Activity enhancement of G-quadruplex/hemin DNAzyme by spermine

Abstract

Biogenic polyamines participate in regulating gene expression, activating DNA synthesis and facilitating DNA–protein interaction through interaction with DNA/RNA. The interaction of polyamines with G-quadruplexes (G4s) has been reported to modulate the structure of G4s. In this paper, we investigate the effects of polyamines on one of the properties of G4s, G4/hemin peroxidase. Three polyamines (spermine, spermidine and putrescine) are found to have positive effects on different G4/hemin DNAzymes, in which spermine exhibits the strongest enhancement efficiency. CD and UV/Vis spectral analysis suggests two reasons for the strong activity enhancement: first, spermine protects hemin from rapid degradation by H₂O₂; second, spermine condenses the G4 structures and provides a favorable microenvironment for the catalytic reaction. Since G4/hemin DNAzymes have been extensively applied in various chemical sensors and biosensors, this finding would be helpful for the design of G4/hemin based sensors and widen the application range of this kind of DNAzyme.

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Introduction

Biogenic polyamines, such as spermine (spm), spermidine (spd) and putrescine (put), are found in a wide variety of organisms and tissues. They participate in modulating gene expression and enzyme activities, activating DNA synthesis, facilitating DNA–protein interactions, as well as protecting DNA molecules from common damaging agents.^1–3 Elevated levels of these polyamines in tissue or blood are associated with many types of cancers.^4–6 Polyamines are multivalent cations with aliphatic hydrocarbon chains separating the charges, which can interact electrostatically with the backbone phosphate groups of DNA/RNA, and consequently modulate the structures of DNA/RNA and condense DNA molecules to a liquid crystalline phase.^7–9 G-quadruplexes (G4s) are four-stranded nucleic acid structures adopted by some repetitive guanine-rich sequences. Putative G-quadruplex-forming sequences are highly prevalent in the human genome.^10,11 Keniry and coworkers¹² have reported the evidence of the formation of complexes between spermine and antiparallel G4s and intermolecular parallel G4. Li and coworker¹³ have reported that spermine can induce the condensation of parallel G4 motifs. Kumar and coworkers¹⁴ have reported that polyamines could drive the c-MYC gene expression by inducing structural transition of c-MYC G4 to a transcriptionally active motif. These results imply that polyamines may play essential roles in the functions of G4s.

G4/hemin peroxidase was first reported by Travascio in 1998, in which the G4 was hemin-binding DNA aptamers (PS2.M and PS5.M).^15,16 Recently, this peroxidase activity has been found to be a general characteristic of many G4s.^17–19 Compared to protein enzymes, G4/hemin DNAzymes have the advantages of ease of chemical synthesis at low cost, high chemical stability, ease of modification and manipulation.^20,21 Thus, G4/hemin DNAzymes have been extensively applied in various chemical sensors and biosensors and have been receiving increasing attention in recent years.^22–29 In previous report, it has been reported that intramolecular parallel G4s possess strong peroxidase activity when they complex with hemin.^17,19,22 However, the catalytic activity of these G4s is still much lower than that of horseradish peroxidase (HRP).³⁰ The relatively low catalytic efficiency of this type of DNAzyme would restrict their further applications. It has been reported that ammonium buffer favour the peroxidation of G4/hemin DNAzymes.^22,31–34 Since polymines have been found to interact with G4s, in this study, we have investigated the effects of polyamines on catalytic efficiency of 14 G4/hemin complexes. Polyamines are found to have positive effects on these DNAzymes, in which spermine exhibits the strongest enhancement efficiency. The mechanism of the enhancement of peroxidase activity has been investigated by UV/Vis and CD spectra.

