Watson-Crick Pairing Research Articles

A general strategy for engineering GU base pairs to facilitate RNA crystallization.

X-ray crystallography is a fundamental technique that provides atomic-level insights into RNA structures. However, obtaining crystals of RNA structures diffracting to high resolution is challenging. We introduce a simple strategy to enhance the resolution limit of RNA crystals by the selective substitution of Watson-Crick pairs by GU pairs within RNA sequences. Our approach has successfully yielded high-resolution structures for eight unique RNA crystals. Notably, six instances showed marked resolution enhancement upon GC/AU to GU base pair substitution, with two cases achieving high-resolution structures from initially poor data. In one case, reverting GU to GC base pairs also improved resolution. Our method facilitated the first structural determinations of the Long Interspersed Nuclear Element-1 and Olfactory Receptor family 4 subfamily K member 15 ribozymes, the 2'-deoxyguanosine-III riboswitch and the Broccoli RNA aptamer. The placement of GU base pairs within the first 5' helical stem of any given RNA species, or in one peripheral stem, is shown to be sufficient. These results offer a simple and effective approach for designing sequences or selecting sequences from homologous sequences, for high-resolution RNA structure determination.

A Self-Assembled Periodic Nanoporous Framework in Aqueous Solutions of the DNA Tetramer GCCG.

The collective behavior of the shortest DNA oligomers in high concentration aqueous solutions is an unexplored frontier of DNA science and technology. Here we broaden the realm of DNA nanoscience by demonstrating that single-component aqueous solutions of the DNA 4-base oligomer GCCG can spontaneously organize into three-dimensional (3D) periodic mesoscale frameworks. This oligomer can form B-type double helices by Watson-Crick (WC) pairing into tiled brickwork-like duplex strands, which arrange into mutually parallel arrays and form the nematic and columnar liquid crystal phases, as is typical for long WC chains. However, at DNA concentrations above 400 mg/mL, these solutions nucleate and grow an additional mesoscale framework phase, comprising a periodic network on a three-dimensional body-centered cubic (BCC) lattice. This lattice is an array of nodes (valence-8, each formed by a pair of quadruplexes of GCCG terminal Gs), connected with a separation of 6.6 nm by struts (6-GCCG-long WC duplexes). This 3D-ordered DNA framework is of low density (DNA volume fraction ∼0.2), but due to its 3D crystal structure, it is osmotically incompressible over its phase range. Atomistic simulations confirm the stability of such structures, which promise to form the basis of families of simply and inexpensively made nanoscale frameworks for templating and selection applications.

Thrombin Nanochannel Logic Gate Inspired by BioMemory.

The process of "reading" and "writing" in biomemory involves the transmission of electrical signals between neurons, with ligand-gated ion channels assuming a key role. The solid-state nanochannels exhibit certain similarities with neurons. Information transmission can be achieved by controlling the flow of ions within nanochannels, rendering them potentially suitable for simulating neuron behavior. Herein, thrombin (Thr) was chosen as the target protein, and a functionalized nanochannel sensing system was successfully constructed using DNA aptamers, enabling a highly sensitive Thr response with a detection limit of 0.221 fM. Simultaneously, based on Watson-Crick base pairing and programmable chain displacement reactions, controlled release and cyclic response of the target molecule were further achieved. This mechanism elucidates the rules governing specific input-output relationships, innovatively linking them with memory storage and recognition through the Thr-nanochannel logic gate, thereby realizing the reading of biomemory at the hardware level. In summary, the biological hybrid nanofluidic control device of this invention converts molecular events into electrical signals, providing potential avenues for establishing connections between the mechanisms of biomemory and solid-state nanochannel biosensing and recognition in the future.

Prime editing: therapeutic advances and mechanistic insights.

