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. 2022 Oct 6;14(1):35–40. doi: 10.1021/acsmedchemlett.2c00361

Physicochemical and antiproliferative characteristics of RNA and DNA sequence-related G-quadruplexes

Weronika Kotkowiak 1, Carolina Roxo 1, Anna Pasternak 1,*
PMCID: PMC9841586  PMID: 36655120

Abstract

graphic file with name ml2c00361_0003.jpg

In this article the physicochemical and biological properties of sequence-related G-quadruplex forming oligonucleotides in RNA and DNA series are analyzed and compared. The intermolecular G-quadruplexes vary in loop length, number of G-tetrads and homogeneity of the core. Our studies show that even slight variations in sequence initiate certain changes of G-quadruplex properties. DNA G-quadruplexes are less thermally stable than their RNA counterparts, more topologically diversified and are better candidates as inhibitors of cancer cells proliferation. The most efficient antiproliferative activity within the studied group of molecules was observed for two DNA G-quadruplexes with unperturbed core and lower content of thymidine residues within the loops leading to reduction of cells viability up to 65% and 33% for HeLa and MCF-7 cell lines, respectively.

Keywords: G-quadruplex, UV melting, circular dichroism, antiproliferative activity, RNA, DNA

Cancer disease constitutes one of the leading causes of death worldwide. The reason for the above can be traced, among others, to the complicated nature of the disease, which entails a constant need to improve currently available therapies. The great improvement of knowledge about a range of changes in cell biology, which lead to cancer development and progression, facilitates the growth of a wide variety of new diagnostic and therapeutic agents. One of the extensively examined research targets is guanosine-rich oligonucleotides (GROs) due to their favorable physicochemical and biological properties such as resistance to nucleases, enhanced cellular uptake, and high affinity toward proteins.1 The main relevant feature of this group of DNA or RNA oligonucleotides is their propensity to form G-quadruplex (G4) structures, that is, structural arrangements that consist of at least two G-tetrads connected by various types of loops: diagonal, lateral, and propeller.2 The G-tetrad itself has planar geometry and is composed of four guanine residues stabilized by Hoogsteen hydrogen bonds.3 Based on the number of forming strands, G-quadruplexes can be divided into two groups: intramolecular, formed by one strand, and intermolecular, containing two or more strands.4 What is more, the orientation of strands within the G4 core results in the appearance of different folding topologies: parallel (all strands have the same orientation), antiparallel (strands are oriented oppositely to each other), and hybrid (one strand has different orientation in relation to the others).5,6 All above-mentioned structural features are relevant and could influence the potential of G4 to act as a therapeutic agent. During the past few years, there was a growing number of short GROs that have been found as biologically active against various cancer cell lines.1,7−12 Their antiproliferative and proapoptotic effects have been often attributed to the ability to form stable G-quadruplex structures, although the details about the desirable spatial arrangement required to obtain efficient biological activity are still not fully recognized.

In this paper we have investigated for the first time the structural aspects of a set of sequence-related RNA G-rich sequences to find subtle correlation between the structure of model intermolecular G-quadruplexes, their thermal stability, and biological activity and compared them with the properties of their DNA counterparts. In detail, we performed a systematic analysis of physicochemical and biological properties of a series of sequentially closely related intermolecular RNA G-quadruplexes with slight variation in loop length or number of G-tetrads building their core. What is more, attempts to find a structurally functional relationship that could be useful during development of potential therapeutic agents with anticancer properties were also made.

As an initial sequence we decided to choose RNA oligonucleotide composed of two 4nt long G-tracts connected by a 4nt long U-tract (OR1, Table 1), which was also treated as a point of reference for all subsequent analyses. This sequence is an RNA counterpart of DNA oligonucleotide with a well-defined structure of intermolecular, antiparallel G-quadruplex in the presence of potassium ions.13 Further alteration in composition of G-tetrads and loop lengths of OR1 led to the setting up of a group of four closely related RNA sequences (Table 1). What is more, the interpretation of the results of the physicochemical and biological analysis was extended to the data concerning DNA oligonucleotides with the same sequences as RNA counterparts, some of which have been already reported by our research group.14 Due to the presented approach, the attempts to draw some general rules useful during designing oligomers with potential therapeutic properties have been made.

Table 1. Thermal Stability of G-Quadruplexesa.

name sequence (5′-3′) TMb (°C)
OR1 GGGGUUUUGGGG 73.8
OR2 GGGUUUUGGGG 67.4
OR3 UGGGGUUGGGGU 75.9
OR4 GGAGGUUUUGGAGG 59.9
OD1c GGGGTTTTGGGG 70.6
OD2c GGGTTTTGGGG 56.6
OD3 TGGGGTTGGGGT 72.9
OD4 GGAGGTTTTGGAGG 20.4
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a

Buffer: 100 mM KCl, 20 mM sodium cacodylate, 0.5 mM EDTA(Na)2, pH 7.0.

b

Calculated for 10–4 M oligonucleotide concentration.

c

TM data according to ref (14).