Experimental section

Spectral analysis of G4/hemin DNAzymes

DNAs (PS2.M, AS1411, VEGF, EAD, CatG4, Bcl2, c-MYC, TBA, Oxy28, HT, ILPR, A4G4A4, A4G8A4, GT12 and T18, see Table 1) (0.5 or 2 μM) were prepared in phosphate buffer (10 mM NaH₂PO₄/Na₂HPO₄, 100 mM KCl, 2 mM MgCl₂, 0.003% (v/v)Triton X-100, pH = 7.0). Polyamine stock solutions (50 mM) were prepared in 10 mM Tris–HCl buffer and adjusted pH to 7.0 with HCl. In order to form the G4 structures, DNAs were heat-denatured prior to use (heated at 95 °C for 10 min, cooled rapidly at 0 °C for 15 min, and kept at 25 °C for 15 min). DNA solutions were then incubated with 1 mM polyamines (spermine, spermidine and putrescine) at 25 °C for 30 min, and held for another 10 min after the addition of hemin (1.0 or 4.0 μM). Then 0.4 mM of ABTS and 0.6 mM of H₂O₂ were added to the mixtures. The color of the reaction mixture was recorded by a digital camera, and the absorbance at 414 nm or the absorption spectra were collected on a 96-well plate in Molecular Devices Spectramax M5 plate reader after 3 min of reaction. The effect of polyamine on DNAzyme-mediated oxidation of TMB was identical to the above description except for that 3 μM DNA/hemin and 0.5 mM TMB was used.

Table 1 The V_i of G4/hemin DNAzymes with different G4 structure in the absence or presence of polyamines

G4 type^a	DNA	V i (×10^−9 M s^−1)
		ctr	spm	spd	put
a The G4 type classification is based upon the CD spectra of each DNA under the buffer condition used for catalytic reaction in this paper. Inter-G4: intermolecular G4s; mixed: intramolecular mixed-parallel G4s; intra-anti: intramolecular anti-parallel G4s; intra-para: intramolecular parallel G4s.
Non-G4	T18	1.21 ± 0.03	1.86 ± 0.08	1.68 ± 0.003	1.59 ± 0.07
Inter-G4	GT12 (G3T3G3T3)	0.91 ± 0.02	3.35 ± 0.13	2.10 ± 0.02	1.53 ± 0.04
	A4G8A4	1.30 ± 0.04	2.67 ± 0.10	1.91 ± 0.01	1.65 ± 0.03
	A4G4A4	1.48 ± 0.008	6.48 ± 0.10	3.68 ± 0.06	1.89 ± 0.05
Mixed	HT ((T2AG3)4)	1.99 ± 0.05	5.17 ± 0.14	2.96 ± 0.24	2.42 ± 0.08
	Oxy28 (G4T4G4T4 G4T4G4)	1.24 ± 0.01	4.99 ± 0.13	2.66 ± 0.03	1.69 ± 0.01
	ILPR (TACAG4TGTG4ACAG4TGTG4)	4.47 ± 0.12	9.21 ± 0.26	5.91 ± 0.18	5.02 ± 0.08
Intra-anti	TBA (G2T2G2TGTG2T2G2)	1.26 ± 0.04	2.73 ± 0.07	1.81 ± 0.06	1.52 ± 0.05
Intra-para	EAD (CTG3TG3TG3TG3A)	7.19 ± 0.05	11.21 ± 0.40	9.02 ± 0.47	7.49 ± 0.09
	PS2.M (GTG3TAG3CG3T2G2)	4.12 ± 0.09	25.9 ± 0.59	8.13 ± 0.41	4.86 ± 0.06
	VEGF (G3CG3CCG5CG3)	4.22 ± 0.03	9.56 ± 0.06	7.14 ± 0.47	5.34 ± 0.33
	CatG4 (TG3TAG3CG3T2G3A3)	7.06 ± 1.00	11.0 ± 0.53	8.05 ± 0.23	7.17 ± 0.12
	c-MYC (TGAG3TG4AG3TG4A2)	5.57 ± 0.01	9.50 ± 0.59	6.19 ± 0.29	5.93 ± 0.32
	Bcl2 (G3CGCG3AG2A2G5CG3)	2.24 ± 0.02	5.10 ± 0.11	3.58 ± 0.18	3.00 ± 0.02
	AS1411 ((G2T)4TGT(G2T)3G2)	4.74 ± 0.13	16.9 ± 0.14	11.9 ± 0.05	5.42 ± 0.24