We are often confronted with a simple question, "which gene editing technique is the best?"; the simple answer is "there isn't one". In 2021, a year after prime editing first made its mark, we evaluated the landscape of this potentially transformative advance in genome engineering towards getting treatments to the clinic [1]. Nearly 20% of the papers we cited were still in pre-print at the time which serves to indicate how early-stage the knowledge base was at that time. Now, three years later, we take a look at the landscape and ask what has been learnt to ensure this tech is broadly accessible, highlighting some key advances, especially those that push this towards the clinic. A big part of the appeal of prime editing is its ability to precisely edit DNA without double stranded breaks, and to install any of the 12 possible single-nucleotide conversion events as well as small insertions and/or deletions, or essentially any combination thereof. Over the last few decades, other transformative and Nobel prize-winning technologies that rely on Watson-Crick base-pairing such as PCR, site-directed mutagenesis, RNA interference, and one might say, "classic" CRISPR, were swiftly adopted across labs around the world because of the speed with which mechanistic rules governing their efficiency were determined. Whilst this perspective focuses on the context of gene therapy applications of prime editing, we also further look at the recent studies which have increased our understanding of the mechanism of PEs and simultaneously improved the efficiency and diversity of the PE toolbox.

A quantitative framework for structural interpretation of DMS reactivity.

Dimethyl sulfate (DMS) chemical mapping is widely used for probing RNA structure, with low reactivity interpreted as Watson-Crick (WC) base pairs and high reactivity as unpaired nucleotides. Despite its widespread use, a quantitative understanding of how DMS reactivity relates to specific RNA 3D structural features remains incomplete. To address this gap, we systematically analyzed DMS reactivity patterns with a massive library of 7,500 RNA constructs containing two-way junctions with known 3D structures. Our results reveal that DMS reactivity exists on a continuous spectrum rather than discrete high and low bins. Approximately 10% overlap in reactivity between WC and non-WC nucleotides demonstrates that simple thresholds cannot accurately determine base-pairing status. In flanking WC pairs, DMS reactivity correlates with base stacking strength and junction dynamics. For non-WC nucleotides, increased hydrogen bonding and decreased solvent accessibility led to WC-like DMS protection. Most significantly, we discover that DMS reactivity in non-canonical pairs strongly correlates with atomic distances and base pair geometry, enabling discrimination between different 3D conformations. These quantitative relationships establish novel metrics for evaluating RNA structural models and provide a new framework for incorporating DMS reactivity patterns into structure prediction algorithms.

A novel all-in-one target-powered entropy-driven dynamic DNA networks to regulate the activity of CRISPR/AsCas12a for enhanced DNA detection

BackgroundThe non-enzyme autonomous DNA nanodevices have been developed to detect various analytes through the programmability of Watson-Crick base pairing. Nevertheless, by comparison with enzymatic biosensors, the usage of enzyme-free DNA networks to create biosensors for testing low amounts of targets is still subject to the finite number of cycles. Besides, these biosensors still require the incorporation of other amplification strategies to improve the sensitivity, which complicates the detection workflow and lacks of a uniform compatible system to respond to the target in one pot. ResultsHere, we put forward a novel way for rapid and sensitive DNA diagnostic via EDN (entropy-driven dynamic network) coupling with CRISPR/AsCas12a-powered amplification. In the absence of the target, the autonomous hybridization among the substrate and crRNA is kinetically hindered by enclosing complementary regions, which leads to the loss of the activation function of Cas12a. On the contrary, the target initiates the EDN, reconfiguring the activator strand from a duplex to branch construction, which provides a valid means to adjust the hybridization with crRNA, thereby controlling the indiscriminate collateral cleavage activities of the CRISPR/AsCas12a. Compared with the traditional EDN, synergistic activation between the EDN and the CRISPR catalyst could dramatically enhance the detection signal of the target in one pot. Furthermore, the proposed approach provides universal platforms through the rational functional and structural design of DNA assembly modules. Significance and noveltyOverall, this target-triggered EDN switches the activator strand to regulate the activity of AsCas12a (called TERA), which showed nearly one orders of magnitude sensitivity than the conventional Cas12a alone assay, resulting in a devisable universal CRISPR sensing platform that favours the fast, robust and one-pot detection of nucleic molecules.