The analysis of physicochemical properties of an analyzed set of oligomers was initiated with thermodynamic studies. To realize this aim the UV melting method has been implemented, which not only allowed us to determine melting temperature (TM) values and confirmed the presence of G-quadruplex structure at physiological temperature but also indicated the molecularity of folding. It has been previously stated that the thermal stability of DNA G4 is strongly dependent on the number of G-tetrads forming the core.14,15 Our studies confirmed that the above rule is also proper for RNA G4. The analysis of the thermal stability of the oligomers revealed that OR1, whose core is formed of four G-tetrads connected by 4nt long loops, has one of the highest TM values (Table 1, TM equal to 73.8 °C). This G4 was also more stable in comparison to its DNA equivalent (Table 1, OD1). Interestingly, the alteration in loop length while the composition of the G4 core is maintained and uridine residues are added at both termini (OR3) resulted in an even more favorable thermal parameter (Table 1, TM was equal to 75.9 °C). The same dependence was observed for its DNA analogues (Table 1, OD3 vs OD1). What is more, the temperature rise in comparison to that of maternal compounds was approximately the same in both cases and equal to 2.1 and 2.3 °C for OR3 and OD3, respectively. The observed tendency points out that, similar to intramolecular RNA16,17 and intermolecular DNA14 G-quadruplexes described formerly, the reduction of loop length causes an increase in the thermal stability of G4. However, previous reports indicate also that the presence of flanking nucleotide residues is rather destabilizing for G4 structures.18,19 Thus, the increase of TM values observed for OR3 and OD3 in reference to OR1 and OD1, respectively, is most probably the sum of a stabilizing effect that originated from loop shortening, which overcomes an unfavorable influence of uridine/thymidine residues, which flank the G-quadruplex motif. The importance of a number of G-tetrads forming the G4 core in assuring its thermal stability was evident for the variant with reduced number of G-tetrads (Table 1, OR2). This G4 is composed of the core formed by three G-tetrads connected by 4nt long loops and one additional guanosine residue at 3′-termini. It was observed that the thermal stability of OR2 was over 6 °C lower than the value determined for OR1 (Table 1, TM was equal to 67.4 °C). Furthermore, the effect of the reduction of a number of G-tetrads was even more noticeable for a DNA variant with the same sequence, and the TM value has been reduced by 14.0 °C in comparison to the TM of the parental DNA compound (Table 1, OD2 vs OD1). Interestingly, equally important as a number of G-tetrads forming the core is also its homogeneity and integrity. The above found its reflection in a significant decrease of thermal stability observed for OR4, in which two adenosine residues are associated into two edges of one G-tetrad leading to formation of the core with one GGGG tetrad and one GGAGGA hexad20,21 (Table 1, TM = 59.9 °C). The lowest thermal stability among the group of studied G-quadruplexes is observed for OD4 (TM = 20.0 °C). In this case, the G-quadruplex contains two single adenosine bulges in the middle of the core, making the structure entirely different from its OR4 RNA counterpart.22 Another important finding, which emerges from the thermodynamic studies, is the molecularity of formed G-quadruplex structures. Due to a versatile analysis of the Tm dependence versus sample concentration, it was possible to conclude that all studied oligomers form intermolecular G-quadruplex structures (see Supporting Information for details).

Based on the data analysis, some general conclusions can be drawn. First of all, RNA G-quadruplexes studied herein are more thermally stable than their DNA counterparts, which is in accordance with previously published observations.23 The above effect can be explained by the presence of 2′-hydroxyl groups, which provide additional stabilization of intramolecular interactions and bind water molecules.23−25 The latter are clustered in grooves and therefore take a part in assuring appropriate G-quadruplex structure.24 What is more, the 2′-hydroxyl groups constitute a spatial hindrance forcing the anti conformation of the nucleic bases, which is preferable during parallel G-quadruplex formation, the mere folding topology presented by RNA GROs.26 It is also worth noting that more compact structures characterized by the presence of longer G-tracts connected by shorter loops exhibited the most favorable thermodynamic effects. Any interferences in this arrangement, either by reducing the number of G-tetrads or disturbance of the integrity of the core through the presence of bulges between G-tetrads or additional residues inside them, resulted in unfavorable influence on the overall G4 thermal stability.

