Nucleic Acid Moieties Research Articles

In vitro selection of a trans aptamer complex for target-responsive fluorescence activation

BackgroundMost biological molecular complexes consist of multiple functional domains, yet rationally constructing such multifunctional complexes is challenging. Aptamers, the nucleic acid-based functional molecules, can perform multiple tasks including target recognition, conformational changes, and enzymatic activities, while being chemically synthesizable and tunable, and thus provide a basis for engineering enhanced functionalities through combination of multiple units. However, the conventional approach of simply combining aptamer units in a serial manner is susceptible to undesired crosstalk or interference between the aptamer units and to false interactions with non-target molecules; besides, the approach would require additional mechanisms to separate the units if they are desired to function independently. It is clearly a challenge to develop multi-aptamer complexes that preserve independent functions of each unit while avoiding undesired interference and non-specific interactions. ResultsBy directly in vitro selecting a ‘trans’ aptamer complex, we demonstrate that one aptamer unit (‘utility module’) can remain hidden or ‘inactive’ until a target analyte triggers the other unit (‘sensing module’) and separates the two aptamers. Since the operation of the utility module occurs free from the sensing module, unnecessary crosstalk between the two units can be avoided. Because the utility module is kept inactive until separated from the complex, non-specific interactions of the hidden module with noncognate targets can be naturally prevented. In our demonstration, the sensing module was selected to detect serotonin, a clinically important neurotransmitter, and the target-binding-induced structure-switching of the sensing module reveals and activates the utility module that turns on a fluorescence signal. The aptamer complex exhibited a moderately high affinity and an excellent specificity for serotonin with ∼16-fold discrimination against common neurotransmitter molecules, and displayed strong robustness to perturbations in the design, disallowing nonspecific reactions against various challenges. SignificanceThis work represents the first example of a trans aptamer complex that was in vitro selected de novo. The trans aptamer complex selected by our strategy does not require chemical modifications or immediate optimization processes to function, because the complex is directly selected to perform desired functions. This strategy should be applicable to a wide range of functional nucleic acid moieties, which will open up diverse applications in biosensing and molecular therapeutics.

Repurposing an antimicrobial peptide for the development of a dual ion channel/molecular receptor-like platform for metal ion detection.

The presence of non-essential metals in the environment as contaminants is prone to cause hazardous health problems following accumulation in the human body and the ensuing toxic effects. This calls for continuous discovery and innovation in the realm of developing easy-to-operate, cheap and sensitive sensors. Herein, we describe the proof of concept approach for designing a molecular receptor-like, chimeric sensor based on the pore-forming peptide alamethicin (Alm), tethered via a linker with an ultrashort peptide nucleic acid (PNA) moiety, capable of generating functional ion channel oligomers in planar lipid membranes. The working principle of the sensor exploits the ability of Hg2+ ions to complex mismatching thymine-thymine sequences between the PNA receptor moiety on Alm oligomers and free, thymine-based, single-stranded DNAs (ssDNAs) in solution, thus creating a stable base pair at the oligomer entrance. This generates a transducing mechanism which converts the metal ion complexation into a specific electrical signature of the self-assembled Alm oligomers, enabling selective Hg2+ ion detection. The platform is programmable, whereby the simple exchange of the PNA sequence and its ssDNA counterpart in solution rendered the system selective for Cu2+ ion detection. With further optimization, the presented solution has the potential to translate into miniaturized, cost-effective biosensors suitable for the real-time, label-free and continuous detection of metal ions or other biomolecules.

TLR9-Selective Therapeutic Targeting of MDS Clones with a CpG Payload-Delivery Strategy