Molecular self-assembly of stable and small branched DNA nanostructures: Higher than a helical turn is enough for hybridization

The Watson-Crick base pairing property of DNA is widely used for fabricating DNA nanostructures with well-defined geometry. Moreover, DNA nanostructures can be easily modified in terms of shape, size and function at the nanoscale level. Therefore, investigation on smaller and stable branched DNA (bDNA) is of critical significance for biomedical applications. In the present communication, we report smaller and stable branched DNA (bDNA) which is of critical significance for biomedical applications. In this study, a novel strategy has been used in identifying stable bDNA nanostructures with a minimum number of Watson-Crick base pairings. The importance of hybridizing regions and helical twists between multiple oligonucleotides has been explored using various biophysical techniques. The electrophoretic analysis demonstrated that hybridizing regions with ≥12 nt nucleotides can form stable bDNA structures. Substantial negative enthalpic contributions determine the significance of base stacking and the length of oligonucleotides in the hybridization process. Finally, thermal melting investigations confirmed the creation of bDNA nanostructures with ≥12 nt long hybridizing regions. In general, our findings indicate that bDNA oligonucleotides do not undergo hybridization if the number of base pairs is lesser for a single helical turn. Furthermore, the yield and stability of smaller bDNA nanostructures in physiological conditions are comparable with the earlier reported higher-order structures. Hence, smaller bDNAs are more stable which may be preferred over conventional bDNA nanostructures for advanced biomedical applications.

Cross-chiral exponential amplification of an RNA enzyme.

An RNA ligase ribozyme that catalyzes the joining of RNA molecules of the opposite chiral handedness was optimized for the ability to synthesize its own enantiomer from two component fragments. The mirror-image D- and L-ligases operate in concert to provide a system for cross-chiral replication, whereby they catalyze each other's synthesis and undergo mutual amplification at constant temperature, with apparent exponential growth and a doubling time of about 1 h. Neither the D- nor the L-RNA components alone can achieve autocatalytic self-replication. Cross-chiral exponential amplification can be continued indefinitely through a serial-transfer process that provides an ongoing supply of the component D- and L-substrates. Unlike the familiar paradigm of semiconservative nucleic acid replication that relies on Watson-Crick pairing between complementary strands, cross-chiral replication relies on tertiary interactions between structured nucleic acids "across the mirror." There are few examples, outside of biology, of autocatalytic self-replication systems that undergo exponential amplification and there are no prior examples, in either biological or chemical systems, of cross-chiral replication enabling exponential amplification.

In silico study of DNA mononucleotide self-assembly.

Recent experiments have demonstrated the self-assembly and long-range ordering of concentrated aqueous solutions of DNA and RNA mononucleotides. These are found to form Watson-Crick pairs that stack into columns that become spatially organized into a columnar liquid-crystalline phase. In this work, we numerically investigate this phase behavior by adopting an extremely coarse-grained model in which nucleotides are represented as semi-disk-like polyhedra decorated with attractive (patchy) sites that mimic the stacking and pairing interactions. We carry out Monte Carlo simulations of these patchy polyhedra by adapting algorithms borrowed from computer graphics. This model reproduces the features of the experimental phase behavior, which essentially depends on the combination of pairing and stacking interactions.

Dynamics of epitranscriptomes uncover translational reprogramming directed by ac4C in rice during pathogen infection.

Messenger RNA modifications play pivotal roles in RNA biology, but comprehensive landscape changes of epitranscriptomes remain largely unknown in plant immune response. Here we report translational reprogramming directed by ac4C mRNA modification upon pathogen challenge. We first investigate the dynamics of translatomes and epitranscriptomes and uncover that the change in ac4C at single-base resolution promotes translational reprogramming upon Magnaporthe oryzae infection. Then by characterizing the specific distributions of m1A, 2'O-Nm, ac4C, m5C, m6A and m7G, we find that ac4Cs, unlike other modifications, are enriched at the 3rd position of codons, which stabilizes the Watson-Crick base pairing. Importantly, we demonstrate that upon pathogen infection, the increased expression of the ac4C writer OsNAT10/OsACYR (N-ACETYLTRANSFERASE FOR CYTIDINE IN RNA) promotes translation to facilitate rapid activation of immune responses, including the enhancement of jasmonic acid biosynthesis. Our study provides an atlas of mRNA modifications and insights into ac4C function in plant immunity.

