Fluoroarabinonucleic Acid Research Articles

Template-independent synthesis and 3'-end labelling of 2'-modified oligonucleotides with terminal deoxynucleotidyl transferases.

Xenobiotic nucleic acids (XNAs) are artificial genetic polymers with altered structural moieties and useful features, such as enhanced biological and chemical stability. Enzymatic synthesis and efficient labelling of XNAs are crucial for their broader application. Terminal deoxynucleotidyl transferases (TdTs) have been exploited for the de novo synthesis and labelling of DNA and demonstrated the capability of recognizing various substrates. However, the activities of TdTs for the synthesis and labelling of commonly used XNAs with 2' modifications have not been systematically explored. In this work, we explored and demonstrated the varied activities of three TdTs (bovine TdT, MTdT-evo and murine TdT) for the template-independent incorporation of 2'-methoxy NTPs, 2'-fluoro NTPs and 2'-fluoroarabino NTPs into the 3' ends of single- and double-stranded DNAs and the extension of 2'-modified XNAs with (d)NTPs containing a natural or unnatural nucleobase. Taking advantages of these activities, we established a strategy for protecting single-stranded DNAs from exonuclease I degradation by TdT-synthesized 2'-modified XNA tails and methods for 3'-end labelling of 2'-modified XNAs by TdT-mediated synthesis of G-quadruplex-containing tails or incorporation of nucleotides with a functionalized nucleobase. A DNA-2'-fluoroarabino nucleic acid (FANA) chimeric hydrogel was also successfully constructed based on the extraordinary activity of MTdT-evo for template-independent FANA synthesis.

Advancing the enzymatic toolkit for 2'-fluoro arabino nucleic acid (FANA) manipulation: phosphorylation, ligation, replication, and templating RNA transcription.

2'-Fluoro arabino nucleic acid (FANA), classified as a xeno nucleic acid (XNA), stands as a prominent subject of investigation in synthetic genetic polymers. Demonstrating efficacy as antisense oligonucleotides (ASOs) and exhibiting the ability to fold into functional structures akin to enzymes and aptamers, FANA holds substantial promise across diverse biological and therapeutic domains. Owing to structural similarities to DNA, the utilization of naturally occurring DNA polymerases for DNA-mediated FANA replication is well-documented. In this study, we explore alternative nucleic acid processing enzymes typically employed for DNA oligonucleotide (ON) phosphorylation, ligation, and amplification, and assess their compatibility with FANA substrates. Notably, T4 polynucleotide kinase (T4 PNK) efficiently phosphorylated the 5'-hydroxyl group of FANA using ATP as a phosphate donor. Subsequent ligation of the phosphorylated FANA with an upstream FANA ON was achieved with T4 DNA ligase, facilitated by a DNA splint ON that brings the two FANA ONs into proximity. This methodology enabled the reconstruction of RNA-cleaving FANA 12-7 by ligating two FANA fragments amenable to solid-phase synthesis. Furthermore, Tgo DNA polymerase, devoid of 3' to 5' exonuclease activity [Tgo (exo-)], demonstrated proficiency in performing polymerase chain reaction (PCR) with a mixture of dNTPs and FANA NTPs (fNTPs), yielding DNA-FANA chimeras with efficiency and fidelity comparable to traditional DNA PCR. Notably, T7 RNA polymerase (T7 RNAP) exhibited recognition of double-stranded fA-DNA chimeras containing T7 promoter sequences, enabling in vitro transcription of RNA molecules up to 649 nt in length, even in the presence of highly structured F30 motifs at the 3' end. Our findings significantly expand the enzymatic toolkit for FANA manipulation, encompassing phosphorylation, ligation, chimeric amplification, and templating T7 RNAP-catalyzed RNA transcription. These advancements are poised to expedite fundamental research, functional evolution, and translational applications of FANA-based XNA agents. They also have the potential to inspire explorations of a broader range of non-natural nucleic acids that can be routinely studied in laboratories, ultimately expanding the repertoire of nucleic acid-based biomedicine and biotechnology.

