Defined folding pattern of poly(rG) supports inherent ability to encode biological information.

The RNA World hypothesis posits that RNA can represent a primitive life form by reproducing itself and demonstrating catalytic activity. However, this hypothesis is incapable of addressing several major origin-of-life (OoL) questions. A recently described paradox-free alternative OoL hypothesis, the Quadruplex (G4) World, is based on the ability of poly(dG) to fold into a stable architecture with an unambiguous folding pattern using G-tetrads as building elements. Because of the folding pattern of three G-tetrads and single-G loops, dG15 is programmable and has the capability to encode biological information. Here, we address two open questions of the G4 World hypothesis: (1) Does RNA follow the same folding pattern as DNA? (2) How do stable quadruplexes evolve into the present-day system of information transfer, which is based on Watson-Crick base pair complementarity? To address these questions, we systematically studied the thermodynamic and optical properties of both DNA and RNA G15- and G3T (GGGTGGGTGGGTGGG)-derived sequences. Our study revealed that similar to DNA sequences, RNAs adopt quadruplexes with only three G-tetrads. Thus, both poly(dG) and poly(rG) possess inherent ability to fold into 3D quadruplex architecture with strictly defined folding pattern. The study also revealed that despite high stability of both DNA and RNA quadruplexes, they are vulnerable to single-nucleotide substitutions, which drop the thermal stability by ~40°C and can facilitate introduction of the complementarity principle into the G4 World.

Which came first: the chicken, the egg, or guanine?

The main goal of the origin of life (OoL) hypothesis is to reconstruct the missing link between the primordial soup and the extant biology. However, the OoL itself is just the initial part of the link representing the bootstrapping operation of Darwinian evolution. The rest of the link is the emergence of the evolution to the present day primary biological system-the ribosome-based translation apparatus. A valid hypothesis must (i) not invoke Darwinian evolution in the bootstrapping and (ii) transform the ab initio life form into the translation apparatus without violating the principle of continuity (i.e., only incremental steps without foresight). Currently, no such hypothesis exists. Here, I discuss the Quadruplex World hypothesis, which fully complies with these requirements and suggests a spontaneous emergence of the ab initio life form. The spontaneity of OoL arises from the physicochemical properties of guanine monomers in a manner of causal determinism: each step of the process (i.e., scaffolding, polymerization, and folding) is caused by the most recent past step such that in the end only the specific 3D architecture forms. The architecture (i) has a length-independent folding pattern; (ii) can play the role of the predecessor of tRNA and single-handedly conduct a primitive form of translation; and (iii) can evolve into the extant translation apparatus without any paradoxes.

Trinity of G-tetrads and origin of translation.

BACKGROUND: The RNA world hypothesis cannot address most of the questions of the origin of life without violating the continuity principle (small Darwinian steps without foresight and miracles). Moreover, the RNA world is an isolated system incapable of accommodating the genetic code and evolving into extant biochemistry. All these problems are rooted in the central assumption of the hypothesis: de novo appearance of the ribozymes, production of which represents a multistep reaction requiring the complementarity principle. Thus, even the basis of the RNA world is at odds with the continuity principle-it uses foresight (multistep reaction) and a miracle (complementarity principle). Can a three-dimensional (3D) architecture, capable of molecular recognition and catalysis, be formed in a single-step reaction without the complementarity or any other preexisting rules? HYPOTHESIS: At first glance, the above question sounds rhetoric since the complementarity principle is the essential feature of the RNA world; it turns an RNA polymer into a genetic material. Without it, the RNA world becomes as shapeless and unconvincing as other hypotheses based on the non-hereditary molecules (i.e., protein world). However, it was suggested recently that the quadruplexes could initiate life and take necessary evolutionary steps before the arrival of the complementarity rules. The hypothesis relies on the unique properties of guanines (Gs) to self-assemble into G-tetrads and efficiently polymerize without any external help or preexisting rules. Interestingly, polyG folds into an unusually stable and well-structured monomolecular architecture that uses the quadruplex domain (QD) assembly. The QD has a strictly defined zigzag-like building pattern to accommodate only three G-tetrads. Since both QD architecture and codon length are based on triplets, the inevitable question arises: are they related? Or could QD play the role of the early adapter and determine the codon length? The current paper is an attempt to answer this question. CONCLUSION: While without translation apparatus most of the steps of the extant translation are physically impossible, the QD-mediated translation is sterically feasible and can be explained by physicochemical properties of the QD and the amino acids without violating the continuity principle. Astonishingly, the quadruplex world hypothesis can address all the shortcomings of the RNA world, including its most significant challenge-step-by-step evolution from the polymerization of the first polynucleotide to the extant biochemistry.