Theoretical Investigation of the structure and Raman scattering properties of Hoogsteen- and Watson-Crick-type Adenine-Thymine base pair

Hoogsteen (HG) and Watson-Crick (WC) adenine–thymine (A-T) base pairs are fundamental to gene expression and genomic stability. Our research applied density functional theory (DFT) calculations to analyze these base pairs, demonstrating that linking them with ribose or deoxyribose and their immersion in solvents markedly enhances the distinctions in their optical properties. DFT results reveal that N···HN hydrogen bonds in both HG and WC pairs exhibit characteristics of both ionic and covalent bonds, playing a crucial role in their stability. While WC base pairs demonstrate stronger orbital interactions, they also experience greater repulsion than HG pairs. This repulsion offsets the positive interactions to a degree, making the interaction energy of WC pairs slightly lower than that of HG pairs. Furthermore, the distinct conformations of these base pairs result in different vibrational modes, suggesting Raman spectroscopy as an effective method for distinguishing between WC and HG base pairs.

Advancing microRNA target site prediction with transformer and base-pairing patterns.

MicroRNAs (miRNAs) are short non-coding RNAs involved in various cellular processes, playing a crucial role in gene regulation. Identifying miRNA targets remains a central challenge and is pivotal for elucidating the complex gene regulatory networks. Traditional computational approaches have predominantly focused on identifying miRNA targets through perfect Watson-Crick base pairings within the seed region, referred to as canonical sites. However, emerging evidence suggests that perfect seed matches are not a prerequisite for miRNA-mediated regulation, underscoring the importance of also recognizing imperfect, or non-canonical, sites. To address this challenge, we propose Mimosa, a new computational approach that employs the Transformer framework to enhance the prediction of miRNA targets. Mimosa distinguishes itself by integrating contextual, positional and base-pairing information to capture in-depth attributes, thereby improving its predictive capabilities. Its unique ability to identify non-canonical base-pairing patterns makes Mimosa a standout model, reducing the reliance on pre-selecting candidate targets. Mimosa achieves superior performance in gene-level predictions and also shows impressive performance in site-level predictions across various non-human species through extensive benchmarking tests. To facilitate research efforts in miRNA targeting, we have developed an easy-to-use web server for comprehensive end-to-end predictions, which is publicly available at http://monash.bioweb.cloud.edu.au/Mimosa.

Unveiling the solution structure of a DNA duplex with continuous silver-modified Watson-Crick base pairs

The challenge of transforming organized DNA structures into their metallized counterparts persists in the scientific field. In this context, utilizing DNA molecules modified with 7-deazapurine, provides a transformative solution. In this study, we present the solution structure of a DNA duplex that can be transformed into its metallized equivalent while retaining the natural base pairing arrangement through the creation of silver-modified Watson-Crick base pairs. Unlike previously documented X-ray structures, our research demonstrates the feasibility of preserving the intrinsic DNA self-assembly while incorporating AgI into the double helix, illustrating that the binding of silver does not disrupt the canonical base-pairing organization. Moreover, in our case, the uninterrupted AgI chain deviates from forming conventional straight linear chains; instead, it adheres to a helical arrangement dictated by the underlying DNA structure. This research challenges conventional assumptions and opens the door to precisely design structures based on the organization of highly stable Ag-DNA assemblies.

Maximal Genetic Code Symmetry Is a Physicochemical Purine-Pyrimidine Symmetry Language for Transcription and Translation in the Flow of Genetic Information from DNA to Proteins.

Until now, research has not taken into consideration the physicochemical purine-pyrimidine symmetries of the genetic code in the transcription and translation processes of proteinogenesis. Our Supersymmetry Genetic Code table, developed in 2022, is common and unique for all RNA and DNA living species. Its basic structure is a purine-pyrimidine symmetry net with double mirror symmetry. Accordingly, the symmetry of the genetic code directly shows its organisation based on the principle of nucleotide Watson-Crick and codon-anticodon pairing. The maximal purine-pyrimidine symmetries of codons show that each codon has a strictly defined and unchangeable position within the genetic code. We discovered that the physicochemical symmetries of the genetic code play a fundamental role in recognising and differentiating codons from mRNA and the anticodon tRNA and aminoacyl-tRNA synthetases in the transcription and translation processes. These symmetries also support the wobble hypothesis with non-Watson-Crick pairing interactions between the translation process from mRNA to tRNA. The Supersymmetry Genetic Code table shows a specific arrangement of the second base of codons, according to which it is possible that an anticodon from tRNA recognises whether a codon from mRNA belongs to an amino acid with two or four codons, which is very important in the purposeful use of the wobble pairing process. Therefore, we show that canonical and wobble pairings essentially do not lead to misreading and errors during translation, and we point out the role of physicochemical purine-pyrimidine symmetries in decreasing disorder according to error minimisation and preserving the integrity of biological processes during proteinogenesis.

