A parallel stranded G‐quadruplex composed of threose nucleic acid (TNA)

G‐rich sequences can adopt four‐stranded helical structures, called G‐quadruplexes, that self‐assemble around monovalent cations like sodium (Na⁺) and potassium (K⁺). Whether similar structures can be formed from xeno‐nucleic acid (XNA) polymers with a shorter backbone repeat unit is an unanswered question with significant implications on the fold space of functional XNA polymers. Here, we examine the potential for TNA (α‐l ‐threofuranosyl nucleic acid) to adopt a four‐stranded helical structure based on a planar G‐quartet motif. Using native polyacrylamide gel electrophoresis (PAGE), circular dichroism (CD) and solution‐state nuclear magnetic resonance (NMR) spectroscopy, we show that despite a backbone repeat unit that is one atom shorter than the backbone repeat unit found in DNA and RNA, TNA can self‐assemble into stable G‐quadruplex structures that are similar in thermal stability to equivalent DNA structures. However, unlike DNA, TNA does not appear to discriminate between Na⁺ and K⁺ ions, as G‐quadruplex structures form equally well in the presence of either ion. Together, these findings demonstrate that despite a shorter backbone repeat unit, TNA is capable of self‐assembling into stable G‐quadruplex structures.     

Simple molecular engineering of glycol nucleic acid: Progression from self-pairing to cross-pairing with cDNA and RNA

The acyclic chiral nucleic acid analogue, Glycol Nucleic Acid (GNA), displayed exceptional structural simplicity and atom economy while forming self-paired duplexes, using canonical Watson–Crick base pairing. We disclose here that the replacement of phosphodiester linker in GNA with somewhat rigid and shorter carbamate linker in Glycol Carbamate Nucleic Acid (GCNA) backbone allows unprecedented stability to the antiparallel self-paired duplexes. The R-GCNA oligomers were further found to form cross-paired antiparallel duplexes with cDNA and RNA following Watson–Crick base pairing. The stability of cross-paired GCNA:DNA and GCNA:RNA duplexes was higher than the corresponding DNA:DNA and DNA:RNA duplexes. The chiral (R) and (S) precursors were easily accessible from naturally occurring l-serine.     

Transliteration of synthetic genetic enzymes

Functional nucleic acids lose activity when their sequence is prepared in the backbone architecture of a different genetic polymer. The only known exception to this rule is a subset of aptamers whose binding mechanism involves G-quadruplex formation. We refer to such examples as transliteration—a synthetic biology concept describing cases in which the phenotype of a nucleic acid molecule is retained when the genotype is written in a different genetic language. Here, we extend the concept of transliteration to include nucleic acid enzymes (XNAzymes) that mediate site-specific cleavage of an RNA substrate. We show that an in vitro selected 2′-fluoroarabino nucleic acid (FANA) enzyme retains catalytic activity when its sequence is prepared as α-l-threofuranosyl nucleic acid (TNA), and vice versa, a TNA enzyme that remains functional when its sequence is prepared as FANA. Structure probing with DMS supports the hypothesis that FANA and TNA enzymes having the same primary sequence can adopt similarly folded tertiary structures. These findings provide new insight into the sequence-structure-function paradigm governing biopolymer folding.     

Influence of threose nucleoside units on the catalytic activity of a hammerhead ribozyme

TNA (alpha-L-threose nucleic acids) is potentially a natural nucleic acid, that might have acted as an evolutionary alternative of RNA. We determined the catalytic activity of hammerhead ribozymes containing a threofuranosyl-modified nucleoside at position U4 and U7, and compared these results with those obtained from HNA (hexitol nucleic acids) insertion into the same ribozyme. Our experiments showed that, although the threofuranosyl-modified ribozymes still cleave the substrate strand, cleavage activity is highly decreased. It, therefore, seems that TNA can play a functional role in the RNA world, but only to a limited extent.     