Inhibition of SARS-CoV-2 infection in human airway epithelium with a xeno-nucleic acid aptamer

BackgroundSARS-CoV-2, the agent responsible for the COVID-19 pandemic, enters cells through viral spike glycoprotein binding to the cellular receptor, angiotensin-converting enzyme 2 (ACE2). Given the lack of effective antivirals targeting SARS-CoV-2, we previously utilized systematic evolution of ligands by exponential enrichment (SELEX) and selected fluoro-arabino nucleic acid (FANA) aptamer R8-9 that was able to block the interaction between the viral receptor-binding domain and ACE2.MethodsHere, we further assessed FANA-R8-9 as an entry inhibitor in contexts that recapitulate infection in vivo.ResultsWe demonstrate that FANA-R8-9 inhibits spike-bearing pseudovirus particle uptake in cell lines. Then, using an in-vitro model of human airway epithelium (HAE) and SARS-CoV-2 virus, we show that FANA-R8-9 significantly reduces viral infection when added either at the time of inoculation, or several hours later. These results were specific to the R8-9 sequence, not the xeno-nucleic acid utilized to make the aptamer. Importantly, we also show that FANA-R8-9 is stable in HAE culture secretions and has no overt cytotoxic effects.ConclusionsTogether, these results suggest that FANA-R8-9 effectively prevents infection by specific SARS-CoV-2 variants and indicate that aptamer technology could be utilized to target other clinically-relevant viruses in the respiratory mucosa.

Chemically modified aptamers for improving binding affinity to the target proteins via enhanced non-covalent bonding.

Nucleic acid aptamers are ssDNA or ssRNA fragments that specifically recognize targets. However, the pharmacodynamic properties of natural aptamers consisting of 4 naturally occurring nucleosides (A, G, C, T/U) are generally restricted for inferior binding affinity than the cognate antibodies. The development of high-affinity modification strategies has attracted extensive attention in aptamer applications. Chemically modified aptamers with stable three-dimensional shapes can tightly interact with the target proteins via enhanced non-covalent bonding, possibly resulting in hundreds of affinity enhancements. This review overviewed high-affinity modification strategies used in aptamers, including nucleobase modifications, fluorine modifications (2'-fluoro nucleic acid, 2'-fluoro arabino nucleic acid, 2',2'-difluoro nucleic acid), structural alteration modifications (locked nucleic acid, unlocked nucleic acid), phosphate modifications (phosphorothioates, phosphorodithioates), and extended alphabets. The review emphasized how these high-affinity modifications function in effect as the interactions with target proteins, thereby refining the pharmacodynamic properties of aptamers.

"Click handle"-modified 2'-deoxy-2'-fluoroarabino nucleic acid as a synthetic genetic polymer capable of post-polymerization functionalization.

The functions of natural nucleic acids such as DNA and RNA have transcended genetic information carriers and now encompass affinity reagents, molecular catalysts, nanostructures, data storage, and many others. However, the vulnerability of natural nucleic acids to nuclease degradation and the lack of chemical functionality have imposed a significant constraint on their ever-expanding applications. Herein, we report the synthesis and polymerase recognition of a 5-(octa-1,7-diynyl)uracil 2′-deoxy-2′-fluoroarabinonucleic acid (FANA) triphosphate. The DNA-templated, polymerase-mediated primer extension using this “click handle”-modified FANA (cmFANA) triphosphate and other FANA nucleotide triphosphates consisting of canonical nucleobases efficiently generated full-length products. The resulting cmFANA polymers exhibited excellent nuclease resistance and the ability to undergo efficient click conjugation with azide-functionalized molecules, thereby becoming a promising platform for serving as a programmable and evolvable synthetic genetic polymer capable of post-polymerization functionalization.

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.

Functional Comparison of Laboratory-Evolved XNA Polymerases for Synthetic Biology.

Artificial genetic polymers (XNAs) have enormous potential as new materials for synthetic biology, biotechnology, and molecular medicine; yet, very little is known about the biochemical properties of XNA polymerases that have been developed to synthesize and reverse-transcribe XNA polymers. Here, we compare the substrate specificity, thermal stability, reverse transcriptase activity, and fidelity of laboratory-evolved polymerases that were established to synthesize RNA, 2'-fluoroarabino nucleic acid (FANA), arabino nucleic acid (ANA), hexitol nucleic acid (HNA), threose nucleic acid (TNA), and phosphonomethylthreosyl nucleic acid (PMT). We find that the mutations acquired to facilitate XNA synthesis increase the tolerance of the enzymes for sugar-modified substrates with some sacrifice to protein-folding stability. Bst DNA polymerase was found to have weak reverse transcriptase activity on ANA and uncontrolled reverse transcriptase activity on HNA, differing from its known recognition of FANA and TNA templates. These data benchmark the activity of current XNA polymerases and provide opportunities for generating new polymerase variants that function with greater activity and substrate specificity.

