Beyond G-Quadruplexes-The Effect of Junction with Additional Structural Motifs on Aptamers Properties.

G-quadruplexes constitute an important type of nucleic acid structure, which can be found in living cells and applied by cell machinery as pivotal regulatory elements. Importantly, robust development of SELEX technology and modern, nucleic acid-based therapeutic strategies targeted towards various molecules have also revealed a large group of potent aptamers whose structures are grounded in G-quadruplexes. In this review, we analyze further extension of tetraplexes by additional structural elements and investigate whether G-quadruplex junctions with duplex, hairpin, triplex, or second G-quadruplex motifs are favorable for aptamers stability and biological activity. Furthermore, we indicate the specific and pivotal role of the G-quadruplex domain and the additional structural elements in interactions with target molecules. Finally, we consider the potency of G-quadruplex junctions in future applications and indicate the emerging research area that is still waiting for development to obtain highly specific and effective nucleic acid-based molecular tools.

G4 Matters-The Influence of G-Quadruplex Structural Elements on the Antiproliferative Properties of G-Rich Oligonucleotides.

G-quadruplexes (G4s) are non-canonical structures formed by guanine-rich sequences of DNA or RNA that have attracted increased attention as anticancer agents. This systematic study aimed to investigate the anticancer potential of five G4-forming, sequence-related DNA molecules in terms of their thermodynamic and structural properties, biostability and cellular uptake. The antiproliferative studies revealed that less thermodynamically stable G4s with three G-tetrads in the core and longer loops are more predisposed to effectively inhibit cancer cell growth. By contrast, highly structured G4s with an extended core containing four G-tetrads and longer loops are characterized by more efficient cellular uptake and improved biostability. Various analyses have indicated that the G4 structural elements are intrinsic to the biological activity of these molecules. Importantly, the structural requirements are different for efficient cancer cell line inhibition and favorable G4 cellular uptake. Thus, the ultimate antiproliferative potential of G4s is a net result of the specific balance among the structural features that are favorable for efficient uptake and those that increase the inhibitory activity of the studied molecules. Understanding the G4 structural features and their role in the biological activity of G-rich molecules might facilitate the development of novel, more potent G4-based therapeutics with unprecedented anticancer properties.

G-Quadruplex-Forming Aptamers-Characteristics, Applications, and Perspectives.

G-quadruplexes constitute a unique class of nucleic acid structures formed by G-rich oligonucleotides of DNA- or RNA-type. Depending on their chemical nature, loops length, and localization in the sequence or structure molecularity, G-quadruplexes are highly polymorphic structures showing various folding topologies. They may be formed in the human genome where they are believed to play a pivotal role in the regulation of multiple biological processes such as replication, transcription, and translation. Thus, natural G-quadruplex structures became prospective targets for disease treatment. The fast development of systematic evolution of ligands by exponential enrichment (SELEX) technologies provided a number of G-rich aptamers revealing the potential of G-quadruplex structures as a promising molecular tool targeted toward various biologically important ligands. Because of their high stability, increased cellular uptake, ease of chemical modification, minor production costs, and convenient storage, G-rich aptamers became interesting therapeutic and diagnostic alternatives to antibodies. In this review, we describe the recent advances in the development of G-quadruplex based aptamers by focusing on the therapeutic and diagnostic potential of this exceptional class of nucleic acid structures.

Homocysteine thiolactone and N-homocysteinylated protein induce pro-atherogenic changes in gene expression in human vascular endothelial cells.

Genetic or nutritional deficiencies in homocysteine (Hcy) metabolism lead to hyperhomocysteinemia (HHcy) and cause endothelial dysfunction, a hallmark of atherosclerosis. In addition to Hcy, related metabolites accumulate in HHcy but their role in endothelial dysfunction is unknown. Here, we examine how Hcy-thiolactone, N-Hcy-protein, and Hcy affect gene expression and molecular pathways in human umbilical vein endothelial cells. We used microarray technology, real-time quantitative polymerase chain reaction, and bioinformatic analysis with PANTHER, DAVID, and Ingenuity Pathway Analysis (IPA) resources. We identified 47, 113, and 30 mRNAs regulated by N-Hcy-protein, Hcy-thiolactone, and Hcy, respectively, and found that each metabolite induced a unique pattern of gene expression. Top molecular pathways affected by Hcy-thiolactone were chromatin organization, one-carbon metabolism, and lipid-related processes [-log(P value) = 20-31]. Top pathways affected by N-Hcy-protein and Hcy were blood coagulation, sulfur amino acid metabolism, and lipid metabolism [-log(P value)] = 4-11; also affected by Hcy-thiolactone, [-log(P value) = 8-14]. Top disease related to Hcy-thiolactone, N-Hcy-protein, and Hcy was 'atherosclerosis, coronary heart disease' [-log(P value) = 9-16]. Top-scored biological networks affected by Hcy-thiolactone (score = 34-40) were cardiovascular disease and function; those affected by N-Hcy-protein (score = 24-35) were 'small molecule biochemistry, neurological disease,' and 'cardiovascular system development and function'; and those affected by Hcy (score = 25-37) were 'amino acid metabolism, lipid metabolism,' 'cellular movement, and cardiovascular and nervous system development and function.' These results indicate that each Hcy metabolite uniquely modulates gene expression in pathways important for vascular homeostasis and identify new genes and pathways that are linked to HHcy-induced endothelial dysfunction and vascular disease.