Proximity-Dependent Activation of Split DNAzyme Kinase.

Binary (also known as split) nucleic acid enzymes have emerged as novel tools in biosensors. We report a new split strategy to split the DNAzyme kinase into two independent and non-functional fragments, denoted Dk1sub and Dk1enz. In the presence of the specific target, their free ends are brought sufficiently close to interact with each other without the formation of Watson-Crick base pairings between Dk1sub and Dk1enz, thus allowing the DNA phosphorylation reaction. We term this approach proximity-dependent activation of split DNAzyme kinase (ProxSDK). The utility of ProxSDK is demonstrated by engineering a biosensing system that is capable of measuring specific DNA-protein interactions. We envision that the approach described herein will find useful applications in biosensing, imaging, and clinical diagnosis.

7-Deaza-2'-deoxyisoguanosine, a Noncanonical Nucleoside for Nucleic Acid Code Expansion and New DNA Constructs: Nucleobase Functionalization of Inverse Watson-Crick and Purine-Purine Base Pairs.

7-Deaza-2'-deoxyisoguanosine forms stable inverse Watson-Crick base pairs with 5-methyl-2'-deoxyisocytidine and purine-purine base pairs with 2'-deoxyguanosine or 5-aza-7-deaza-2'-deoxyguanosine. Both base pairs expand the genetic coding system. The manuscript reports on the functionalization of these base pairs with halogen atoms and clickable side chains introduced at 7-position of the 7-deazapurine base. Oligonucleotides containing the functionalized base pairs were prepared by solid-phase synthesis. To this end, a series of phosphoramidites were synthesized and clickable side chains with short and long linkers were incorporated in oligonucleotides. Fluorescent pyrene conjugates were obtained by postmodification. Functionalization of DNA with a single inverse Watson-Crick base pair by halogens or clickable residues has only a minor impact on duplex stability. Pyrene click adducts increase (long linker) or decrease (short linker) the double helix stability. Stable hybrid duplexes were constructed containing three consecutive purine-purine pairs of 7-functionalized 7-deaza-2'-deoxyisoguanine with guanine or 5-aza-7-deazaguanine in the center and Watson-Crick pairs at both ends. The incorporation of a hybrid base pair tract of 7-deaza-2'-deoxyisoguanosine/5-aza-7-deaza-2'-deoxyguanosine pairs stabilizes the double helix strongly. Fluorescence intensity of pyrene short linker adducts increased when the 7-deazapurine base was positioned opposite to 5-methylisocytosine (inverse base pair) compared to purine-purine base pairs with guanine or 5-aza-7-deazaguanine in opposite positions. For long liker adducts, the situation is more complex. Circular dichroism (CD) spectra of purine DNA differ to those of Watson-Crick double helices and are indicative for the new DNA constructs. The impact of 7-deaza-2'-deoxyisoguanine base pair functionalization is studied for the first time and all experimental details are reported to prepare DNA functionalized at the 7-deazaisoguanine site. The influence of single and multiple incorporations on DNA structure and stability is shown. Clickable residues introduced at the 7-position of the 7-deazaisoguanine base provide handles for Huisgen-Sharpless-Meldal click cycloadditions without harming the stability of purine-pyrimidine and purine-purine base pairs. Other chemistries might be used for bioconjugation. Our investigation paves the way for the functionalization of a new DNA related recognition system expanding the common Watson-Crick regime.

Design of novel broad-spectrum antiviral nucleoside analogues using natural bases ring-opening strategy.