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The effect of spermine on the dynamic process of G4/hemin DNAzyme reaction under different pH condition

The dynamic process of G4/hemin DNAzyme reaction was measured on a 96-well plate in Molecular Devices Spectramax M5 plate reader. 1 mM spermine was incubated with 0.5 μM PS2.M at different pH (pH = 4, 5, 6, 7, 8, 9) for 30 min at room temperature, then 1 μM hemin was added and incubated for 10 min, finally 0.4 mM ABTS and 0.4 mM H₂O₂ were added. The absorbance of ABTS˙⁻ at 414 nm was collected as a function of time after the reaction had started for 5 min. The control samples were incubated without spermine.

Determination of the initial velocity of G4/hemin DNAzyme reaction

The initial velocities (V_i) of G4/hemin DNAzyme reaction in the presence and absence of 1 mM polyamines were determined in phosphate buffer (10 mM NaH₂PO₄/Na₂HPO₄, 100 mM KCl, 2 mM MgCl₂, 0.003% (v/v)Triton X-100, pH = 7.0) that contained 1 μM G4/hemin, ABTS (0.6 mM) and H₂O₂ (0.1 mM). Reactions were initiated by the addition of H₂O₂. The increase of absorbance at 414 nm was measured immediately after the addition of H₂O₂ and monitored over 5 min on SpectraMax M5 (Molecular Devices Corporation, USA). V_i was calculated from the slope of the initial linear portion of the absorbance increase and the Δε value of ABTS at 414 nm (36 000 M⁻¹ cm⁻¹).¹⁷

CD spectra assay

Circular dichroism (CD) spectra were collected on a Jasco J-815 circular dichroism spectropolarimeter (JASCO, Japan) at a rate of 500 nm min⁻¹ in a quartz cell with an optical path length of 10 mm. Measurements were taken in sodium phosphate buffer (10 mM NaH₂PO₄/Na₂HPO₄, 100 mM KCl, 2 mM MgCl₂, pH = 7.0). All the DNA samples (3 μM) were heated at 95 °C for 10 min, cooled on ice for 15 min. Then spermine (1 and 2 mM), spermidine (1 mM) or putrescine (1 mM) was added to samples. All samples were stored at 4 °C for over 4 hours before CD scan. To facilitate comparisons, the CD spectra were background subtracted, smoothed, and calibrated for concentration so that molar ellipticities could be obtained. The UV/Vis spectra were gained with CD scan simultaneously.

The activity enhancement of low concentration of DNAzymes

The PS2.M solutions with different concentration (0.05, 0.1, 0.2, 0.4, 0.6, 0.8 and 1 μM) were heat-denatured and renatured. Then 1 mM spermine was added to these samples. The mixtures were allowed to incubate at 25 °C for 30 min, and held for another 10 min after the addition of hemin (1 μM). Then 0.2 mM of ABTS and 0.4 mM of H₂O₂ were added to the mixtures. The absorption spectra were collected on a 96-well plate in a Molecular Devices Spectramax M5 plate reader after 3 min of reaction.