Unlocked nucleic acids: implications of increased conformational flexibility for RNA/DNA triplex formation.

Unlocked nucleic acids (UNAs) have been introduced at specific positions in short model DNA hairpins and RNA/DNA triplexes for the first time. UNA residues destabilize the hairpins and decrease triplex thermodynamic stability or suppress triplex formation for most of the evaluated structures. Nevertheless, the incorporation of UNA residues at certain positions of dsDNA was found to be energetically favourable or at least did not affect triplex stability. Notably, the most thermodynamically stable UNA-modified triplexes exhibited improved stability at both acidic and physiological pH. The specificity of the interactions between the triplex-forming oligonucleotide and dsDNA was characterized using EMSA for the most thermodynamically stable structures, and triplex dissociation constants were determined. One of the modified triplexes exhibited an improved Kd in comparison with the unmodified triplex. CD and thermal difference spectra indicated that UNA residues do not alter the overall structure of the most thermodynamically stable triplexes. In addition, incubation of the modified oligonucleotides with human serum indicated that the UNAs demonstrate the potential to improve the biological stability of nucleic acids.

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

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.

A Comprehensive Analysis of the Thrombin Binding Aptamer Containing Functionalized Pyrrolo-2'-deoxycytidines.

Aptamers constitute an answer for the growing need for targeted therapy development. One of the most well-known representatives of this group of compounds is thrombin binding aptamers (TBA) targeted towards thrombin. The TBA inhibitory activity is determined by its spatial arrangement, which consists of two G-tetrads linked by two shorter TT loops and one longer TGT loop and folds into a unimolecular, antiparallel G-quadruplex structure. Interesting properties of the aptamer can be further improved via the introduction of a number of chemical modifications. Herein, a comprehensive analysis of the influence of pyrrolo-2'-deoxycytidine (Py-dC) and its derivatives on TBA physicochemical and biological properties has been presented. The studies have shown that the presence of modified residues at the T7 position of the TGT loop has only minor effects on TBA thermodynamic stability without affecting its folding topology. All analyzed oligomers exhibit anticoagulant properties, but only aptamer modified with a decyl derivative of Py-dC was able to inhibit thrombin activity more efficiently than unmodified, parental compounds. Importantly, the same compound also possessed the potential to effectively restrain HeLa cell line growth.

Improved RE31 Analogues Containing Modified Nucleic Acid Monomers: Thermodynamic, Structural, and Biological Effects.

RE31 is a 31-nt DNA aptamer, consisting of the G-quadruplex and a duplex domain, which is able to effectively prolong thrombin time. This article reports on the influence of certain modified nucleotide residues on thermodynamic and biological properties as well as the folding topology of RE31. Particularly, the effect of the presence of nucleosides in unlocked nucleic acid (UNA), locked nucleic acid (LNA), or ß-l-RNA series was evaluated. The studies presented herein show that all modified residues can influence thermal and biological stabilities of G-quadruplex in a position-dependent manner. The aptamers modified simultaneously with UNA at the T15 position and LNAs in the duplex part possess the highest value of melting temperature and a 2-fold higher anticoagulant effect. Importantly, RE31 variants modified with nucleosides in UNA, LNA, or ß-l-RNA series exhibit unchanged G-quadruplex folding topology. Crucially, introduction of any of the modified residues into RE31 causes prolongation of aptamer stability in human serum.

Novel isoguanine derivative of unlocked nucleic acid-Investigations of thermodynamics and biological potential of modified thrombin binding aptamer.

Thrombin binding aptamer (TBA), is a short DNA 15-mer that forms G-quadruplex structure and possesses anticoagulant properties. Some chemical modifications, including unlocked nucleic acids (UNA), 2'-deoxy-isoguanosine and 2'-deoxy-4-thiouridine were previously found to enhance the biological activity of TBA. In this paper, we present thermodynamic and biological characteristics of TBA variants that have been modified with novel isoguanine derivative of UNA as well as isoguanosine. Additionally, UNA-4-thiouracil and 4-thiouridine were also introduced simultaneously with isoguanine derivatives. Thermodynamic analysis indicates that the presence of isoguanosine in UNA or RNA series significantly decreases the stability of G-quadruplex structure. The highest destabilization is observed for substitution at one of the G-tetrad position. Addition of 4-thiouridine in UNA or RNA series usually decreases the unfavorable energetic cost of the presence of UNA or RNA isoguanine. Circular dichroism and thermal denaturation spectra in connection with thrombin time assay indicate that the introduction of UNA-isoguanine or isoguanosine into TBA negatively affects G-quadruplex folding and TBA anticoagulant properties. These findings demonstrate that the highly-ordered structure of TBA is essential for inhibition of thrombin activity.