Structure of Tetrahelical DNA Homopolymers Supports Quadruplex World Hypothesis.

We previously reported a tetrahelical monomolecular architecture of DNA, tmDNA, which employs G-quartets and an all-parallel GGGTGGGTGGGTGGG (G3T) quadruplex as the repeating unit. Based on thermodynamic and kinetic studies, we proposed that covalently joined (G3T) n units formed an uninterrupted programmable homopolymer; however, structural evidence for the tmDNA architecture was lacking. Here, we used NMR spectroscopy of wild-type and single-inosine-substituted constructs to characterize both monomolecular (G3T)2 and bimolecular quadruplex-Mg-coupled versions of tmDNA. The NMR results support an architecture consisting of uninterrupted stacked G-tetrads in both the monomolecular constructs and bimolecular assemblies. Taken together, these data support the formation of a stable programmable homopolymeric tmDNA architecture, which may have been a precursor to the modern-day Watson-Crick DNA duplex.

A Chimeric DNA/RNA Antiparallel Quadruplex with Improved Stability.

Nucleic acid quadruplexes are proposed to play a role in the regulation of gene expression, are often present in aptamers selected for specific binding functions and have potential applications in medicine and biotechnology. Therefore, understanding their structure and thermodynamic properties and designing highly stable quadruplexes is desirable for a variety of applications. Here, we evaluate DNA→RNA substitutions in the context of a monomolecular, antiparallel quadruplex, the thrombin-binding aptamer (TBA, GGTTGGTGTGGTTGG) in the presence of either K+ or Sr2+ . TBA predominantly folds into a chair-type configuration containing two G-tetrads, with G residues in both syn and anti conformation. All chimeras with DNA→RNA substitutions (G→g) at G residues requiring the syn conformation demonstrated strong destabilization. In contrast, G→g substitutions at Gs with anti conformation increased stability without affecting the monomolecular chair-type topology. None of the DNA→RNA substitutions in loop positions affected the quadruplex topology; however, these substitutions varied widely in their stabilizing or destabilizing effects in an unpredictable manner. This analysis allowed us to design a chimeric DNA/RNA TBA construct that demonstrated substantially improved stability relative to the all-DNA construct. These results have implications for a variety of quadruplex-based applications including for the design of dynamic nanomachines.

Quadruplex World.

The RNA world hypothesis relies on the double-helix complementarity principle for both replication and catalytic activity of RNA. However, the de novo appearance of the complementarity rules, without previous evolution steps, is doubtful. Another major problem of the RNA world is its isolated nature, making it almost impossible to accommodate the genetic code and transform it into modern biochemistry. These and many other unanswered questions of the RNA world led to suggestions that some simpler molecules must have preceded RNA. Most of these alternative hypotheses proposed the double-helical polymers with different backbones but used the same complementarity principle. The current paper describes a fundamentally different idea: the de novo appearance of a nucleic acid polymer without any preexisting rules or requirements. This approach, coined as the quadruplex world hypothesis, is based on (i) the ability of guanines to form stable G-tetrads that facilitate polymerization; and (ii) the unique property of polyguanines to fold into a monomolecular tetrahelix with a strictly defined building pattern and tertiary structure. The tetrahelix is capable of high-affinity intermolecular interactions and catalytic activities. The quadruplex world hypothesis has the potential to address almost all the shortcomings of the RNA world.

HIV-1 genomic RNA U3 region forms a stable quadruplex-hairpin structure.

The U3 promoter region of the HIV-1 long terminal repeat (LTR) has previously been shown to fold into a series of dynamic G-quadruplex structures. Among the G-quadruplexes identified in the LTR sequence, LTR-III was shown to be the most stable in vitro. NMR studies of this 28-nucleotide (nt) DNA revealed a unique quadruplex-hairpin structure. Whether the hairpin forms in RNA element is unknown and the role of the hairpin in the structure and stability of quadruplexes has not been characterized. Here, we used optical and thermodynamic studies to address these questions. The wild-type LTR-III RNA formed a monomolecular quadruplex with a parallel topology using only propeller loops, including the hairpin loop element. By comparison to the WT and variant RNAs, LTR-III DNA structures were more heterogeneous and less stable. Increased stability of the RNA suggests that the RNA quadruplex-hairpin structure may be a more attractive therapeutic target than the analogous DNA element.

Quadruplex-Templated and Catalyzed Ligation of Nucleic Acids.