2'-Fluoro-arabinonucleic Acid (FANA): A Versatile Tool for Probing Biomolecular Interactions.

This Account highlights the structural features that render 2'-deoxy-2'-fluoro-arabinonucleic acid (FANA) an ideal tool for mimicking DNA secondary structures and probing biomolecular interactions relevant to chemical biology.The high binding affinity of FANA to DNA and RNA has had implications in therapeutics. FANA can hybridize to complementary RNA, resulting in a predominant A-form helix stabilized by a network of 2'F-H8(purine) pseudohydrogen bonding interactions. We have shown that FANA/RNA hybrids are substrates of RNase H and Ago2, both implicated in the mechanism of action of antisense oligonucleotides (ASOs) and siRNA, respectvely. This knowledge has helped us study the conformational preferences of ASOs and siRNA as well as crRNA in CRISPR-associated Cas9, thereby revealing structural features crucial to biochemical activity.Additionally, FANA is of particular use in stabilizing noncanonical DNA structures. For instance, we have taken advantage of the anti N-glycosidic bond conformation of FANA monomers to induce a parallel topology in telomeric G-quadruplexes. Subsequent single-molecule FRET studies elucidated the mechanism by which these parallel G-quadruplexes are recognized and extended by telomerase. Similarly, we have utilized FANA to stabilize elusive telomeric i-motifs in the presence of concomitant parallel G-quadruplexes and under physiological conditions, thereby reinforcing their potential relevance to telomere biology. In another study, we adapted microarray technology and used FANA substitutions to enhance the binding affinity of the G-quadruplex thrombin-binding aptamer to its thrombin target.Finally, we discovered that DNA polymerases can synthesize FANA strands from DNA templates. On the basis of this property, other groups demonstrated that FANA, like DNA, can store hereditary information. They did so by engineering polymerases to efficiently transfer genetic information from DNA to FANA and retrieve it back into DNA. Subsequent studies showed that FANA could be evolved to acquire ribozyme-like endonuclease or ligase activity and to form high-affinity aptamers.Overall, the implications of these studies are remarkable because they promise a deeper understanding of human biochemistry for innovative therapeutic avenues. This Account summarizes past achievements and provides an outlook for inspiring the increased use of FANA in biological applications and fostering interdisciplinary collaborations.

A biologically stable DNAzyme that efficiently silences gene expression in cells.

Efforts to use RNA-cleaving DNA enzymes (DNAzymes) as gene-silencing agents in therapeutic applications have stalled due to their low efficacy in clinical trials. Here we report a xeno-nucleic-acid-modified version of the classic DNAzyme 10-23 that achieves multiple-turnover activity under cellular conditions and resists nuclease digestion. The new reagent, X10-23, overcomes the problem of product inhibition, which limited previous 10-23 designs, using molecular chemotypes with DNA, 2'-fluoroarabino nucleic acid and α-L-threofuranosyl nucleic acid backbone architectures that balance the effects of enhanced biological stability with RNA hybridization and divalent metal ion coordination. In cultured mammalian cells, X10-23 facilitates persistent gene silencing by efficiently degrading exogenous and endogenous messenger RNA transcripts. Together, these results demonstrate that new molecular chemotypes can improve the activity and stability of DNAzymes, and may provide a new route for nucleic acid enzymes to reach the clinic.

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.

Evaluating the Catalytic Potential of a General RNA-Cleaving FANA Enzyme.

The discovery of synthetic genetic polymers (XNAs) with catalytic activity demonstrates that natural genetic polymers are not unique in their ability to function as enzymes. However, all known examples of in vitro selected XNA enzymes function with lower activity than their natural counterparts, suggesting that XNAs might be limited in their ability to fold into structures with high catalytic activity. To explore this problem, we evaluated the catalytic potential of FANAzyme 12-7, an RNA-cleaving catalyst composed entirely of 2'-fluoroarabino nucleic acid (FANA) that was evolved to cleave RNA at a specific phosphodiester bond located between an unpaired guanine and a paired uracil in the substrate recognition arm. Here, we show that this activity extends to chimeric DNA substrates that contain a central riboguanosine (riboG) residue at the cleavage site. Surprisingly, FANAzyme 12-7 rivals known DNAzymes that were previously evolved to cleave chimeric DNA substrates under physiological conditions. These data provide convincing evidence that FANAzyme 12-7 maintains the catalytic potential of equivalent DNAzymes, which has important implications for the evolution of XNA catalysts and their contributions to future applications in synthetic biology.