Thermodynamic, Anticoagulant, and Antiproliferative Properties of Thrombin Binding Aptamer Containing Novel UNA Derivative.

Thrombin is a serine protease that plays a crucial role in hemostasis, fibrinolysis, cell proliferation, and migration. Thrombin binding aptamer (TBA) is able to inhibit the activity of thrombin molecule via binding to its exosite I. This 15-nt DNA oligonucleotide forms an intramolecular, antiparallel G-quadruplex structure with a chair-like conformation. In this paper, we report on our investigations on the influence of certain modified nucleotide residues on thermodynamic stability, folding topology, and biological properties of TBA variants. In particular, the effect of single incorporation of a novel 4-thiouracil derivative of unlocked nucleic acid (UNA), as well as single incorporation of 4-thiouridine and all four canonical UNAs, was evaluated. The studies presented herein have shown that 4-thiouridine in RNA and UNA series, as well as all four canonical UNAs, can efficiently modulate G-quadruplex thermodynamic and biological stability, and that the effect is strongly position dependent. Interestingly, TBA variants containing the modified nucleotide residues are characterized by unchanged folding topology. Thrombin time assay revealed that incorporation of certain UNA residues may improve G-quadruplex anticoagulant properties. Noteworthy, some TBA variants, characterized by decreased ability to inhibit thrombin activity, possess significant antiproliferative properties reducing the viability of the HeLa cell line even by 95% at 10 µM concentration.

Studies on Transcriptional Incorporation of 5'-N-Triphosphates of 5'-Amino-5'-Deoxyribonucleosides.

In this study, several RNA polymerases were used for the first time to examine the possibility of transcriptional incorporation of 5'-N-triphosphates of 5'-amino-5'-deoxyribonucleosides (5'NH NTPs). The T3, T7, Sp6 and T7 Y639F RNA polymerases were employed to show that the full-length transcript cannot be synthesized. The results suggest that the application of 5'NH NTPs could decrease transcription reaction rates. What is more, the modification of transcription conditions had no influence on the rate of 5'NH NTPs incorporation. Based on experimental data it is postulated that 5'NH NTPs can be used as potential transcription inhibitors. Our findings expand the knowledge on suitable uses of the 5'-N-triphosphates of 5'-amino-5'-deoxyribonucleoside and the exact mechanism of transcriptional inhibition.

Thermodynamic Features of Structural Motifs Formed by ß-L-RNA.

This is the first report to provide comprehensive thermodynamic and structural data concerning duplex, hairpin, quadruplex and i-motif structures in ß-L-RNA series. Herein we confirm that, within the limits of experimental error, the thermodynamic stability of enantiomeric structural motifs is the same as that of naturally occurring D-RNA counterparts. In addition, formation of D-RNA/L-RNA heterochiral duplexes is also observed; however, their thermodynamic stability is significantly reduced in reference to homochiral D-RNA duplexes. The presence of three locked nucleic acid (LNA) residues within the D-RNA strand diminishes the negative effect of the enantiomeric, complementary L-RNA strand in the formation of heterochiral RNA duplexes. Similar behavior is also observed for heterochiral LNA-2'-O-methyl-D-RNA/L-RNA duplexes. The formation of heterochiral duplexes was confirmed by 1H NMR spectroscopy. The CD curves of homochiral L-RNA structural motifs are always reversed, whereas CD curves of heterochiral duplexes present individual features dependent on the composition of chiral strands.

Hybridization Properties of RNA Containing 8-Methoxyguanosine and 8-Benzyloxyguanosine.

Modified nucleobase analogues can serve as powerful tools for changing physicochemical and biological properties of DNA or RNA. Guanosine derivatives containing bulky substituents at 8 position are known to adopt syn conformation of N-glycoside bond. On the contrary, in RNA the anti conformation is predominant in Watson-Crick base pairing. In this paper two 8-substituted guanosine derivatives, 8-methoxyguanosine and 8-benzyloxyguanosine, were synthesized and incorporated into oligoribonucleotides to investigate their influence on the thermodynamic stability of RNA duplexes. The methoxy and benzyloxy substituents are electron-donating groups, decreasing the rate of depurination in the monomers, as confirmed by N-glycoside bond stability assessments. Thermodynamic stability studies indicated that substitution of guanosine by 8-methoxy- or 8-benzyloxyguanosine significantly decreased the thermodynamic stability of RNA duplexes. Moreover, the presence of 8-substituted guanosine derivatives decreased mismatch discrimination. Circular dichroism spectra of modified RNA duplexes exhibited patterns typical for A-RNA geometry.