Template-guided chemical reactions between nucleic acid strands are an important process in biomedical research. However, almost all of these reactions employ an oligonucleotide-templated approach that is based on the double-helix alignment. The moderate stability of the double helix makes this approach unsuitable for many chemical reactions, so alternative nucleic acid alignment mechanisms, demonstrating higher thermal and chemical stability, are desirable. Earlier, we described a noncovalent coupling mechanism between DNA strands through a quadruplex-and-Mg2+ connection (QMC). QMC is based on G-quadruplexes and allows unusually stable and specific interactions. Herein, a novel catalytic nucleic acid reaction, based on QMC, is described. This approach uses G-tetrads as a structural and recognition element without employing Watson-Crick complementarity rules at any stage of substrate/catalyst formation or interaction between them. Quadruplex-templated ligation can be achieved through the self-ligation of two nucleic acid strands, or through a quadruplex catalyst, which forms a G-triplex and specifically connects the strands. The process is extraordinarily robust and efficient. For instance, the ligation of carbodiimide-activated substrates can proceed in boiling solutions, and complete ligation is demonstrated within a minute. The quadruplex-templated and catalyzed reactions will create new opportunities for chemical reactions requiring harsh experimental conditions.

Quadruplex-Based Reactions for Dynamic DNA Nanotechnology.

The stringent base-pairing rules of DNA make it an exceptionally powerful bottom-up nanoscale material for dynamic nanotechnologies. However, current nanomachines are based on rearrangements between DNA duplexes, which limits structural versatility and introduces detrimental background activity. Specifically, to make the reactions unidirectional, product duplexes are designed to be thermodynamically more favorable than their respective substrate duplexes. As a result, the reactions are thermodynamically driven, which represents the main source for the background activity. Here we test an alternative approach based on a structural transformation (ST) between a monomolecular DNA substrate and a quadruplex product. The quadruplex sequence is incorporated into a hairpin substrate. ST reaction is initiated by an addition of a target molecule, which through toehold-mediated strand displacement releases the quadruplex-forming sequence. The liberated sequence folds into a stable quadruplex and stays folded after dissociation of the target molecule. This Article analyzes the thermodynamic principles of ST reactions and demonstrates that the unidirectional nonenzymatic reaction can be run without thermodynamic favorability by transforming thermodynamically stable substrates into metastable products. In other words, ST is capable to drive nanomachines using thermodynamically uphill reactions. This allows for (i) running nanodevices without detrimental background activity and (ii) charging the product molecules with potential energy, which could be used in downstream endergonic activities.

Stability Factors of the Parallel Quadruplexes: DNA Versus RNA.

One of the most stable quadruplexes is formed by the G3T sequence (GGGTGGGTGGGTGGG) that folds into a parallel quadruplex with three G-tetrads and chain-reversal T-loops. For example, in 1 mM K+, it unfolds at 75 °C and at physiological conditions, it unfolds above 100 °C. The RNA analogue, ggguggguggguggg (g3u), which employs exactly same folding topology, demonstrates even higher thermal stability. Here, we performed melting experiments of G3T, g3u, and more than 30 chimeric constructs (G3T with RNA nucleotides at certain positions). Although the g3u quadruplex is 13 °C more stable than G3T, majority of G → g (DNA-for-RNA) substitutions destabilize G3T. Only three G → g and loop T → u substitutions stabilize the structure. However, stabilization effects of these six substitutions overcome destabilization of other nine G → g, resulting in higher stability of all-RNA g3u. The present work clearly indicates that the stacking interactions are more favorable in parallel DNA quadruplexes, whereas the chain-reversal loops play an important role in higher stability of RNA quadruplexes. In addition, we have shown that the 5'-end of RNA quadruplexes represents a more favorable target for stacking interactions than the 3'-end. Based on the current study, rational design of the quadruplexes for particular biotechnological applications and drugs, targeting the quadruplexes, may be envisaged.

Isothermal amplification of long DNA segments by quadruplex priming amplification.

Amplification of long DNA segments with the highest possible specificity and lowest bias is one of the main goals of modern genomics. Quadruplex priming amplification (QPA) is a single-primer isothermal method, which employs the free energy of quadruplex structures as the driving force for DNA amplification without any extra reaction components. As a result, QPA represents one of the simplest isothermal assays and was previously shown to be suitable for amplification of relatively short DNA sequences. The current study reveals that single-primer QPA can be used for both exponential and linear amplification of relatively long DNA segments (>100 nt), and switching between these modes can be accomplished by simple re-design of the primer used. While exponential amplification resulted in production of some undesired higher molecular weight species, linear QPA demonstrated highly specific amplification of the target molecules without any side products.

Monomolecular tetrahelix of polyguanine with a strictly defined folding pattern.