The further physicochemical characterization of analyzed G4s was continued by conducting structural studies. The implementation of circular dichroism (CD) spectroscopy made possible the investigation of the influence of the core and loop alterations within G-quadruplexes on their folding topology. In general, there have been identified three folding topologies of G-quadruplexes: antiparallel (positive ellipticity near 240 and 295 nm; negative ellipticity around 265 nm), parallel (positive ellipticity near 260 nm and negative ellipticity near 240 nm), and hybrid (positive ellipticity near 260 and 295 nm; negative ellipticity near 245 nm).27 Due to the fact that all three types of structures display distinct CD profiles, it is possible to distinguish which G-quadruplex spatial arrangement appears in the case of analyzed oligomers.27−30

According to the above and in order to assess the range of changes in folding topology induced by the alteration of G-quadruplex structure in physiological temperature, we decided to conduct the CD analysis for all analyzed oligomers at 37 °C. As it was previously mentioned, the RNA G-quadruplexes, due to the presence of 2′-hydroxyl groups of ribose residues, fold into parallel structures, and we expected to observe this specific pattern of the CD spectra for OR1–OR4. The results revealed that this observation concerns only three oligomers, that is, OR1, OR2, and OR3, the spectral patterns of which possessed a negative peak around 245 nm and a positive peak around 265 nm (Figure 1). The CD spectrum shape of OR4, apart from two peaks characteristic for parallel G-quadruplexes, had also additional positive signals around 295 nm and above 300 nm (Figure 1). According to previously reported studies OR4 contains one conservative G-tetrad, which stacks over the GGAGGA hexad.20,21 Moreover, two OR4 molecules stack over each other via a hexad–hexad interface with intrastrand parallel orientation and interstrand antiparallel polarity. The disturbance of G-quadruplex core homogeneity and opposite orientation of both dimer units most probably affects the CD pattern of OR4 and results in its changing away from a spectrum shape typical for parallel strand polarity. The folding topology of OD1 and OD2 has been previously determined by our research group as antiparallel and hybrid, respectively14 (Figure 1). Interestingly, the shortening of the loop length of DNA G4 and the presence of two flanking thymidine residues without affecting the G4 core caused the shift in G4 folding topology. The CD spectrum shape for OD3 exhibited a pattern characteristic for parallel G-quadruplex with the negative peak around 245 nm and positive peak around 265 nm (Figure 1). This observation is consistent with previously published data indicating that the presence of shorter loops predisposes G-quadruplexes to adopt a parallel folding topology.31,32 The potential reason for the above changes could be traced to the restriction of structural flexibility caused by a reduction of loop length, which forces the appearance of a parallel G4 structure, being more compact due to the presence of medium width grooves.26 The low-intensity, broad, and positive peak is observed for OD4 at the wavelength range that is rather characteristic for parallel G-quadruplex structures. This observation is seemingly contradictory to the antiparallel G-quadruplex structure reported by the Uesugi group,21,22 and it is probably due to a very minor CD spectrum intensity, which might be attributed to the low thermal stability of OD4 (TM = 20 °C). Additionally, as a supplement of CD spectra the thermal difference spectra (TDS) for all studied oligonucleotides were also analyzed; however, the observed patterns were not conclusive (see Supporting Information).

Figure 1.

Figure 1

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Spectral analysis of G-quadruplex folding topology. Circular dichroism spectra performed at 37 °C of OR1 (black, solid lines), OR2 (red, solid line), OR3 (blue, solid line), OR4 (green, solid line), OD114 (black, dashed line), OD214 (red, dashed line), OD3 (blue, dashed line), and OD4 (green, dashed line).

After determination of the thermal stability and folding topology, we analyzed the antiproliferative potential of studied G-quadruplexes and verified whether any correlation between structure and biological function appears. The above experiments were based on recent findings concerning inhibitory properties of guanosine-rich oligonucleotides toward cancer cell lines. Interestingly, the detailed mechanism of action of GROs remains unclear. One of the theories simply assumes toxicity of G4 degradation products, whereas another indicates a restraint of cancer cell growth via interaction with specific proteins involved in the cell cycle.14,33−37 In order to determine the antiproliferative potential of analyzed G-quadruplexes, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on cervical (HeLa) and breast (MCF-7) cancer cell lines was performed. The basis of the method relies on the ability of mitochondria of living cells to reduce the water-soluble yellow tetrazole salt (MTT) into insoluble dark blue formazan. The quantity of the latter is directly proportional to cell viability. The test for both cell lines was carried out in the same oligonucleotide concentration (10 μM) for 7 d.