Inflammatory molecules and their receptors serve as regulatory cues driving the proliferation of hematopoietic stem and progenitors (HSPC) in myelodysplastic syndromes (MDS). Recent studies indicate that inflammaging (inflammation in aging) in the bone marrow (BM) microenvironment contributes to hematopoietic impairment and MDS clone expansion. However, attempts to develop therapeutics targeting these processes have been hampered by potential off target effects due to the lack of identification of selective biomarkers to specifically target the MDS malignant clone. Therefore, a better understanding of the mechanisms of MDS inflammaging processes for selection and target of malignant clones remains a key goal. Several groups, including ours, have found that innate immune receptors are over expressed in MDS BM HSPC, including Toll-like receptors (TLRs). Overexpression of these receptors was associated with secretion of inflammatory cytokines and mediators including S100A9. S100A9, a Damage-Associated Molecular Pattern (DAMPs) molecule, causes pyroptosis of HSPC and induces genomic instability leading to MDS clonal expansion in an autocrine amplification loop. However, most of the TLRs and associated factors are hard to target due to their wide expression on healthy immune cells. We found that TLR9, usually an intracellular receptor, is upregulated and translocated to the surface of MDS progenitors, which thereby presents an opportunity for disease-specific targeting through its natural ligand, CpG oligonucleotides. TLR9 is one of the main pathogen recognition receptors expressed intracellularly in innate immune cells and recognizes bacterial nucleic acid moieties. Its gene expression has been previously found to be upregulated in MDS and now our work demonstrates a functional upregulation and translocation in low-risk disease. Flow cytometric sorting of low risk MDS BM cells with labeled CpG separated cells based on mutational burden suggesting that surface TLR9 is specific to malignant clones. Furthermore, pyroptotic release of nucleic acid DAMPs, such as RNA:DNA hybrids and our recently published biomarker oxidized mitochondrial DNA, is linked to the translocation of TLR9 from intracellular endosomes to the surface of these cells. This overexpression was validated in our unique inflammaging animal model, the S100A9 transgenic (Tg) mice, that phenocopies human MDS. Moreover, treatment of human BM mononuclear cells (MNC) with recombinant human S100A9 induced surface expression of TLR9, through the accumulation of S100A9-induced RNA:DNA hybrids and oxidized mitochondrial DNA. Therefore, this paradoxical surface expression of TLR9 in MDS BM HSPCs provides an opportunity to selectively target malignant cells through the targeted delivery of cytotoxins using our newly developed CpG-linked payload deliver strategy. CpG has been used safely in phase I and Phase II clinical trials where it has been shown to be well tolerated, although not effective as a stand-alone therapy. Therefore, we linked CpG to dendrimer particles (CpG-Den) providing a cationic lipid surface structure capable of disrupting cells only after internalization through TLR9. The CpG-Den linkage ratio can be manipulated to modify cytotoxicity strength and the particle size avoids leukocyte engulfment. Using both primary MDS BM MNC and BM MNC from S100A9Tg mice we demonstrate that treatment in vitro and in vivo with CpG-Den selectively targets surface TLR9+ HSPC, increasing healthy hematopoiesis in colony forming assays, and BM expression of HSPC, as well ameliorating anemia in vivo in the S100A9Tg mice. Use of this therapy also led to a decrease in suppressive myeloid cells likely aiding in the reduction of the inflammaging cycle. Localized targeting was confirmed in vivo by labeling CpG-Den with IR800 revealing specific accumulation in the mice BM. Our data supports TLR9-targeting potential in a proof-of-concept strategy to selectively target primary MDS malignant cells to restore and enhance hematopoiesis while avoiding toxicities.

Chimeric Peptide-Based Biomolecular Constructs for Versatile Nucleic Acid Biosensing.

Nucleic acid biomarkers hold great potential as key indicators for the diagnosis and monitoring of diseases. Herein we design and implement bifunctional chimeric biomolecules composed of a solid-binding peptide (SBP) domain that specifically adsorbs onto solid sensor surfaces and a peptide nucleic acid (PNA) moiety that facilitates anchoring of antisense oligonucleotide (ASO) probes for the detection of nucleic acid targets. A gold-binding peptide, AuBP1, previously selected by directed evolution to specifically bind to gold, served as the basis for immobilizing nucleic acid probes onto gold substrates. Using surface plasmon resonance (SPR) spectroscopy and quartz crystal microbalance (QCM) analyses, we demonstrate the sequential biomolecular assembly of the heterofunctional solid-binding peptide-antisense oligomer (SBP-ASO) construct onto a sensor surface and the subsequent detection of DNA in an aqueous environment. The effect of steric hindrance on optimal probe assembly is observed, establishing that less packing density results in greater target capture efficacy. In addition, an adsorbed layer of chimeric solid-binding peptide-peptide nucleic acid (SBP-PNA) undergoes viscoelastic changes at the solid-liquid interface upon probe immobilization and DNA target capture, whereby the rigid biofunctional layer becomes more flexible. The dual nature of the chimeric construct is highly amenable to a variety of platforms allowing for both specific recognition and probe immobilization on the sensor surface, while the modular design of the solid-binding peptide-antisense oligonucleotide provides facile functionalization of a wide diversity of solid substrates.

