-Dex promotes DNA association. The combination of PLL-g-Dex with DNA chemical modification such as oligo-N3′-N5′ phosphoramidate modification [14] or a 2′-O,4′-C-methylene-bridge [15] synergistically stabilizes a triplex by increasing the association rate and decreasing the dissociation rate. The synergistic stabilization effect resulted in an increase of 4 orders of magnitude in the thermodynamic stability of DNA triplexes.

Fig. 4
figure 4

PLL-g-Dex enhances the DNA hybridization rate. The TAMRA-labeled T25P strand is complementary to the DABCYL-labeled D25T strand, whereas the DABCYL-labeled D25S strand is a control without complementarity to T25P. Hybridization was monitored as a function of time by fluorescence resonance energy transfer (FRET). The fluorescence emission from the TAMRA-labeled strand is quenched when it hybridizes with the complementary DABCYL-labeled strand [12]. Reproduced by permission of The Royal Society of Chemistry

Reduction of the counterion condensation effect

The thermodynamic stability of DNA duplexes and triplexes increases with increasing salt concentration. The effect of ion screening on the thermal stability of DNA helices is explained by the counterion condensation theory proposed by Manning [16], which states that the counterion condensation that occurs in the local environment of DNA partially neutralizes the phosphate anionic charges and stabilizes the DNA structure. The extent of counterion condensation increases with increasing negative charge density. Hence, triplex formation requires condensation of more counterions around DNA strands than duplex and single-stranded DNA formation. Counterion condensation contributes to the entropic barrier to DNA hybridization. An increase in salt concentration effectively reduces this entropic barrier and increases the Tm of a DNA duplex. In the presence of PLL-g-Dex, this salt dependence is not observed (Fig. 5a) [17], suggesting that the counterion condensation effect is eliminated in the presence of PLL-g-Dex. Upon interaction of the cationic comb-type copolymer with negatively charged nucleic acids, counterions are released into the bulk solution (Fig. 5b). This effect increases the entropy of the system, which contributes to the enhanced thermal stability [17] and accelerated hybridization rate [12].

Fig. 5
figure 5

PLL-g-Dex reduces the counterion condensation effect and reduces the entropic penalty for DNA structure formation. a Tm of a DNA duplex at various Na+ concentrations in the absence (square) and presence (circle) of PLL-g-Dex. Reprinted from ref. [17]. Copyright 1999, with permission from Elsevier. b Illustration of the counterion condensation effect eliminated by PLL-g-Dex

Acceleration of DNA strand exchange reaction

The strand exchange reaction (SER) occurs when single-stranded DNA displaces a homologous strand of a prehybridized duplex. The sequential-displacement mechanism of the SER initiates with the partial melting of the prehybridized duplex, followed by the formation of a branched nucleation intermediate that forms as the homologous single strand invades the duplex. The invader strand gradually replaces the initially hybridized strand through branch migration until a new duplex product is formed (Fig. 6a) [18]. As a variety of DNA nanodevices involve the SER [19], fast and efficient strand exchange is desirable. Nucleic acid chaperone proteins such as NCp7 catalyze the SER by destabilizing the duplex, which decreases the energy barrier to strand displacement [20]. The cationic comb-type copolymer accelerates the SER by promoting the formation of three-stranded nucleation complexes by reducing counterion condensation. The formation of the three-stranded intermediate, which is accompanied by a thermodynamically unfavorable accumulation of counterions, is the rate-limiting step of the SER. PLL-g-Dex accelerates SER more than 10,000 times compared to the reaction in the absence of copolymer (Fig. 6b) [21, 22], a considerably higher level of acceleration than that observed with the natural nucleic acid chaperone protein NCp7. The ability to catalyze the SER demonstrates the nucleic acid chaperone feature of the cationic comb-type copolymer. Furthermore, the incorporation of guanidino or ureido groups into the copolymer structure can improve its nucleic acid chaperone activity [23,24,25].