Cations as Mediators of the Adsorption of Nucleic Acids on Clay Surfaces in Prebiotic Environments

Monovalent ([Na⁺] > 10 mM) and divalent ([Ca²⁺], [Mg²⁺] > 1.0 mM) cations induced the precipitationof nucleic acid molecules. In the presence of clay minerals (montmorillonite and kaolinite), there was adsorption instead of precipitation. The cation concentration needed for adsorption depended on both the valence of the cations and the chemical nature of the nucleic acid molecules. Double-stranded nucleic acids needed higher cation concentrations than single-stranded ones to be adsorbed to the same extent on clay. Divalent cations were more efficient than monovalent ones in mediating adsorption. Adsorption to the clay occurred only when both nucleic acids and cations were present. However, once the complexes were formed, the cations could not be removed from the system by washing, indicating that they are directly involved in the association between nucleic acids and mineral surfaces.These observations indicate that cations take part directly in the formation of nucleic acid-clay complexes, acting as a `bridge' between the negative charges on the mineral surface and those of the phosphate groups of the genetic polymer. The relatively low cation concentrations needed for adsorption and the ubiquitous presence of clay minerals in the environment suggest that the adsorption of nucleic acids on mineral surfaces could have taken place in prebiotic habitats. This may have played an important role in the formation and preservation of nucleic acids and/or their precursor polymers.     

Chemical etiology of nucleic acids: aminopropyl nucleic acids (APNAs)

Aminopropyl nucleic acids (APNAs) are constitutionally simple nucleic acid alternatives with one stereogenic center per nucleotide, and with the potential to hybridize with RNA and to exert catalytic functions. We have developed a protecting group strategy to synthesize APNAs, although in a not very efficient way. Isolation and purification of APNAs proved to be difficult. Their structures might be more suited to function as potential catalytic polymers than as information systems that may evolve into RNA.     

Synthetic polymers and their potential as genetic materials

DNA and RNA are the only known natural genetic materials. Systematic modification of each of their chemical building blocks (nucleobase, sugar, and phosphate) has enabled the study of the key properties that make those nucleic acids genetic materials. All three moieties contribute to replication and, significantly, all three moieties can be replaced by synthetic analogs without loss of function. Synthetic nucleic acid polymers capable of storing and propagating information not only expand the central dogma, but also highlight that DNA and RNA are not unique chemical solutions for genetic information storage. By considering replication as a question of information transfer, we propose that any polymer that can be replicated could serve as a genetic material. Editor's suggested further reading in BioEssays Xenobiology: A new form of life as the ultimate biosafety tool Abstract     

The proto-Nucleic Acid Builder: a software tool for constructing nucleic acid analogs

The helical structures of DNA and RNA were originally revealed by experimental data. Likewise, the development of programs for modeling these natural polymers was guided by known structures. These nucleic acid polymers represent only two members of a potentially vast class of polymers with similar structural features, but that differ from DNA and RNA in the backbone or nucleobases. Xeno nucleic acids (XNAs) incorporate alternative backbones that affect the conformational, chemical, and thermodynamic properties of XNAs. Given the vast chemical space of possible XNAs, computational modeling of alternative nucleic acids can accelerate the search for plausible nucleic acid analogs and guide their rational design. Additionally, a tool for the modeling of nucleic acids could help reveal what nucleic acid polymers may have existed before RNA in the early evolution of life. To aid the development of novel XNA polymers and the search for possible pre-RNA candidates, this article presents the proto-Nucleic Acid Builder (https://github.com/GT-NucleicAcids/pnab), an open-source program for modeling nucleic acid analogs with alternative backbones and nucleobases. The torsion-driven conformation search procedure implemented here predicts structures with good accuracy compared to experimental structures, and correctly demonstrates the correlation between the helical structure and the backbone conformation in DNA and RNA.     

