Base-pairing Properties Research Articles

Effect of N7-methylation on base pairing patterns of guanine: a DFT study.

In this paper, we aim to determine whether the N7-methylation can influence the base pairing properties of guanine by promoting the formation of guanine enol-tautomers. The keto- to -enol-tautomerization of N7-methylguanine (N7mG) and its base pairing patterns with all the canonical DNA bases have been investigated at the M06-2X/6-311+G(d,p) level of density functional theory. The barrier free energy calculations reveal that N7-methylation does not promote the keto- to enol- tautomerization of guanine. The Watson-Crick-like enol-N7mG:T1 or enol-N7mG:T2 base pair similar to what is observed experimentally is found to be energetically more stable than the keto-N7mG:T base pairs. However, the keto-N7mG:C1 which is structurally similar to the canonical G:C base pair is the most stable base pair among all the base pairs studied here. Thus, our calculations predict that N7mG would pair preferably with cytosine during DNA replication but there is also a probability that it can cause mutation through mispairing with thymine, in agreement with experimental observations.

A Threose Nucleic Acid Enzyme with RNA Ligase Activity.

Threose nucleic acid (TNA) has been considered a potential RNA progenitor in evolution due to its chemical simplicity and base pairing property. Catalytic TNA sequences with RNA ligase activities might have facilitated the transition to the RNA world. Here we report the isolation of RNA ligase TNA enzymes by in vitro selection. The identified TNA enzyme T8-6 catalyzes the formation of a 2'-5' phosphoester bond between a 2',3'-diol and a 5'-triphosphate group, with a kobs of 1.1 × 10-2 min-1 (40 mM Mg2+, pH 9.0). For efficient reaction, T8-6 requires UA|GA at the ligation junction and tolerates variations at other substrate positions. Functional RNAs such as hammerhead ribozyme can be prepared by T8-6-catalyzed ligation, with site-specific introduction of a 2'-5' linkage. Together, this work provides experimental support for TNA as a plausible pre-RNA genetic polymer and also offers an alternative molecular tool for biotechnology.

Genomic Uracil and Aberrant Profile of Demethylation Intermediates in Epigenetics and Hematologic Malignancies.

DNA of all living cells undergoes continuous structural and chemical alterations resulting from fundamental cellular metabolic processes and reactivity of normal cellular metabolites and constituents. Examples include enzymatically oxidized bases, aberrantly methylated bases, and deaminated bases, the latter largely uracil from deaminated cytosine. In addition, the non-canonical DNA base uracil may result from misincorporated dUMP. Furthermore, uracil generated by deamination of cytosine in DNA is not always damage as it is also an intermediate in normal somatic hypermutation (SHM) and class shift recombination (CSR) at the Ig locus of B-cells in adaptive immunity. Many of the modifications alter base-pairing properties and may thus cause replicative and transcriptional mutagenesis. The best known and most studied epigenetic mark in DNA is 5-methylcytosine (5mC), generated by a methyltransferase that uses SAM as methyl donor, usually in CpG contexts. Oxidation products of 5mC are now thought to be intermediates in active demethylation as well as epigenetic marks in their own rights. The aim of this review is to describe the endogenous processes that surround the generation and removal of the most common types of DNA nucleobase modifications, namely, uracil and certain epigenetic modifications, together with their role in the development of hematological malignances. We also discuss what dictates whether the presence of an altered nucleobase is defined as damage or a natural modification.

A non-radioactive, improved PAR-CLIP and small RNA cDNA library preparation protocol.