Selection of 2'-Deoxy-2'-Fluoroarabino Nucleic Acid (FANA) Aptamers That Bind HIV-1 Integrase with Picomolar Affinity.

Systematic Evolution of Ligands by Exponential Enrichment (SELEX) is the iterative process by which nucleic acids that can bind with high affinity and specificity (termed aptamers) to specific protein targets are selected. Using a SELEX protocol adapted for Xeno-Nucleic Acid (XNA) as a suitable substrate for aptamer generation, 2′-fluoroarabinonucleic acid (FANA) was used to select several related aptamers to HIV-1 integrase (IN). IN bound FANA aptamers with equilibrium dissociation constants (KD,app) of ∼50–100 pM in a buffer with 200 mM NaCl and 6 mM MgCl2. Comparisons to published HIV-1 IN RNA and DNA aptamers as well as IN genomic binding partners indicated that FANA aptamers bound more than 2 orders of magnitude more tightly to IN. Using a combination of RNA folding algorithms and covariation analysis, all strong binding aptamers demonstrated a common four-way junction structure, despite significant sequence variation. IN aptamers were selected from the same starting library as FA1, a FANA aptamer that binds with pM affinity to HIV-1 Reverse Transcriptase (RT). It contains a 20-nucleotide 5′ DNA sequence followed by 59 FANA nucleotides. IN-1.1 (one of the selected aptamers) potently inhibited IN activity and intasome formation in vitro. Replacing the FANA nucleotides of IN-1.1 with 2′-fluororibonucleic acid (F-RNA), which has the same chemical formula but with a ribose rather than arabinose sugar conformation, dramatically reduced binding, suggesting that FANA adopts unique structural conformations that promote binding to HIV-1 IN.

2'-Deoxy-2'-fluoro-arabinonucleic acid: a valid alternative to DNA for biotechnological applications using charge transport.

The non-biological 2'-deoxy-2'-fluoro-arabinonucleic acid (2'F-ANA) may be used as a valid alternative to DNA in biomedical and electronic applications because of its higher resistance to hydrolysis and nuclease degradation. However, the advantage of using 2'F-ANA in such applications also depends on its charge-transfer properties compared to DNA. In this study, we compare the charge conduction properties of model 2'F-ANA and DNA double-strands, using structural snapshots from MD simulations to calculate the electronic couplings and reorganization energies associated with the hole transfer steps between adjacent nucleobase pairs. Inserting these charge-transfer parameters into a kinetic model for charge conduction, we find similar conductive properties for DNA and 2'F-ANA. Moreover, we find that 2'F-ANA's enhanced chemical stability does not correspond to a reduction in the nucleobase π-stack structural flexibility relevant to both electronic couplings and reorganization free energies. Our results promote the use of 2'F-ANA in applications that can be based on charge transport, such as biosensing and chip technology, where its chemical stability and conductivity can advantageously combine.

Direct sequencing of 2'-deoxy-2'-fluoroarabinonucleic acid (FANA) using nanopore-induced phase-shift sequencing (NIPSS).

2'-deoxy-2'-fluoroarabinonucleic acid (FANA), which is one type of xeno-nucleic acid (XNA), has been intensively studied in molecular medicine and synthetic biology because of its superior gene-silencing and catalytic activities. Although urgently required, FANA cannot be directly sequenced by any existing platform. Nanopore sequencing, which identifies a single molecule analyte directly from its physical and chemical properties, shows promise for direct XNA sequencing. As a proof of concept, different FANA homopolymers show well-distinguished pore blockage signals in a Mycobacterium smegmatis porin A (MspA) nanopore. By ligating FANA with a DNA drive-strand, direct FANA sequencing has been demonstrated using phi29 DNA polymerase by Nanopore-Induced Phase Shift Sequencing (NIPSS). When bound with an FANA template, the phi29 DNA polymerase shows unexpected reverse transcriptase activity when monitored in a single molecule assay. Following further investigations into the ensemble, phi29 DNA polymerase is shown to be a previously unknown reverse transcriptase for FANA that operates at room temperature, and is potentially ideal for nanopore sequencing. These results represent the first direct sequencing of a sugar-modified XNA and suggest that phi29 DNA polymerase could act as a promising enzyme for sustained sequencing of a wide variety of XNAs.