Fig. 6
figure 6

The cationic copolymer accelerates DNA strand exchange. a Schematic of a DNA strand exchange reaction. b Analysis of % strand exchange as a function of time in the presence of PLL-g-Dex (CCC(+)) and in the absence of PLL-g-Dex (CCC(−)) as monitored using FRET. Reproduced with permission from ref. [22]. Copyright 2002 American Chemical Society

Selective stabilization of noncanonical DNA structures

The most familiar DNA structure is the right-handed double-helix DNA (B-form), which was first characterized by Watson and Crick [26]. Noncanonical DNA structures have also been discovered [27], such as the A-form duplex, a right-handed duplex having a shorter distance between base pairs than the B-form duplex [28]; the left-handed Z-form duplex [29]; the triplex, which is a B-form duplex with a third strand folded into the major groove [30]; and the guanine-rich G-quadruplex having a four-stranded helical structure [31]. The formation of these noncanonical structures is influenced by nucleotide sequences, hydration, solvent, coexisting cationic species, pH, and specific proteins. We found that cationic comb-type copolymers with certain backbone structures and grafted chains selectively stabilize different nucleic acid structures.

The conformational transition of the poly(dG-dC)·poly(dG-dC) B-form duplex to the Z-form is triggered by PLL-g-Dex [32, 33]. The presence of PLL-g-Dex at 10−5 M stabilizes Z-DNA to an extent comparable to that of 10−5 M of the cationic compound [Co(NH3)6]Cl3, which is known as a general B–Z transition inducer [34]. In addition to the shielding of electrostatic repulsion between DNA strands, reduction in water activity when DNA merges into the dextran-enriched phase is another factor favorable for the B–Z transition. Microenvironmental factors such as water activity and dielectric constant can be adjusted by varying the graft chain residues. Z-DNA was more effectively stabilized by the heterograft copolymer having dextran and poly(ethylene glycol) side chains than the homograft copolymer [33].

A cationic comb-type copolymer with a polyallylamine backbone (PAA-g-Dex) (Fig. 7) induces the B-form to A-form transition of a GC-rich DNA duplex, whereas PLL-g-Dex does not [35]. The binding of PAA-g-Dex, which has a more hydrophobic backbone than PLL-g-Dex, leads to DNA strand dehydration, making the environment more favorable for A-form structure formation. Relatively low concentrations of PAA-g-Dex more effectively drive the B–A transition than other triggering reagents such as ethanol, methanol, or trifluoroethanol.

Fig. 7
figure 7

Structure of a cationic comb-type copolymer having a polyallylamine backbone (PAA-g-Dex)

A cationic comb-type copolymer with a PAA backbone also selectively stabilizes the DNA triplex relative to the duplex [36], while PLL-g-Dex stabilizes both duplex and triplex structures. Dehydration caused by the PAA backbone but not the PLL backbone provides this unique stabilizing effect. It was previously reported that dehydration drives triplex formation [37]. Dehydration by PAA-g-Dex thermodynamically stabilizes triplex formation but destabilizes duplex formation. The binding constant for triplex formation in the presence of PAA-g-Dex is more than 10-fold higher than that in the presence of PLL-g-Dex. This phenomenon is because the triplex stabilization by PAA-g-Dex involves two mechanisms: electrostatic repulsion shielding and dehydration, whereas the triplex stabilization by PLL-g-Dex involves only shielding of electrostatic repulsion.

Chaperoning the formation of DNA quadruplex structures

In the four-stranded G-quadruplex, planar G-quartets stabilized by Hoogsteen hydrogen bonds are stacked on top of one another and coordinate metal ions. There are several topological variants of the G-quadruplex [38]. An intramolecular G-quadruplex is formed from one DNA strand (unimolecular), and intermolecular G-quadruplexes can be formed from two (bimolecular) or four (tetramolecular) separate DNA strands. G-quadruplex structures may be classified according to the strand orientation. If all the strands are oriented in the same direction, the G-quadruplex is parallel. In contrast, if strands have an opposite orientation, the quadruplex is antiparallel.

G-quadruplex formation is retarded by the electrostatic repulsion between the phosphates on the DNA backbone. Moreover, G-rich DNA strands frequently assemble into a mixture of metastable conformations, and rearrangement to the most thermodynamically stable structure is extremely slow. The nucleic acid chaperone activity of PLL-g-Dex significantly increases both the association rate and the dissociation rate of tetramolecular quadruplex DNA and promotes the SER between quadruplex and single-stranded DNA [39]. Accordingly, the copolymer also chaperones intermolecular quadruplex folding into a monomorphic structure from a polymorphic mixture by accelerating the rearrangement of metastable heteroquadruplexes into the thermodynamically most stable homoquadruplex (Fig. 8) [40].