Crosslinking and immunoprecipitation (CLIP) methods are powerful techniques to interrogate direct protein-RNA interactions and dissect posttranscriptional gene regulatory networks. One widely used CLIP variant is photoactivatable ribonucleoside enhanced CLIP (PAR-CLIP) that involves in vivo labeling of nascent RNAs with the photoreactive nucleosides 4-thiouridine (4SU) or 6-thioguanosine (6SG), which can efficiently crosslink to interacting proteins using UVA and UVB light. Crosslinking of 4SU or 6SG to interacting amino acids changes their base-pairing properties and results in characteristic mutations in cDNA libraries prepared for high-throughput sequencing, which can be computationally exploited to remove abundant background from non-crosslinked sequences and help pinpoint RNA binding protein binding sites at nucleotide resolution on a transcriptome-wide scale. Here we present a streamlined protocol for fluorescence-based PAR-CLIP (fPAR-CLIP) that eliminates the need to use radioactivity. It is based on direct ligation of a fluorescently labeled adapter to the 3'end of crosslinked RNA on immobilized ribonucleoproteins, followed by isolation of the adapter-ligated RNA and efficient conversion into cDNA without the previously needed size fractionation on denaturing polyacrylamide gels. These improvements cut the experimentation by half to 2 days and increases sensitivity by 10-100-fold.

Structural insights into the bypass of the major deaminated purines by translesion synthesis DNA polymerase.

The exocyclic amines of nucleobases can undergo deamination by various DNA damaging agents such as reactive oxygen species, nitric oxide, and water. The deamination of guanine and adenine generates the promutagenic xanthine and hypoxanthine, respectively. The exocyclic amines of bases in DNA are hydrogen bond donors, while the carbonyl moiety generated by the base deamination acts as hydrogen bond acceptors, which can alter base pairing properties of the purines. Xanthine is known to base pair with both cytosine and thymine, while hypoxanthine predominantly pairs with cytosine to promote A to G mutations. Despite the known promutagenicity of the major deaminated purines, structures of DNA polymerase bypassing these lesions have not been reported. To gain insights into the deaminated-induced mutagenesis, we solved crystal structures of human DNA polymerase η (polη) catalyzing across xanthine and hypoxanthine. In the catalytic site of polη, the deaminated guanine (i.e., xanthine) forms three Watson-Crick-like hydrogen bonds with an incoming dCTP, indicating the O2-enol tautomer of xanthine involves in the base pairing. The formation of the enol tautomer appears to be promoted by the minor groove contact by Gln38 of polη. When hypoxanthine is at the templating position, the deaminated adenine uses its O6-keto tautomer to form two Watson-Crick hydrogen bonds with an incoming dCTP, providing the structural basis for the high promutagenicity of hypoxanthine.

2'-Fluoroarabinonucleic Acid Nanostructures as Stable Carriers for Cellular Delivery in the Strongly Acidic Environment.

DNA nanotechnology is powerful in constructing programmable nanostructures with distinct dimensions, sizes, and shapes. However, natural DNA molecules are prone to nuclease degradation, thus limiting the in vivo applications of such DNA nanostructures. 2'-Fluoroarabinonucleic acid (FANA) is a chemically modified oligonucleotide with similar base pairing properties to DNA and exhibits superior physical and chemical stabilities. In this work, FANA molecules were used to construct double crossover nanostructures, and it was demonstrated that incorporation of FANA conferred nucleic acid nanostructures with increased thermal stability and stronger nuclease resistance. More importantly, FANA nanostructures were able to maintain the structural integrity in the strongly acidic environment (pH 1.2). Last, such FANA nanostructures functioned well in acting as stable carriers of small-molecule cargoes for cellular delivery in simulated gastric fluid, while the DNA counterparts were mostly degraded. Collectively, these results demonstrated that FANA self-assembly was not only a substantial complement to the structural DNA nanotechnology but also an appealing molecular tool for in vivo biomedical applications.

Engineering Aptamer Switches for Multifunctional Stimulus-Responsive Nanosystems.

Although RNA and DNA are best known for their capacity to encode biological information, it has become increasingly clear over the past few decades that these biomolecules are also capable of performing other complex functions, such as molecular recognition (e.g., aptamers) and catalysis (e.g., ribozymes). Building on these foundations, researchers have begun to exploit the predictable base-pairing properties of RNA and DNA in order to utilize nucleic acids as functional materials that can undergo a molecular "switching" process, performing complex functions such as signaling or controlled payload release in response to external stimuli including light, pH, ligand-binding and other microenvironmental cues. Although this field is still in its infancy, these efforts offer exciting potential for the development of biologically based "smart materials". Herein, ongoing progress in the use of nucleic acids as an externally controllable switching material is reviewed. The diverse range of mechanisms that can trigger a stimulus response, and strategies for engineering those functionalities into nucleic acid materials are explored. Finally, recent progress is discussed in incorporating aptamer switches into more complex synthetic nucleic acid-based nanostructures and functionalized smart materials.