Evolution of a General RNA-Cleaving FANA Enzyme

The isolation of synthetic genetic polymers (XNAs) with catalytic activity demonstrates that catalysis is not limited to natural biopolymers, but it remains unknown whether such systems can achieve robust catalysis with Michaelis-Menten kinetics. Here, we describe an efficient RNA-cleaving 2’-fluoroarabino nucleic acid enzyme (FANAzyme) that functions with a rate enhancement of >106-fold over the uncatalyzed reaction and exhibits substrate saturation kinetics typical of most natural enzymes. The FANAzyme was generated by in vitro evolution using natural polymerases that were found to recognize FANA substrates with high fidelity. The enzyme comprises a small 25 nucleotide catalytic domain flanked by substrate-binding arms that can be engineered to recognize diverse RNA targets. Substrate cleavage occurs at a specific phosphodiester bond located between an unpaired guanine and a paired uracil in the substrate recognition arm. Our results expand the chemical space of nucleic acid enzymes to include nuclease-resistant scaffolds with strong catalytic activity.

Abstract A57: Antisense oligonucleotide therapeutics with receptor-targeted delivery in triple-negative breast cancer cells via microRNA blockade without passenger strand side effects

Abstract Triple-negative breast cancer (TNBC) attacks >40,000 young women annually in the US, and has no molecular targeted therapies. MicroRNAs drive many forms of cancer, and they modulate multiple gene pathways simultaneously. Highly expressed microRNA miR-17-5p is distinctive in TNBC and contributes to cancer cell survival. We hypothesized that reduction of miR-17-5p activity by delivering oligonucleotide-based antagomiRs specifically to breast cancer cells via receptor-mediated endocytosis would inhibit metastatic behavior of TNBC cells. Using a luciferase reporter system harboring a miR-17-5p binding site in the 3′ untranslated region of the luciferase gene, we assessed the efficacy of various antagomiRs. Contrary to conventional wisdom, that only one of the two strands in a pre-miRNA duplex is active, such as miR-17-5p, we previously observed that a full-length miR-17-5p DNA-LNA blocker created additional off-target effects by mimicking miR-17-3p, implying that therapeutic microRNA blockers should be designed to avoid resemblance to the opposing strands. We designed guide strand specific blockers of miR-17-5p composed of gapmers with anionic backbone derivatives 2′-fluoro-arabino nucleic acid (FANA), and 2′-aminomethyl-bridged nucleic acid (NC-BNA). Since most breast cancer cells overexpress insulin-like growth factor receptor (IGF1R), a peptide derivative of the IGF1 was conjugated to anti-miR-17-5p antagomiRs to achieve breast cancer cell-specific delivery. Our results showed that in multiple TNBC cell lines, the NC-BNA 15mers displayed high efficacy with sub-nM activity for miR-17-5p blockade and significant inhibition of cell growth. Anti-miR-17-5p-IGF1 peptide conjugate successfully inhibited miR-17-5p activity in the luciferase reporter system. We are further examining the effects of NC-BNA-peptides in multiple TNBC cell lines. Our study provides a good basis for the development of a new TNBC targeted therapy. Supported by Bound Therapeutics LLC. Conflict of interest: pending TJU PCT/US2015/015681 patent application licensed to Bound Therapeutics LLC. Citation Format: Yuan-Yuan Jin, Eric Wickstrom. Antisense oligonucleotide therapeutics with receptor-targeted delivery in triple-negative breast cancer cells via microRNA blockade without passenger strand side effects [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A57.