The G3TG3TG3TG3 (G3T) sequence folds into a monomolecular quadruplex with all-parallel G3 segments connected to each other by chain-reversal loops. The homopolymer consisting of n number of G3T domains directly conjugated to each other folds into an uninterrupted and unusually stable polymer, tetrahelical monomolecular DNA (tmDNA). It was demonstrated that the tmDNA architecture has strong potential in nanotechnologies as highly programmable building material, high affinity coupler and the driving force for endergonic reactions. Here, we explore capability of analogous DNA sequences (i.e., monomolecular quadruplexes with G2 or G4 segments) to construct tmDNA architecture. The study demonstrates that tmDNA can have only one building pattern based on a quadruplex domain with three G-tetrads and single-nucleotide loops, G3N (N = G, A, C and T); all other domains demonstrate antiparallel topologies unsuitable for tmDNA. The present study also suggests that polyguanine is capable of tmDNA formation with strictly defined building pattern; G3 segments connected to each other by chain-reversal G-loops. These findings can have significant impact on (i) DNA nanotechnologies; (ii) structure prediction of G-rich sequences of genome; and (iii) modeling of abiogenesis.

Sr(2+) induces unusually stable d(GGGTGGGTGGGTGGG) quadruplex dimers.

Guanine-rich sequences are able to form quadruplexes consisting of G-quartet structural units. Quadruplexes play an important role in the regulation of gene expression and have therapeutic and biotechnological potential. The HIV-1 integrase inhibitor, (GGGT)4 , and its variants demonstrate unusually high thermal stability. This property has been exploited in the use of quadruplex formation to drive various endergonic reactions of nucleic acids such as isothermal DNA amplification. Quadruplex stability is mainly determined by cations, which specifically bind into the inner core of the structure. In the present work, we report a systematic study of a variant of the HIV-1 integrase inhibitor, GGGTGGGTGGGTGGG (G3T), in the presence of alkali and alkaline-earth cations. We show that Sr(2+) -G3T is characterized by the highest thermal stability and that quadruplex formation requires only one Sr(2+) ion that binds with low micromolar affinity. These concentrations are sufficient to drive robust isothermal quadruplex priming DNA amplification reaction. The Sr(2+) -quadruplexes are also able to form unusually stable dimers through end-to-end stacking. The multimerization can be induced by a combination of quadruplex forming cations (i.e., K(+) or Sr(2+) ) and non-specific Mg(2+) .

Stable Domain Assembly of a Monomolecular DNA Quadruplex: Implications for DNA-Based Nanoswitches.

In the presence of K(+) ions, the 5'-GGGTGGGTGGGTGGG-3' (G3T) sequence folds into a monomolecular quadruplex with unusually high thermal stability and unique optical properties. In this study we report that although single G3T molecules unfold and fold rapidly with overlapping melting and refolding curves, G3T multimers (G3T units covalently attached to each other) demonstrate highly reproducible hysteretic behavior. We demonstrate that this behavior necessitates full-length tandem G3T monomers directly conjugated to each other. Any modification of the tandem sequences eliminates the hysteresis. The experimentally measured kinetic parameters and equilibrium transition profiles suggest a highly specific two-state transition in which the folding and unfolding of the first G3T monomer is rate-limiting for both annealing and melting processes. The highly reproducible hysteretic behavior of G3T multimers has the potential to be used in the design of heat-stimulated DNA switches or transistors.

Quadruplex-and-Mg(2+) Connection (QMC) of DNA.

This work highlights a novel method of coupling of nucleic acids through formation of an extraordinary stable, specific and fully reversible quadruplex-and-Mg(2+) connection (QMC). QMC employs the monomolecular tetrahelical architecture of DNA and has two key components: (i) shape complementarity between QMC partners, which is introduced by specific modifications of the quadruplexes, and (ii) Mg(2+) ions. The on-rate of QMC formation is between 10(5)-10(6) M(-1) s(-1), while the off-rate is undetectable even at 80 °C. However, QMC dissociates rapidly upon removal of Mg(2+) ions (i.e., by EDTA). QMC is characterized by high specificity, as even a single-nucleotide modification of one of the connectors inhibits complex-formation. QMC has the potential to revolutionize biotechnology by introducing a new class of capture molecules with major advantages over traditional systems such as streptavidin-biotin. The advantages include reversibility, multiplexing, higher stability and specificity, longer shelf life and low cost.

Exponential quadruplex priming amplification for DNA-based isothermal diagnostics.