Amino Acid Modified RNA Bases as Building Blocks of an Early Earth RNA-Peptide World.

Fossils of extinct species allow us to reconstruct the process of Darwinian evolution that led to the species diversity we see on Earth today. The origin of the first functional molecules able to undergo molecular evolution and thus eventually able to create life, are largely unknown. The most prominent idea in the field posits that biology was preceded by an era of molecular evolution, in which RNA molecules encoded information and catalysed their own replication. This RNA world concept stands against other hypotheses, that argue for example that life may have begun with catalytic peptides and primitive metabolic cycles. The question whether RNA or peptides were first is addressed by the RNA‐peptide world concept, which postulates a parallel existence of both molecular species. A plausible experimental model of how such an RNA‐peptide world may have looked like, however, is absent. Here we report the synthesis and physicochemical evaluation of amino acid containing adenosine bases, which are closely related to molecules that are found today in the anticodon stem‐loop of tRNAs from all three kingdoms of life. We show that these adenosines lose their base pairing properties, which allow them to equip RNA with amino acids independent of the sequence context. As such we may consider them to be living molecular fossils of an extinct molecular RNA‐peptide world.

Mechanisms of nucleotide selection by telomerase.

Telomerase extends telomere sequences at chromosomal ends to protect genomic DNA. During this process it must select the correct nucleotide from a pool of nucleotides with various sugars and base pairing properties, which is critically important for the proper capping of telomeric sequences by shelterin. Unfortunately, how telomerase selects correct nucleotides is unknown. Here, we determined structures of Tribolium castaneum telomerase reverse transcriptase (TERT) throughout its catalytic cycle and mapped the active site residues responsible for nucleoside selection, metal coordination, triphosphate binding, and RNA template stabilization. We found that TERT inserts a mismatch or ribonucleotide ~1 in 10,000 and ~1 in 14,000 insertion events, respectively. At biological ribonucleotide concentrations, these rates translate to ~40 ribonucleotides inserted per 10 kilobases. Human telomerase assays determined a conserved tyrosine steric gate regulates ribonucleotide insertion into telomeres. Cumulatively, our work provides insight into how telomerase selects the proper nucleotide to maintain telomere integrity.

Molecular and structural characterization of disease-associated APE1 polymorphisms

Under conditions of oxidative stress, reactive oxygen species (ROS) continuously assault the structure of DNA resulting in oxidation and fragmentation of the nucleobases. When the nucleobase structure is altered, its base-pairing properties may also be altered, promoting mutations. Consequently, oxidative DNA damage is a major source of the mutation load that gives rise to numerous human maladies, including cancer. Base excision repair (BER) is the primary pathway tasked with removing and replacing mutagenic DNA base damage. Apurinic/apyrimidinic endonuclease 1 (APE1) is a central enzyme with AP-endonuclease and 3′ to 5′ exonuclease functions during BER, and therefore is key to maintenance of genome stability. Polymorphisms, or SNPs, in the gene encoding APE1 (APEX1) have been identified among specific human populations and result in variants of APE1 with modified function. These defects in APE1 potentially result in impaired DNA repair capabilities and consequently an increased risk of disease for individuals within these populations. In the present study, we determined the X-ray crystal structures of three prevalent disease-associated APE1 SNPs (D148E, L104R, and R237C). Each APE1 SNP results in unique localized changes in protein structure, including protein dynamics and DNA binding contacts. Combined with comprehensive biochemical characterization, including pre-steady-state kinetic and DNA binding analyses, variant APE1:DNA complex structures with both AP-endonuclease and exonuclease substrates were analyzed to elucidate how these SNPs might perturb the two major repair functions employed by APE1 during BER.