Fig. 8
figure 8

Schematic of the free energy of G-quadruplex folding. PLL-g-Dex facilitates refolding of metastable DNA quadruplexes into the thermodynamically most stable structure by reducing the energy barrier between metastable and stable states. The solid line is the free energy in the presence of PLL-g-Dex, and the dashed line is the free energy in the absence of PLL-g-Dex

Applications of the cationic comb-type copolymer in nucleic acid-based diagnostics and nanotechnology

The copolymer enhances the performance of several DNA-based analytical tools. For example, the chaperone activity of PLL-g-Dex enabled the development of a precise method for the discrimination of single-nucleotide polymorphisms (SNPs). The acceleration of the SER by the copolymer shifted the rate-limiting step from heteroduplex nucleation to branch migration, which is retarded when there is a mismatch. A single-base mismatch could be discriminated from a fully matched 20-mer DNA in the presence of the copolymer but not in its absence [21]. Molecular beacon probes were also shown to have enhanced sensitivity in the presence of the copolymer [41]. Molecular beacons are single-stranded oligonucleotides held in a stem-loop conformation and labeled with a fluorescence donor and quencher at the 3′- and 5′-termini. When the probe forms a duplex with a complementary target nucleic acid strand, the stem loop is disrupted, and fluorescence is observed, as the quencher is no longer in proximity to the fluorescent donor. The copolymer suppresses background emission by stabilizing the stem-loop conformation and increases the signal by accelerating the SER and stabilizing the target/probe duplex. Furthermore, the activity of the copolymer can be “switched off” by the addition of an anionic polymer, resulting in a reversible transformation between the stem loop and duplex [42]. The performance of DNA-driven quadruplex and tweezer nanomachines is also boosted by the addition of the copolymer. The transformations that operate these nanomachines are triggered by a DNA “fuel” strand, and the copolymer both enhances SER and stabilizes the duplex formed with the fuel [43]. In other applications, the acceleration of the SER markedly increases turnover in a DNA-templated fluorophore transfer reaction [44], accelerates photodriven DNA strand displacement reactions [45], and reduces the operation time of DNA computing from hours to minutes [46]. The copolymer also promotes strand displacement without hindering enzyme activity when utilized in a photoregulated DNA amplification system [47]. The therapeutic applicability of the copolymer has also been demonstrated. The copolymer functions as a triplex stabilizer for inhibiting protein-DNA interaction [48] and as a therapeutic nucleic acid carrier for prolonging blood circulation time and increasing accumulation in cancer tissue [49, 50].

Deoxyribozymes (DNAzymes) are a novel class of functional DNAs that catalytically act on specific substrates. DNAzymes are easy to prepare, are inexpensive, have high chemical and biochemical stability, and can be chemically modified. To improve the performance of these functional nucleic acids, techniques for increasing the stability of the nucleic acid structures necessary for enzymatic activity and for increasing the assembly rate are required.

The allosteric control of peroxidase-like DNAzyme activity using the cationic comb-type copolymer was recently reported [51]. The peroxidase-like DNAzyme is a G-quadruplex structure that binds hemin and has higher peroxidase activity than the uncomplexed hemin [52, 53]. The peroxidase activity of G-quadruplex DNAzyme strongly depends on its conformation. An intramolecular parallel G-quadruplex structure has higher peroxidase activity than antiparallel and intermolecular parallel structures [54]. The addition of PLL-g-Dex increases the parallel G-quadruplex content of an otherwise heteromorphic sample and results in a more than 30-fold increase in peroxidase activity, which indicates that the polymer acts as a positive allosteric effector of the peroxidase-like G-quadruplex DNAzyme [51]. Moreover, reversible allosteric control of the DNAzyme was achieved by adding the anionic polymer poly(vinylsulfonic acid) (PVS). Once PLL-g-Dex was neutralized by PVS, the DNAzyme returned to its inactive state (Fig. 9).

Fig. 9
figure 9

Copolymers enable reversible allosteric control of the peroxidase-like G-quadruplex DNAzyme. Peroxidase activity in the absence of PLL-g-Dex is slow. The addition of PLL-g-Dex accelerates the reaction, and the addition of an excess amount of PVS turns off the reaction. The effect of PVS is reversed upon the addition of PLL-g-Dex. Reproduced with permission from ref. [51]. Copyright 2018 American Chemical Society