Results and discussion

Effects of polyamines on G4/hemin DNAzymes

In previous research, some G4s were found to bind amino group rich peptide and materials stronger than double-stranded DNAs or single-stranded DNAs do.^35–37 The interaction of polyamines with G4s was also reported.^13,14,38 Additionally, ammonium buffer has also been report to favour the G4/hemin DNAzymes.^22,31–34 All these inspired us to investigate whether polyamines have any effect on G4/hemin DNAzymes. The G4/hemin catalyzed ABTS–H₂O₂ system was used to perform this investigation. The reaction product, a colored radical cation ABTS˙⁻, exhibits absorption maxima at wavelengths 414 nm, 660 nm, 734 nm and 815 nm, in which the absorption at 414 nm is commonly used to indicate the reaction.³⁹ Three common biogenic polyamines, spermine, spermidine and putrescine were tested. As shown in Fig. 1, polyamines did not have significant effect on the non-G4/hemin (T18/hemin) system, but had positive effect on the G4/hemin (PS2.M/hemin) catalytic system. In the absence of polyamine, the absorption bands of ABTS˙⁻ in PS2.M/hemin system were weak (control); whereas in the presence of spermine and spermidine these bands greatly increased, and the reaction solution became deep green (Fig. 1b). These results suggest that spermine and spermidine indeed enhanced the catalytic efficiency of PS2.M/hemin DNAzyme, and could not catalyze the oxidation of ABTS in the absence of G4s. Putrescine only has very slightly effect on the catalytic efficiency of PS2.M/hemin DNAzyme, suggesting that the catalysis enhancement may related to the length of polyamine chain and the number of amine groups. The efficiency of catalysis enhancement of PS2.M/hemin DNAzyme was observed to increase with the increase of the concentration of spermine and reached a plateau at 1.0 mM (Fig. 1c), which is much smaller than the concentration of ammonium used for enhancing the G4/hemin DNAzyme activity.^22,31–34 It is well known that ammonium cation locate between the G-quartet planes, this set of results indicates that the enhancement of PS2.M/hemin DNAzyme activity by spermine and spermidine has different mechanism from that of ammonium cation.

Fig. 1 (a and b) Effects of polyamines (1.0 mM) on the catalytic activity of T18/hemin (a) and PS2.M/hemin DNAzyme (b) on the catalytic oxidation of ABTS, [T18] = 2.0 μM, [PS2.M] = 0.5 μM, [hemin] = 1.0 or 4.0 μM, [ABTS] = 0.4 mM, [H₂O₂] = 0.6 mM. (c) Plot of absorbance at 414 nm of PS2.M/hemin DNAzyme reaction solution versus the concentration of spermine (0.1, 0.3, 0.5, 0.8, 1.0, 3.0, 5.0 mM), [PS2.M] = 0.5 μM, [hemin] = 1.0 μM, [ABTS] = 0.2 mM, [H₂O₂] = 0.5 mM. (d) Effects of polyamines on the catalytic oxidation of TMB by PS2.M/hemin DNAzyme, [PS2.M] = 3.0 μM, [hemin] = 3.0 μM. All the measurements were performed at 3 min after the reaction started. “ctr” is control sample without polyamine addition.

To exclude the possibility that the catalytic activity enhancement may only take place in ABTS–H₂O₂ system, the enhancement efficiency was also tested by using another commonly used substrate, TMB instead of ABTS. The absorbance at 650 nm was used to indicate the oxidation of TMB.^40,41 As shown in Fig. 1d, polyamines also enhanced the catalytic activity of PS2.M/hemin DNAzyme on the oxidation of TMB; the order of the enhancement extent is comparable with that on the oxidation of ABTS. These results suggest that the positive effect on G4/hemin peroxidase is not limited to the specific substrate, ABTS.

As the pH value can influence the catalytic oxidation of ABTS by G4/hemin DNAzymes, we also measured the dynamic process of the reaction in the presence or absence of spermine under different pH condition. As shown in Fig. 2, spermine enhanced the catalytic activity of PS2.M/hemin DNAzyme at all the tested pH values (4.0–9.0), and the largest enhancement was observed at the pH range from 6–7.

Fig. 2 Effect of spermine on the dynamic process of G4/hemin DNAzyme reaction under different pH conditions. [PS2.M] = 0.5 μM, [hemin] = 1.0 μM, [ABTS] = 0.4 mM, [H₂O₂] = 0.4 mM.