Question 1: Peptide Nucleic Acids and the Origin and Homochirality of Life

The possibilities of pseudo peptide DNA mimics like PNA (peptide nucleic acid) having a role for the prebiotic origin of life prior to an RNA world is discussed. In particular a scenario is proposed in which protocells with an achiral genetic material through several generations stepwise is converted into a chiral genetic material, e.g., by incorporation of RNA units. Provided that a sufficiently large sequence space is occupied, a selection process based on catalytic function in which a single cell (first common ancestor) has a definite evolutionary advantage, selection of this cell would by contingency also lock it into homochirality. Presented at: International School of Complexity – 4th Course: Basic Questions on the Origins of Life; “Ettore Majorana” Foundation and Centre for Scientific Culture, Erice, Italy, 1–6 October 2006.     

Selenium Derivatization of Nucleic Acids for X‐Ray Crystal‐Structure and Function Studies

It is estimated that over two thirds of all new crystal structures of proteins are determined via the protein selenium derivatization (selenomethionine (Se‐Met) strategy). This selenium derivatization strategy via MAD (multi‐wavelength anomalous dispersion) phasing has revolutionized protein X‐ray crystallography. Through our pioneer research, similarly, Se has also been successfully incorporated into nucleic acids to facilitate the X‐ray crystal‐structure and function studies of nucleic acids. Currently, Se has been stably introduced into nucleic acids by replacing nucleotide O‐atom at the positions 2′, 4′, 5′, and in nucleobases and non‐bridging phosphates. The Se derivatization of nucleic acids can be achieved through solid‐phase chemical synthesis and enzymatic methods, and the Se‐derivatized nucleic acids (SeNA) can be easily purified by HPLC, FPLC, and gel electrophoresis to obtain high purity. It has also been demonstrated that the Se derivatization of nucleic acids facilitates the phase determination via MAD phasing without significant perturbation. A growing number of structures of DNAs, RNAs, and protein–nucleic acid complexes have been determined by the Se derivatization and MAD phasing. Furthermore, it was observed that the Se derivatization can facilitate crystallization, especially when it is introduced to the 2′‐position. In addition, this novel derivatization strategy has many advantages over the conventional halogen derivatization, such as more choices of the modification sites via the atom‐specific substitution of the nucleotide O‐atom, better stability under X‐ray radiation, and structure isomorphism. Therefore, our Se‐derivatization strategy has great potentials to provide rational solutions for both phase determination and high‐quality crystal growth in nucleic‐acid crystallography. Moreover, the Se derivatization generates the nucleic acids with many new properties and creates a new paradigm of nucleic acids. This review summarizes the recent developments of the atomic site‐specific Se derivatization of nucleic acids for structure determination and function study. Several applications of this Se‐derivatization strategy in nucleic acid and protein research are also described in this review.     

DNA and RNA Syntheses by Intraerythrocytic Stages of Plasmodium knowlesi*

Incorporation of the nucleic acid precursors, orotic acid, adenosine, thymidine, and uridine, was studied in various stages of intraerythrocytic Plasmodium knowlesi from infected rhesus monkeys. Incubation of the parasitized erythrocytes with the precursors was for 3 hr periods using a plasma-free culture medium. The samples containing primarily rings, early trophozoites, or late trophozoites incorporated orotic acid, adenosine, and uridine into RNA; however, these stages exhibited negligible or very low levels of incorporation of any of the precursors into DNA. The sample containing late trophozoite and schizont stages incorporated orotic acid, adenosine, and uridine into RNA, and orotic acid, adenosine, and very low levels of thymidine into DNA. These results indicate that DNA synthesis (the S phase of the cell cycle) occurs very close to the time of nuclear division, and that either the G1 or G2 phase is very short in P. knowlesi. It was also observed that adenosine and orotic acid, 2 precursors which are incorporated into both DNA and RNA, are utilized differently by the intraerythrocytic parasites. Incorporation of orotic acid into RNA and DNA and adenosine incorporation into DNA were continuous for the entire incubation period, whereas incorporation of adenosine into RNA was very low during the last 2 hr of each period. It was further demonstrated that the parasites utilized exogenous uridine for synthesis of RNA, and that the older parasite stages incorporated thymidine into DNA.     