DNAzymes having RNA cleavage activity (that is, ribonuclease activity) were first reported by Breaker and Joyce [55]. DNAzymes have several advantages over RNA or protein enzymes, such as higher stability [56, 57] and resource-effectiveness. However, the catalytic activity of DNAzymes is limited by insufficient association and low turnover efficiency. Unlike other approaches used to enhance turnover, the use of PLL-g-Dex stabilizes DNA duplexes by increasing association rather than by decreasing dissociation. The copolymer considerably increases DNAzyme activity and widens the temperature range in which the enzyme has catalytic activity [58]. Furthermore, copolymers enabled isothermal nucleic acid detection by a DNAzyme. PLL-g-Dex chaperones assembly of the allosteric DNAzyme components, including substrate, two partial DNAzymes, and target, into a catalytically active structure (Fig. 10a) and results in a 200-fold increase in enzymatic activity relative to the reaction performed in the absence of the copolymer (Fig. 10b) [59]. In the presence of PLL-g-Dex, a target at a picomolar concentration can be detected at physiological temperature [59].

Fig. 10
figure 10

a Illustration of a typical DNAzyme and an allosteric DNAzyme. The allosteric DNAzyme only adopts the enzymatically active structure when the target is bound. Cleavage separates the fluorophore and quencher conjugated to the substrate, resulting in fluorescence. b The percent cleavage of the substrate by the allosteric DNAzyme is enhanced by the addition of PLL-g-Dex [59]. Published by The Royal Society of Chemistry

Peptide structure and function control by the cationic comb-type copolymer

Compared to protein medicines, therapeutic peptides are highly selective, effective, and inexpensive; however, they have the propensity to aggregate and denature [60]. The cationic comb-type copolymer has been utilized to manipulate the folding, solubility, and activity of the functional peptide E5 (Fig. 11a). The E5 peptide is an amphipathic anionic peptide that mimics the lipid membrane-disrupting activity of the influenza virus. In an acidic environment, the E5 peptide folds into an α-helix structure due to an anisotropic arrangement of hydrophobic and hydrophilic amino acid residues. This amphiphilic helix structure exhibits membrane-disrupting activity. However, the E5 peptide has poor solubility due to its hydrophobicity and tends to aggregate under acidic conditions, which inhibits its activity.

Fig. 11
figure 11

PAA-g-Dex enhances the membrane-disrupting activity of the E5 peptide. a E5 peptide folding under acidic and neutral conditions. b Membrane-disrupting activity of E5 measured by analysis of leakage of fluorescein di(β-d-galactopyranoside) from a vesicle with and without PAA-g-Dex at acidic and neutral pH. Adapted from ref. [61]. Copyright 2015, with permission from Elsevier

The cationic comb-type copolymer PAA-g-Dex promotes folding of the E5 peptide from a random coil to an active helix structure without loss of solubility. The copolymer has an ionic charge opposite to that of the E5 peptide and shields electrostatic repulsion between glutamic acid side chains of E5. In the presence of PAA-g-Dex, E5 folds into the active conformation, and the complex of PAA-g-Dex and E5 is soluble even under acidic conditions. Interestingly, the polymer significantly enhances the membrane-disrupting activity of the E5 peptide not only under acidic conditions but also at neutral pH, at which the E5 peptide without polymer has no activity (Fig. 11b) [61]. The complex of the E5 peptide and PAA-g-Dex has potential for use in the intracellular delivery of therapeutic molecules.

Conclusion

Cationic comb-type copolymers consist of a polycationic backbone and polysaccharide side chains. These copolymers form soluble interpolyelectrolyte complexes with DNA via electrostatic interactions and exhibit nucleic acid chaperone activity. The activity of natural nucleic acid chaperone proteins involves destabilizing nucleic acid base pairing, but a cationic comb-type copolymer stabilizes nucleic acid hybrids. The mechanism underlying the chaperone activity of the cationic comb-type copolymer most likely results from shielding of the electrostatic repulsion along negatively charged DNA strands. The chaperone activity of the polymer promotes association; stabilizes DNA duplex, triplex, and quadruplex structures; accelerates strand exchange reactions; and facilitates the conversion from a metastable structure to the most stable structure. The unique properties of the cationic comb-type copolymers enhance the potential of several DNA-based analytical tools and nanodevices. A cationic comb-type copolymer has also been shown to chaperone the folding of a peptide into its active structure under physiological conditions. In future work, we will seek to obtain a comprehensive understanding of the molecular mechanism of cationic comb-type copolymers and will use these reagents in practical applications. We anticipate that the cationic comb-type copolymer will find application in multidisciplinary research areas.