Xenobiotic Nucleic Acid (XNA) Synthesis by Phi29 DNA Polymerase

Phi29 DNA polymerase (DNAP) is the replicative enzyme of the Bacillus subtilis bacteriophage Phi29. Its extraordinary processivity and its ability to perform isothermal amplification of DNA are central to many molecular biology applications, including high-sensitivity detection and large-scale production of DNA. We present here Phi29 DNAP as an efficient catalyst for the production of various artificial nucleic acids (XNAs) carrying backbone modifications such as 1,5-anhydrohexitol nucleic acid (HNA), 2'-deoxy-2'-fluoro-arabinonucleic acid (FANA), and 2'-fluoro-2'-deoxyribonucleic acid (2'-fluoro-DNA). A full protocol for the synthesis of HNA polymers by an exonuclease-deficient variant (D12A) of Phi29 DNAP plus a detailed guide for the design and test of novel XNA synthetase reactions performed by Phi29 DNAP are provided. © 2018 by John Wiley & Sons, Inc.

Electron transfer characteristics of 2'-deoxy-2'-fluoro-arabinonucleic acid, a nucleic acid with enhanced chemical stability.

The non-biological nucleic acid 2'-deoxy-2'-fluoro-arabinonucleic acid (2'F-ANA) may be of use because of its higher chemical stability than DNA in terms of resistance to hydrolysis and nuclease degradation. In order to investigate the charge transfer characteristics of 2'F-ANA, of relevance to applications in nucleic acid-based biosensors and chip technologies, we compare the electronic couplings for hole transfer between stacked nucleobase pairs in DNA and 2'F-ANA by carrying out density functional theory (DFT) calculations on geometries taken from molecular dynamics simulations. We find similar averages and distribution widths of the base-pair couplings in the two systems. On the basis of this result, 2'F-ANA is expected to have charge transfer properties similar to those of DNA, while offering the advantage of enhanced chemical stability. As such, 2'F-ANA may serve as a possible alternative to DNA for use in a broad range of nanobiotechnological applications. Furthermore, we show that the (experimentally observed) enhanced chemical stability resulting from the backbone modifications does not cause reduced fluctuations of the base-pair electronic couplings around the values found for "ideal" B-DNA (with standard step parameter values). Our study also supports the use of a DFT implementation, with the M11 functional, of the wave function overlap method to compute effective electronic couplings in nucleic acid systems.

2'-Fluoroarabinonucleic acid modification traps G-quadruplex and i-motif structures in human telomeric DNA.

Human telomeres and promoter regions of genes fulfill a significant role in cellular aging and cancer. These regions comprise of guanine and cytosine-rich repeats, which under certain conditions can fold into G-quadruplex (G4) and i-motif structures, respectively. Herein, we use UV, circular dichroism and NMR spectroscopy to study several human telomeric sequences and demonstrate that G4/i-motif-duplex interconversion kinetics are slowed down dramatically by 2′-β-fluorination and the presence of G4/i-motif-duplex junctions. NMR-monitored kinetic experiments on 1:1 mixtures of native and modified C- and G-rich human telomeric sequences reveal that strand hybridization kinetics are controlled by G4 or i-motif unfolding. Furthermore, we provide NMR evidence for the formation of a hybrid complex containing G4 and i-motif structures proximal to a duplex DNA segment at neutral pH. While the presence of i-motif and G4 folds may be mutually exclusive in promoter genome sequences, our results suggest that they may co-exist transiently as intermediates in telomeric sequences.

2'-Fluoroarabinonucleic acid modification traps G-quadruplex and i-motif structures in human telomeric DNA.

Human telomeres and promoter regions of genes fulfill a significant role in cellular aging and cancer. These regions comprise of guanine and cytosine-rich repeats, which under certain conditions can fold into G-quadruplex (G4) and i-motif structures, respectively. Herein, we use UV, circular dichroism and NMR spectroscopy to study several human telomeric sequences and demonstrate that G4/i-motif-duplex interconversion kinetics are slowed down dramatically by 2′-β-fluorination and the presence of G4/i-motif-duplex junctions. NMR-monitored kinetic experiments on 1:1 mixtures of native and modified C- and G-rich human telomeric sequences reveal that strand hybridization kinetics are controlled by G4 or i-motif unfolding. Furthermore, we provide NMR evidence for the formation of a hybrid complex containing G4 and i-motif structures proximal to a duplex DNA segment at neutral pH. While the presence of i-motif and G4 folds may be mutually exclusive in promoter genome sequences, our results suggest that they may co-exist transiently as intermediates in telomeric sequences.