The global prevalence of RNA virus infections has presented significant challenges to public health in recent years, necessitating the expansion of its alternative therapeutic library. Due to its evolutional conservation, RNA-dependent RNA polymerase (RdRp) has emerged as a potential target for broad-spectrum antiviral nucleoside analogues. However, after over half a century of structural modification, exploring unclaimed chemical space using frequently-used structural substitution methods to design new nucleoside analogues is challenging. In this study, we explore the use of the "ring-opening" strategy to design new base mimics, thereby using these base mimics to design new nucleoside analogues with broad-spectrum antiviral activities. A total of 29 compounds were synthesized. Their activity against viral RdRp was initially screened using an influenza A virus RdRp high-throughput screening model. Then, the antiviral activity of 38a was verified against influenza virus strain A/PR/8/34 (H1N1), demonstrating a 50% inhibitory concentration (IC50) value of 9.95 μM, which was superior to that of ribavirin (the positive control, IC50 = 11.43 μM). Moreover, 38a also has inhibitory activity against coronavirus 229E with an IC50 of 30.82 μM. In addition, compounds 42 and 46f exhibit an 82% inhibition rate against vesicular stomatitis virus at a concentration of 20 μM and hardly induce cytotoxicity in host cells. This work demonstrates the feasibility of designing nucleoside analogues with "ring-opening" bases and suggests the "ring-opening" nucleosides may have greater polarity, and designing prodrugs is an important aspect of optimizing their antiviral activity. Future research should focus on enhancing the conformational restriction of open-loop bases to mimic Watson-Crick base pairing better and improve antiviral activity.

Towards combining backbone and sugar constraint in 3'-3' bis-phosphonate tethered 2'-4' bridged LNA oligonucleotide trimers.

Therapeutic oligonucleotides are chemically modified to enhance their drug-like properties - including binding affinity for target RNA. Many nucleic acid analogs that enhance RNA binding affinity constrain the furanose sugar in an RNA-like sugar pucker. The improvements in binding affinity result primarily from increased off-rates with minimal effects on on-rates for hybridization. To identify alternate chemical modification strategies that can modulate on- and off-rates for oligonucleotide hybridization, we hypothesized that extending conformational restraint across multiple nucleotides could modulate hybridization kinetics by restricting rotational freedom of the sugar-phosphate backbone. As part of that effort, we recently reported that using hydrocarbon tethers to bridge adjacent phosphodiester linkages as phosphonate tethered bridges can pre-organize nucleic acids in conformations conducive for Watson-Crick base-pairing and modulate hybridization kinetics. In this report, we describe the synthesis of locked nucleic acid (LNA) trimers linked through alkylphosphonate tethers which restrict conformation of the furanose sugar in addition to restricting conformational mobility of the sugar-phosphate backbone across three nucleotide units.

Rational Design of Chimeric Antisense Oligonucleotides on a Mixed PO-PS Backbone for Splice-Switching Applications.

Synthetic antisense oligonucleotides (ASOs) are emerging as an attractive platform to treat various diseases. By specifically binding to a target mRNA transcript through Watson-Crick base pairing, ASOs can alter gene expression in a desirable fashion to either rescue loss of function or downregulate pathogenic protein expression. To be clinically relevant, ASOs are generally synthesized using modified analogs to enhance resistance to enzymatic degradation and pharmacokinetic and dynamic properties. Phosphorothioate (PS) belongs to the first generation of modified analogs and has played a vital role in the majority of approved ASO drugs, mainly based on the RNase H mechanism. In contrast to RNase H-dependent ASOs that bind and cleave target mature mRNA, splice-switching oligonucleotides (SSOs) mainly bind and alter precursor mRNA splicing in the cell nucleus. To date, only one approved SSO (Nusinersen) possesses a PS backbone. Typically, the synthesis of PS oligonucleotides generates two types of stereoisomers that could potentially impact the ASO's pharmaco-properties. This can be limited by introducing the naturally occurring phosphodiester (PO) linkage to the ASO sequence. In this study, towards fine-tuning the current strategy in designing SSOs, we reported the design, synthesis, and evaluation of several stereo-random SSOs on a mixed PO-PS backbone for their binding affinity, biological potency, and nuclease stability. Based on the results, we propose that a combination of PO and PS linkages could represent a promising approach toward limiting undesirable stereoisomers while not largely compromising the efficacy of SSOs.

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.