DNA/TNA mesoscopic modeling of melting temperatures suggests weaker hydrogen bonding of CG than in DNA/RNA

TNA/DNA hybrids share several similarities to RNA/DNA, such as the tendency to form A-type helices and a strong dependency of their thermodynamic properties on purine/pyrimidine ratio. However, unlike RNA/DNA, not much is known about the base-pair properties of TNA. Here, we use a mesoscopic analysis of measured melting temperatures to obtain an estimate of hydrogen bonds and stacking interactions. Our results reveal that the AT base pairs in TNA/DNA have nearly identical hydrogen bond strengths than their counterparts in RNA/DNA, but surprisingly CG turned out to be much weaker despite similar stability.

Synthesis and base-pairing properties of pyrazine nucleic acids

The diversity of backbone modifications in the study of primitive informational polymers is partly limited by the plausible formation of their prebiotic starting components. In this paper, we synthesize pyrazine nucleic acid, an acyclic polymer, with the nucleoside derivable from a prebiotic one-pot synthesis containing alanine amide and D-ribose. Pyrazine nucleic acid (PzNA) which has a backbone structurally similar to glycerol nucleic acid (GNA), contain pyrazine derived nucleosides as informational elements that may exhibit base pairing properties similar to the pyrimidines present in RNA.[1] We found that insertion of pyrazinone nucleotides into DNA oligonucleotide sequences is not well-tolerated, and that homogenous sequences of PzNA are unable to form duplexes with RNA or DNA. Reasons for our results may be attributed to the pyrazine-2-one moiety, which is purposed to be a thymine analog, but has a lower pKa (pKa ∼ 8.5) than thymine and uracil. Additionally, we discovered an “apparent” regioselective protection of pyrazine-2-one derivatives in the presence of a secondary hydroxyl group that proved crucial in the preparation of the pyrazine-2-one phosphoramidite. The regioselectivity observed is proposed to be of general interest in the context of heterocyclic chemistry. In the larger context of origins of life studies, it points to the importance of keto-enol preferences of the canonical nucleobases versus pyrazine heterocycles in functioning as recognition elements.

Oxidation and alkylation stresses activate ribosome-quality control

Oxidation and alkylation of nucleobases are known to disrupt their base-pairing properties within RNA. It is, however, unclear whether organisms have evolved general mechanism(s) to deal with this damage. Here we show that the mRNA-surveillance pathway of no-go decay and the associated ribosome-quality control are activated in response to nucleobase alkylation and oxidation. Our findings reveal that these processes are important for clearing chemically modified mRNA and the resulting aberrant-protein products. In the absence of Xrn1, the level of damaged mRNA significantly increases. Furthermore, deletion of LTN1 results in the accumulation of protein aggregates in the presence of oxidizing and alkylating agents. This accumulation is accompanied by Hel2-dependent regulatory ubiquitylation of ribosomal proteins. Collectively, our data highlight the burden of chemically damaged mRNA on cellular homeostasis and suggest that organisms evolved mechanisms to counter their accumulation.

N8-Glycosylated 8-Azapurine and Methylated Purine Nucleobases: Synthesis and Study of Base Pairing Properties.

In this report, we present the synthesis of N8-glycosylated 8-aza-2-methylhypoxanthine and 8-aza-6-thiohypoxanthine 2'-deoxynucleosides as well as methylated 2'-deoxynebularine derivatives. In vitro base pairing properties between each modified and canonical nucleobase were studied. As demonstrated by Tm, incorporation of the modified bases in DNA resulted, with few exceptions, in low stability of duplexes. Modified bases studied in this report are preferentially recognized by T (for N8-glycosylated 8-aza-2-methylhypoxanthine and methylated purines) and G (N8-glycosylated 8-aza-2-methylhypoxanthine). The base pair formed between N8-glycosylated 8-aza-6-thiohypoxanthine and N9-glycosylated 2-methyl-6-thiohypoxanthine (X2:X6) showed, to some extent, an orthogonal interaction. Based on Tm studies, the only potential self-pairing system is formed by the N8-glycosylated 8-aza-6-thiohypoxanthine nucleoside (X2) but only in the absence of canonical G and T. This study indicated that the canonical thymine base is the preferential base partner of methylated purine bases.