To determine the universality of the positive effect of polyamines on G4/hemin DNAzymes, we investigated the catalytic reaction by other 13 G4/hemin DNAzymes (Fig. S1†). The structures of these G4s include intermolecular parallel, intramolecular parallel, intramolecular anti-parallel and intramolecular mixed-parallel types. Since intramolecular parallel G4s possess strong peroxidase activity when they complex with hemin,^17,19,22 the concentration of intramolecular parallel G4s used for this study was 0.5 μM, and that of other types was 2 μM. Similar to the effect on PS2.M/hemin DNAzyme, three polyamines enhanced the catalytic activity of all the tested G4/hemin DNAzymes; and the order of the enhancement efficiency was spermine > spermidine > putrescine, which is consistent with the number of amine groups on each polyamine. For purposes of comparison, we further measured the initial velocity (V_i) of 14 G4/hemin DNAzyme reactions in the presence and absence of polyamines (Table 1). It can be found that the catalytic activity of G4/hemin DNAzymes mainly depended on the G4 structures, in which intramolecular parallel G4s have stronger catalytic activity than other G4s. However the extent (or ratio) of activity enhancement by polyamines did not depend on whether G4 adopted parallel or anti-parallel structures. The activity enhancements of the G4s with high peroxidase activity (such as EAD, CatG4 and c-MYC, <2-fold) were weaker than that of G4s with lower activity. PS2.M and AS1411 exhibited relatively weak peroxidase activity among the parallel G4s, but their activity enhancement arrived 6-fold (the largest) and 3.6-fold.

Protective effect of spermine on the degradation of hemin

In previous work, we have demonstrated that the inactivation of G4/hemin DNAzyme is mainly attributed to the degradation of hemin by H₂O₂.³⁰ Inhibition of hemin degradation would enhance the apparent efficiency of G4/hemin DNAzyme, therefore we investigated whether spermine prevents the degradation of hemin. The absorption bands of hemin in the range of 350–700 nm reflect the environment and binding state of hemin. Oxidative degradation of hemin leads to the elimination of the Soret band (370–420 nm).^30,42 As shown in Fig. 3a, the addition of PS2.M to hemin caused significant increase of Soret band, indicating the formation of PS2.M/hemin complex.³⁰ Further addition of spermine to PS2.M/hemin complex caused significant broadening of the Soret band and hyperchromicity of the visible spectra, which suggests that spermine changed the binding state of hemin to PS2.M, and the changed binding state may be in favor of the catalytic oxidation of G4/hemin DNAzyme. The addition of H₂O₂ to the PS2.M/hemin system (without spermine) caused a sharp decrease (54.2%) of the Soret band, indicating the degradation of hemin. This Soret band decrease was also observed in the PS2.M/hemin system in the presence of spermine, but the extent of the decrease (21.8%) was much smaller than that in the absence of spermine. As shown in Fig. 3b, the addition of spermine to hemin solution (without G4) only caused a slight change of the UV/Vis spectra of hemin. The addition of H₂O₂ to hemin solutions (without G4) in the presence and absence of spermine also caused decrease of the Soret bands, and the extent of the decrease in the presence of spermine (17.9%) was also smaller than that in the absence of spermine (26.4%). These results suggest that spermine has the suppression effect on the degradation of hemin by H₂O₂ and could prolong the active time of the G4/hemin DNAzymes. The interaction between biogenic polyamines and DNA has been shown to protect DNA from reactive oxygen species;^2,43 similar protection mechanism of spermine may also apply to hemin, especially to hemin that binds tightly to the G4 DNA.

Fig. 3 The effect of spermine on the degradation of hemin. For samples containing PS2.M, PS2.M were incubated with/without spermine at 25 °C for 30 min, and then incubated with hemin for another 10 min before measuring. For samples containing H₂O₂, the spectra were recorded at 3 min after H₂O₂ addition.