Modulation of DNA and RNA by PNA

Peptide nucleic acid (PNA), a synthetic DNA mimic that is devoid of the (deoxy)ribose-phosphate backbone yet still perfectly retains the ability to recognize natural nucleic acids in a sequence-specific fashion, can be employed as a tool to modulate gene expressions via several different mechanisms. The unique strength of PNA compared to other oligonucleotide analogs is its ability to bind to nucleic acid targets with secondary structures such as double-stranded and quadruplex DNA as well as RNA. This digest aims to introduce general readers to the advancement in the area of modulation of DNA/RNA functions by PNA, its current status and future research opportunities, with emphasis on recent progress in new targeting modes of structured DNA/RNA by PNA and PNA-mediated gene editing.     

The evolution of bat nucleic acid‐sensing Toll‐like receptors

We characterized the nucleic acid‐sensing Toll‐like receptors (TLR) of a New World bat species, the common vampire bat (Desmodus rotundus), and through a comparative molecular evolutionary approach searched for general adaptation patterns among the nucleic acid‐sensing TLRs of eight different bats species belonging to three families (Pteropodidae, Vespertilionidae and Phyllostomidae). We found that the bat TLRs are evolving slowly and mostly under purifying selection and that the divergence pattern of such receptors is overall congruent with the species tree, consistent with the evolution of many other mammalian nuclear genes. However, the chiropteran TLRs exhibited unique mutations fixed in ligand‐binding sites, some of which involved nonconservative amino acid changes and/or targets of positive selection. Such changes could potentially modify protein function and ligand‐binding properties, as some changes were predicted to alter nucleic acid binding motifs in TLR 9. Moreover, evidence for episodic diversifying selection acting specifically upon the bat lineage and sublineages was detected. Thus, the long‐term adaptation of chiropterans to a wide variety of environments and ecological niches with different pathogen profiles is likely to have shaped the evolution of the bat TLRs in an order‐specific manner. The observed evolutionary patterns provide evidence for potential functional differences between bat and other mammalian TLRs in terms of resistance to specific pathogens or recognition of nucleic acids in general.     

PNA technology

Peptide nucleic acids (PNA) are deoxyribonucleic acid (DNA) mimics with a pseudopeptide backbone. PNA is an extremely good structural mimic of DNA (or of ribonucleic acid [RNA]), and PNA oligomers are able to form very stable duplex structures with Watson-Crick complementary DNA and RNA (or PNA) oligomers, and they can also bind to targets in duplex DNA by helix invasion. Therefore, these molecules are of interest in many areas of chemistry, biology, and medicine, including drug discovery, genetic diagnostics, molecular recognition, and the origin of life. Recent progress in studies of PNA properties and applications is reviewed.     

Locked Nucleic Acids: Promising Nucleic Acid Analogs for Therapeutic Applications

Locked Nucleic Acid (LNA) is a unique nucleic‐acid modification possessing very high binding affinity and excellent specificity toward complementary RNA or DNA oligonucleotides. The remarkable properties exhibited by LNA oligonucleotides have been employed in different nucleic acid‐based therapeutic strategies both in vitro and in vivo. Herein, we highlight the applications of LNA nucleotides for controlling gene expression.     

Polyethyleneimine derivative for nucleic acid model

Water-soluble polyethyleneimine (PE) derivatives containing nucleic acid bases and hydrophilic amino acids such as homoserine (Hse) and serine were prepared by the activated ester method as nucleic acid models. From spectroscopic measurements, the polymers were found to interact with DNA accompanied by an induction of conformational change. Hypochromicity in UV spectra indicated that a stable polymer complex was formed between poly (A) with PEI-Hse-Ura by complementary hydrogen bonding with equimolar nucleic base units (adenine∶uracil=1∶1). The induced conformation of DNA by the interaction with the polymer containing uracil and homoserine (PEI-Hse-Ura) was concluded to be a super triple helical structure. The formation of the polymer complex, DNA:PEI-Hse-Ura, was found to be affected by the presence of metal ions such as Ca²⁺ and Cu²⁺.