Electronic properties for detection of DNA methylation

Detection of DNA methylation is of great significance for early diagnosis and precise treatments of related cancers. We calculated electronic properties of base pairs including cytosine (C), 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) with guanine (G). Calculation results show that modifications can regulate the energy gap of base pairs mainly by changing distributions of frontier orbitals and would not reduce the efficiency of charge transfer in DNA. 5fCG and 5caCG have better electrical transport properties due to the narrower gap and higher electron affinity compared with that of CG. Modified base pairs would remain stable when charge transport and not notably strengthen or weaken hydrogen bonding interactions of paired bases. Moreover, transverse electronic transport properties of benzamide with C and 5mC were studied by using the density functional theory (DFT) combined with the non-equilibrium green function (NEGF). The obtained current values of two systems have obvious difference under 0-0.2V biases, thus realizing the distinction of methylated cytosine.

Broadening and Enhancing Functions of Antibodies by Self-Assembling Multimerization at Cell Surface.

Monoclonal antibody therapy has offered treatment benefits. Nonetheless, a lack of efficacy still exists, partially because monovalent binding of antibodies to specific receptors fails to translate into an active response. Here, we report a pretargeting–postassembly approach that exploits the selective Watson–Crick base pairing properties of oligonucleotides and multivalently tethers receptor-prebound antibodies to albumin at the cell surface. We demonstrate that this two-step self-assembling strategy allows sequential actions of receptor binding and clustering that broadens and strengthens the functions of antibodies. We show that anti-CD20 obinutuzumab (OBN) modified with one morpholino oligonucleotide (OBN-MORF1) maintains the feature of naked OBN antibody upon CD20 binding, and results in actin redistribution, homotypic adhesion, and lysosome-mediated cell death. Consecutive treatment with albumin grafted with multiple copies of a complementary morpholino oligonucleotide (HSA-(MORF2)x) hybridizes with surface-attached OBN-MORF1, manipulates CD20 clustering, and engages additional signals to induce calcium influx and caspase-related apoptosis. With the two types of different mechanisms collaborating in one system, the simple design exerted a notable survival extension of mice bearing disseminated B-cell lymphomas.

Computational Insight into the Differential Mutagenic Patterns of O-Methylthymine Lesions.

Differential mutagenic patterns were recently reported for O-methylated thymine lesions, which indicate that O4-methylthymine (O4-Me-T) frequently leads to G misinsertions, whereas O2-methylthymine (O2-Me-T) is primarily nonmutagenic. The reasons for these differences are unclear since both lesions similarly alter the Watson-Crick binding face of T. To rationalize these replication outcomes at a molecular level, this work uses density functional theory calculations and molecular dynamics simulations to probe the lesion base-pairing properties as well as lesion accommodation by human polymerase η (pol η) and post-extension DNA duplexes. O4-Me-T forms two strong hydrogen bonds with an opposing G in the active site of pol η, which rationalizes the observed lesion mutagenicity. Nevertheless, dATP insertion opposite O4-Me-T can proceed through water-mediated hydrogen bonding, which is similar to the pathway previously proposed for pol η bypass of abasic sites and other T alkylation lesions. In contrast, the position of O2-Me-T in the pol η active site is dynamic due to the presence of the aberrant methyl group on the minor groove side of DNA. In fact, the experimental replication outcomes can only be rationalized when the syn glycosidic orientation of O2-Me-T is considered, which stabilizes the pre-insertion complex by placing the damage in the polymerase open pocket on the major groove side of DNA. Although dATP insertion can occur opposite syn-O2-Me-T through a water-mediated pathway similar to O4-Me-T replication, rotation about the glycosidic bond precludes a stable pol η ternary complex corresponding to dGTP insertion, which correlates with the reported nonmutagenic bypass of O2-Me-T. In addition to providing structural insights into the differential mutagenicity of methylated T adducts, our data highlight an emerging theme in the literature for the replication of pyrimidine alkylation products in noncanonical glycosidic orientations and sets the stage for future work on the replication of other alkylated lesions by TLS polymerases.