Effects of spermine on the structure of PS2.M

It has been reported that spermine can influence the structure of G4s and i-motifs.¹³ In order to understand the enhancement mechanism of the spermine on the G4/hemin DNAzyme, we investigated the CD and UV/Vis spectral change of PS2.M in the presence and absence of spermine. As shown in Fig. 4a, the addition of spermine caused significant change of the CD spectra of PS2.M, including a slight red-shift and sharp decrease of the characteristic bands of parallel G4s around 240 nm and 262 nm, and a significant increase of the CD signal from 295–450 nm. The extent of CD signal change enhanced with the increase of spermine concentration. Meanwhile the UV/Vis spectra showed that the peak at 255 nm decreased sharply, and the absorbance from 300 nm to 450 nm increased upon the addition of spermine; the extent of absorbance change enhanced with the increase of spermine concentration (Fig. 4b). These results are consistent well with the results reported by Li and coworkers about the effect of spermine on a parallel G4, c-Kit2. They have demonstrated that the decrease of the characteristic CD signal of G4 and the increase of the absorbance in long wavelength region (300–450 nm) are due to the condensation of parallel G4s into microaggregates induced by spermine.¹³ It has been reported that the surface of guanine quartets provide the hydrophobic binding sites for hemin to form the hexacoordinate heme catalytic center.^15,30 Monchaud and coworkers⁴⁴ have reported that multimeric quadruplexes have higher peroxidatic activity than monomeric quadruplex at the same concentration of quadruplex units, which is attributed to the formation of high-activity hemin-binding sites (more hydrophobic) between two quadruplex units. D'Agostino and coworkers⁴⁵ have reported that polyamines self-assemble with phosphate ions in phosphate buffer and form a cyclic structure aggregates. The aggregates interact with DNA and form a tube-like arrangement around DNA. Therefore, it can be considered that the interaction of G4 with spermine induces the formation of G4-spermine (micro)aggregates, the aggregates provide a more hydrophobic binding site for hemin, and a better microenvironment for the formation and stabilization of ferryl heme intermediates, and thus results in the enhancement of catalytic activity.

Fig. 4 Effects of spermine on the CD (a) and UV/Vis (b) spectra of PS2.M. [PS2.M] = 3 μM, [spermine] = 1 and 2 mM. The UV/Vis spectra were gained with CD scan simultaneously.

The effects of spermine on the CD and UV/Vis spectra of the non-G4 DNA (T18) and other G4s were also investigated (Fig. S2†). Spermine did not show significant effect on the CD and UV/Vis spectra of non-G4 DNA (T18), but decreased the characteristic signals of the CD and UV/Vis spectra of other G4s. A correlation could be found between the enhancement of catalytic activity and the change of the CD and UV/Vis spectra of G4s, the higher the ratio of V_i in the presence and absence of spermine, the larger the change of CD and UV/Vis signals. These results suggest that the enhancement of catalytic activity of G4s may relate to their structure change induced by spermine. The G4 condensation or aggregation induced by spermine may be one of the reasons of the enhancement of G4/hemin DNAzyme activity. Unlike spermine, spermidine and putrescine did not show significant effect on the CD and UV/Vis spectra of PS2.M (Fig. S3†), which corresponded to their weak enhancement efficiency on the catalytic activity.

The enhancement efficiency on low concentration of G4/hemin DNAzyme

Peroxidases are powerful tools used for signal amplification in sensor design. Because of the advantages of G4/hemin DNAzyme, different G4 forming sequences have been extensively used in the design of various chemical sensors and biosensors. In many sensors, the concentration of the resulting DNAzyme is very low when they response to a low concentration of analyte.^23,46 Enhancement of the catalytic activity of low concentration of DNAzyme will increase the sensitivity of sensors. As shown in Fig. 5, the signal strength of 1.0 μM PS2.M reaction system with 1.0 mM spermine was about four-fold higher than that of the reaction system without spermine. And the signal strength of 0.1 μM PS2.M reaction system with 1.0 mM spermine reached the same degree of 1.0 μM PS2.M reaction system without spermine. The activity enhancement of low concentration of DNAzyme by spermine suggests that the addition of spermine may increase the sensitivity of the G4/hemin based sensors and may cut down the amount of used G4 DNAs in sensor development. In addition, the activity enhancement of G4/hemin DNAzymes by spermine greatly increased the reaction rate of the catalytic reaction (Table 1), which would greatly increase the response speed of detection and save the testing time.