Polymerase chain reaction (PCR) is a method of choice for molecular diagnostics. However, PCR relies on thermal cycling, which is not compatible with the goals of point-of-care diagnostics. A simple strategy to turn PCR into an isothermal method would be to use specific primers, which upon polymerase elongation can self-dissociate from the primer-binding sites. We recently demonstrated that a monomolecular DNA quadruplex, GGGTGGGTGGGTGGG, meets these requirements, which led to the development of the linear versions of quadruplex priming amplification (QPA). Here we demonstrate exponential version of isothermal QPA, which allows an unprecedented 10(10)-fold amplification of DNA signal in less than 40 min.

Quadruplex priming amplification combined with nicking enzyme for diagnostics.

Quadruplex priming amplification (QPA) is a straightforward assay that allows isothermal amplification of DNA and possesses an intrinsic real-time detection mechanism. QPA can be employed as a diagnostic tool for both linear and exponential signal amplification. The linear QPA, which is less prone to background activity characteristics of exponential systems, suffers from low sensitivity. To increase the sensitivity, here we introduce specific probe molecules that are designed for combined activities of Bst 2.0 polymerase and Nt.BstNBI nicking enzyme. The current assay, which is suitable for single-tube isothermal signal amplification, has increased sensitivity of plain linear QPA by three orders of magnitude to levels of low femtomolar concentration of target molecules.

Tetrahelical monomolecular architecture of DNA: a new building block for nanotechnology.

DNA nanotechnology typically relies on Watson-Crick base pairing as both a recognition and structural element. This limits structural versatility and introduces errors during self-assembly of DNA. Guanine (G) quartet motifs show promise as an alternative to DNA duplexes, but the synthesis of long, precisely defined molecules is a significant challenge. Here we demonstrate a continuous tetrahelical DNA architecture capable of programmed self-assembly. We report that the homopolymer consisting of (G3T)3G3 monomeric units has the capability to fold into a monomolecular DNA tetrahelix with unprecedented speed and stability. For instance, in the presence of 1 mM K(+) ions the dimer, (G3T)2, folds readily and melts above 100 °C. These findings have the potential to revolutionize DNA nanotechnology by introducing fast and error-free self-assembly of long and extraordinarily stable molecules.

Isothermal amplification of DNA using quadruplex primers with fluorescent pteridine base analogue 3-methyl isoxanthopterin.

We previously developed a method, known as quadruplex priming amplification (QPA), which greatly simplifies DNA amplification and quantification assays. QPA employs specific primers based on GGGTGGGTGGGTGGG (G3T) sequence, which upon polymerase elongation spontaneously dissociates from the target and folds into a stable quadruplex. Fluorescent nucleotide analogs, when incorporated into these primers, emit light upon quadruplex formation and permit simple, specific, and sensitive quantification without the attachment of probe molecules. Here, we studied optical [fluorescence and circular dichroism (CD)] and thermodynamic properties of the G3T sequence and variants incorporating 3-methylisoxanthopterin (3MI), a highly fluorescent nucleotide analog suitable for QPA. CD studies demonstrate that the incorporation of 3MI does not change the overall tertiary structure of G3T; however, thermal unfolding experiments revealed that it significantly destabilizes the quadruplex. Enzymatic studies revealed that Taq and Bst are practically unable to incorporate any nucleotides opposite to template 3MI. Based on this knowledge, we designed QPA assays with truncated targets that demonstrate efficient amplification around 55°C. Overall, these studies suggest that 3MI-based QPA is a useful assay for DNA amplification and detection.

Thermal stability of quadruplex primers for highly versatile isothermal DNA amplification.

Quadruplex priming amplification (QPA) allows isothermal amplification of nucleic acids with improved yield and simplified detection. This assay is based on a DNA quadruplex, GGGTGGGTGGGTGGG (G3T), which in the presence of specific cations possesses unusually high thermal stability. QPA employs truncated G3T sequences as primers, which upon polymerase elongation, self-dissociate from the binding site and allow the next round of priming without thermal unfolding of amplicons. The rate of amplification strongly depends on the thermal stability of the primer/primer binding site (PBS) complex and to date QPA has been demonstrated to work over a narrow temperature range. To expand the capabilities of QPA, in the present study, we studied the fold and thermodynamic properties of the wild-type G3T and variants containing sequence modifications or extensions at the 5'-end. Circular dichroism studies demonstrate that the substitution of thymidines by other nucleotides or GC addition at the 5'-end does not change the parallel fold of G3T. Thermal unfolding experiments revealed that purine bases incorporated at loop positions and 5'-end dinucleotide extension significantly destabilize the quadruplex, while loop pyrimidines have almost no effect. Overall, the results of these studies suggest that linear isothermal QPA can be performed over a wide temperature range to accommodate both thermophilic and mesophilic DNA polymerases.