A Search for Structural Alternatives of RNA

This account describes a search for potentially primordial informational oligomers; the work is the direct outcome of the research program that was initiated by the Eschenmoser group —at ETH Zürich about 20 years ago and was continued at The Scripps Research Institute since 1996— in order to understand the chemical basis on which nucleic acids were chosen by nature as the molecular foundation of its genetic apparatus. The investigation began with the study of the base-pairing properties of structural alternatives of nucleic acids —constructed from different sugars (hexo- & pentopyranoses and tetrofuranose) retaining the canonical nucleobases and phosphates. The outcome from these studies led to the conclusion that Watson-Crick type base-pairing is not unique to RNA/DNA, and that it can be compatible with a wide variety of backbone edifice. This provided the motivation to map the landscape of potentially primordial informational oligomer systems that may contain backbones, recognition elements and linker groups structurally quite different from those known so far. The oligomer systems chosen for study are, conceptually, deemed to be (a) potentially primordial (based on the nature of the starting materials and reaction conditions considered to be prebiotically realistic) and (b) informational (based on their ability to adopt a repetitive conformation such that the information encoded by the recognition elements can be transmitted intermolecularly). Though such studies suggest the possibility of finding informational systems that could lay claim as functional ancestors of RNA —they are more likely to generate results that provide the opportunity to assess the structural and functional uniqueness of nature’s choice. The experimental investigation described here deals with the base-pairing properties of oligomer systems derived from 2,4-disubsituted -triazines, -5-aminopyrimdines and -6-carboxy pyrimidines as recognition elements that are tagged to oligo-dipeptide backbones via different linker groups. The results from the inter- and intra-system cross-pairing studies reveal that there is, on first approximation, a direct correlation between the magnitude of the difference in DpKa of the recognition elements and their base-pairing strength —smaller the Dpka between the base-pairing partners, weaker is the base-pairing strength (in aqueous medium at near neutral pH). These results exemplify the inherent singularity of the canonical nucleobases— their ability to remain un-ionized under physiological conditions based on their constitution – emphasizing the relationship between their physicochemical properties and their functional competence in connection with their role in informational base-pairing.

Chimeric XNA: An Unconventional Design for Orthogonal Informational Systems.

The paradigm of homogenous-sugar-backbone of RNA and DNA has reliably guided the construction of many functional and useful xeno nucleic acid (XNA) systems to date. Deviations from this monotonous and canonical design, in many cases, results in oligonucleotide systems that lack base pairing with themselves, or with RNA or DNA. Here we show that nucleotides of two such compromised XNA systems can be combined with RNA and DNA in specific patterns to produce chimeric-backbone oligonucleotides, which in certain cases demonstrate base pairing properties comparable to-or stronger than-canonical systems, while also altering the conventional Watson-Crick pairing behavior. The unorthodox pairing properties generated from these chimeric sugar-backbone oligonucleotides suggest a counterintuitive approach of creating modules consisting of non-base pairing XNAs with RNA/DNA in a set pattern. This strategy has the potential to increase the diversity of unconventional nucleic acids leading to orthogonal backbone-sequence-controlled informational systems.

Methylated Nucleobases: Synthesis and Evaluation for Base Pairing In Vitro and In Vivo.

The synthesis, base pairing properties and in vitro (polymerase) and in vivo (E. coli) recognition of 2'-deoxynucleotides with a 2-amino-6-methyl-8-oxo-7,8-dihydro-purine (X), a 2-methyl-6-thiopurine (Y) and a 6-methyl-4-pyrimidone (Z) base moiety are described. As demonstrated by Tm measurements, the X and Y bases fail to form a self-complementary base pair. Despite this failure, enzymatic incorporation experiments show that selected DNA polymerases recognize the X nucleotide and incorporate this modified nucleotide versus X in the template. In vivo, X is mainly recognized as a A/G or C base; Y is recognized as a G or C base and Z is mostly recognized as T or C. Replacing functional groups in nucleobases normally involved in W-C recognition (6-carbonyl and 2-amino group of purine; 6-carbonyl of pyrimidine) readily leads to orthogonality (absence of base pairing with natural bases).