Fig. 5 Effects of spermine on different concentration of PS2.M/hemin DNAzyme. [PS2.M] = 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 μM, [spermine] = 1.0 mM, [hemin] = 1.0 μM, [ABTS] = 0.2 mM, [H₂O₂] = 0.4 mM, 1.0 μM PS2.M in the absence of spermine was used as control. “+”: in the presence of spermine; “−”: in the absence of spermine.

Above results have demonstrated that spermine has a strong enhancement efficiency on the catalytic oxidation activity of G4/hemin complexes. Spermine is widely distributed in cells and involves in cellular metabolism and growth regulation.⁴⁷ Hemin is the oxidized form of heme that serves as the prosthetic group of numerous hemoproteins (e.g., hemoglobin, myoglobin, cytochromes, guanylate cyclase, catalase and nitric oxide synthase, etc.).⁴⁸ Hemin has also been reported to release from hemoglobin during several pathological states.^49,50 Genome-wide bioinformatic searches have revealed that putative G-quadruplex-forming sequences are highly prevalent in human genes.^10,11 The activity enhancement of G4/hemin DNAzymes by spermine implies that spermine may play a potential role in hemin-mediated cellular events (e.g., cellular injury). Furthermore because of the great promise of G4/hemin DNAzymes on the sensor development and signal amplification in bioanalysis, this finding may provide new option for the design of the G4/hemin based assays. This finding may also widen the applications of G4/hemin DNAzyme in other areas besides analysis and testing technology.

Conclusions

The effects of biogenic polyamines (spermine, spermidine and putrescine) on G4/hemin peroxidase activity were studied. Three polyamines were found to have positive effects on different G4/hemin peroxidases, in which spermine exhibits the strongest enhancement efficiency. UV/Vis spectral analysis of hemin in the presence of G4s and H₂O₂ showed that spermine could protect hemin from rapid degradation by H₂O₂. CD and UV/Vis spectral analysis of G4s showed that spermine could condense G4 into orderly microaggregates and provide favorable microenvironment for the catalytic reactions. This finding of the enhancement effect of spermine on G4/hemin DNAzyme would improve the performance of this kind of DNAzyme, and make the G4/hemin based chemical sensors more efficiency.

Abbreviations

G4s	G-quadruplexes
Spm	Spermine
Spd	Spermidine
Put	Putrescine
CD	Circular Dichroism
TMB	3,3′,5,5′-Tetramethylbenzidine
ABTS	2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt
T18	TTT TTT TTT TTT TTT TTT
GT12	GGG TTT GGG TTT
A4G8A4	AAA AGG GGG GGG AAA A
A4G4A4	AAA AGG GGA AAA
HT	TTA GGG TTA GGG TTA GGG TTA GGG
Oxy28	GGG GTT TTG GGG TTT TGG GGT TTT GGG G
ILPR	TAC AGG GGT GTG GGG ACA GGG GTG TGG GG
TBA	GGT TGG TGT GGT TGG
EAD	CTG GGT GGG TGG GTG GGA
PS2.M	GTG GGT AGG GCG GGT TGG
VEGF	GGG CGG GCC GGG GGC GGG
CatG4	TGG GTA GGG CGG GTT GGG AAA
c-MYC	TGA GGG TGG GGA GGG TGG GGA A
Bcl2	GGG CGC GGG AGG AAG GGG GCG GG
AS1411	GGT GGT GGT GGT TGT GGT GGT GGT GG.

Acknowledgements

We gratefully acknowledge the financial support from Grant 973 Program (2011CB935800 and 2013CB33700) and NSF of China (21275149, 21205124, 21375135 and 21321003).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Reagents and instruments; effects of polyamines on the catalytic activity of G-quadruplex-hemin DNAzymes; effects of spermine on the CD and UV/Vis spectra of different types of G-quadruplexs; effects of spermidine and putrescine on the CD and UV/Vis spectra of PS2.M. See DOI: 10.1039/c3ra45429k

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