The data analysis of the results for RNA G4s indicated that no antiproliferative effect in both cell lines could be observed for OR1 and OR3 (Figure 2, mean cell viability was equal to 100%). The common feature of both G-quadruplexes is the presence of four G-tetrads in the core and high thermal stability, but the latter possesses shorter, 2nt long loops and uridine residues that flank the G4 motif. Importantly, oligomer OR4, which was characterized by the lowest value of TM among all studied RNA G4s and possess a GGAGGA hexagon-modified G-tetrad core, exhibited also poor activity toward cancer cells. A relative improvement in the antiproliferative effect was achieved only via reduction of the number of G-tetrads in the G4 core without affecting loop length and G-quadruplex core homogeneity. The OR2, which possesses the above-mentioned alteration, was able to restrain the HeLa cells’ viability by approximately 30%, but in contrast, in case of MCF-7 cells this value was less favorable and equal to 12% (Figure 2). A similar tendency has been observed among DNA G-quadruplexes. The OD1, which possesses four G-tetrads in the core, has almost no antiproliferative effect in the MCF-7 cell line (Figure 2) with the ability to restrain HeLa cell growth by around 11%14 (Figure 2). The reduction of the G4 core to three G-quartets significantly improved inhibition of HeLa cell line proliferation and resulted in over 65% decrease in cell viability14 (OD2, Figure 2). A noticeable advance was also observed for the MCF-7 cell line, in case of which around 33% of cell growth was inhibited in the presence of the OD2 (Figure 2). Surprisingly, in contrast to RNA G-quadruplexes, the reduction of loop length and the presence of thymidine residues flanking the unaffected G4 core (OD3) resulted in a meaningful increase of antiproliferative activity toward the HeLa cell line (Figure 2, cell viability was equal to 40%). A slight improvement was also noticed for MCF-7, but the decrease in cell viability was only at the level of 14% (Figure 2). The isosequential DNA counterpart of OR4 (OD4), which folds into a G-quadruplex structure with bulged adenosine residues in the middle of the core, exhibited also poor antiproliferative activity reducing HeLa and MCF-7 cell viability by 10% and 19.5%, respectively. This might be attributed to the loss of G-quadruplex core integrity but is rather due to the lack of the G-quadruplex structure formation in physiological temperature as indicated by melting temperature of this variant.

Figure 2.

Figure 2

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Antiproliferative activity of G-quadruplexes. The viability of HeLa (solid bars) and MCF-7 (patterned bars) cells in the presence of OR1 (gray), OR2 (red), OR3 (blue), OR4 (magenta), OD114 (olive), OD214 (navy), OD3 (violet), and OD4 (yellow). All data are presented as the mean ± standard error of measurement from two independent experiments.

Based on the above data analysis it could be concluded that particular DNA G-quadruplexes possess substantial antiproliferative activity in comparison to RNA counterparts. What is more, herein, the former conclusions, indicating that G4 with shorter G-tetrad core and longer loops exhibited greater potential to restrain cancer cell growth,14 were also confirmed and are reflected in our experimental results. However, it was observed that not necessarily thermal stability itself but folding topology in a DNA series might be a factor, which should be considered to achieve substantial antiproliferative properties of G-quadruplexes. The most beneficial capability of inhibiting cancer cell viability was noticed for DNA G-quadruplexes with parallel and hybrid types of G4 folding. The above could be justified by previously published observations indicating that parallel G-quadruplexes exhibited superior potential of binding to cell surface in comparison to antiparallel G4s.38

In summary, our investigations showed that G-quadruplex physicochemical and biological properties are strongly dependent on slight variations in sequences. RNA G-quadruplexes are characterized by higher thermal stability in comparison to DNA counterparts, wherein a longer core provides higher values of TM. It is also worth noting that a more favorable antiproliferative effect was observed for DNA G-quadruplexes and surprisingly in this group not thermal stability but folding topology was a more significant factor determining the oligonucleotide inhibitory properties. The comprehensive investigations presented herein constitute a useful background, which could facilitate the designing of G-quadruplex-based drugs with promising anticancer properties in the future.

Acknowledgments

This work was supported by the National Science Center grants 2017/25/B/NZ7/00127 and 2020/37/B/NZ7/02008 to A.P. and 2019/35/N/NZ7/02777 to C.R.

Glossary

Abbreviations

GROs

guanosine-rich oligonucleotides

G4

G-quadruplex

TM

melting temperature

CD

circular dichroism

TDS

thermal difference spectra

HeLa

cervical cancer cell line

MCF-7

breast cancer cell line

MTT

yellow tetrazole salt (chemical name: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsmedchemlett.2c00361.

  • Materials and methods section, MALDI-MS data, graphs of Tm dependence versus sample concentration of oligonucleotides (PDF)

Author Contributions

W.K. performed experiments for RNA G-quadruplexes, wrote the paper, and reviewed and edited the article draft; C.R. performed experiments for DNA G-quadruplexes; A.P. performed oligonucleotide synthesis, conceptualized and supervised the experiments, and reviewed and edited the article draft.

The authors declare no competing financial interest.

Supplementary Material

ml2c00361_si_001.pdf (312.5KB, pdf)

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