Acetyl‐CoA Electron Density: Acyl‐CoA Reactivity and Crystallographic Data

Acetyl‐CoA and other acyl‐CoAs are the building blocks used in carbon‐carbon bond forming reactions underlying fatty acid and polyketide biosynthesis and many other biological activities. Since many enzymes use acetyl‐CoA, understanding how the substrate binds in the active site is key to determining interactions leading to enzymatic catalysis and substrate selectivity. We examined all of the PDB deposited crystal structures that report acetyl‐CoA bound and report stats for the reactive acetyl‐thioester moiety. We quantified this data by generating the real‐space R‐factor (RSR) and real‐space correlation coefficient (RSCC) by running a few rounds of refinement with Refmac to normalize the data. We examined the modeled thioester ends in comparison to the rest of CoA by creating a Linux script that splits acetyl‐CoA into the nucleic acid, pantetheine, and acetyl‐thioester moieties. We also analyzed the 3’phosphate of acetyl‐CoA to examine how splitting the molecule affects the RSR/RSCC values of small portions of the molecules along with the ribonucleotide containing substrates NAD+, NADH, NADP, and NADPH. Preliminary analysis of the output data reveals the nucleic acid and pantetheine RSR/RSCC values indicate good fits of models to electron density, but there is a portion of structures where RSR/RSCC values indicate the acetyl‐thioester moiety is poorly fit. We also find that the RSR method of calculation shows a significant difference between the acetyl‐CoA thioester and the 3’phosphate where the 3’phosphate is better than the average of the entire structure while the thioester is poorly fit. This is also consistent with the RSR values of NADP and NADPH 3’ phosphates. Conversely, the RSCC values of the acetyl‐CoA thioester end and the 3’phosphate were both equally lower than the entire structure. It appears there is an overfitting of the acetyl‐thioester fitting in crystal structures, likely due to a pressure to claim mechanistic inference from the structures.

DNA polymerases η and κ bypass N2-guanine-O6-alkylguanine DNA alkyltransferase cross-linked DNA-peptides

DNA-protein cross-links are formed when proteins become covalently trapped with DNA in the presence of exogenous or endogenous alkylating agents. If left unrepaired, they inhibit transcription as well as DNA unwinding during replication and may result in genome instability or even cell death. The DNA repair protein O6-alkylguanine DNA-alkyltransferase (AGT) is known to form DNA cross-links in the presence of the carcinogen 1,2-dibromoethane, resulting in G:C to T:A transversions and other mutations in both bacterial and mammalian cells. We hypothesized that AGT-DNA cross-links would be processed by nuclear proteases to yield peptides small enough to be bypassed by translesion (TLS) polymerases. Here, a 15-mer and a 36-mer peptide from the active site of AGT were cross-linked to the N2 position of guanine via conjugate addition of a thiol containing a peptide dehydroalanine moiety. Bypass studies with DNA polymerases (pols) η and κ indicated that both can accurately bypass the cross-linked DNA peptides. The specificity constant (kcat/Km) for steady-state incorporation of the correct nucleotide dCTP increased by 6-fold with human (h) pol κ and 3-fold with hpol η, with hpol η preferentially inserting nucleotides in the order dC > dG > dA > dT. LC-MS/MS analysis of the extension product also revealed error-free bypass of the cross-linked 15-mer peptide by hpol η. We conclude that a bulky 15-mer AGT peptide cross-linked to the N2 position of guanine can retard polymerization, but that overall fidelity is not compromised because only correct bases are inserted and extended.

Functionalizing DNA nanostructures for therapeutic applications.

Recent advances in nanotechnology have enabled rapid progress in many areas of biomedical research, including drug delivery, targeted therapies, imaging, and sensing. The emerging field of DNA nanotechnology, in which oligonucleotides are designed to self-assemble into programmable 2D and 3D nanostructures, offers great promise for further advancements in biomedicine. DNA nanostructures present highly addressable and functionally diverse platforms for biological applications due to their ease of construction, controllable architecture and size/shape, and multiple avenues for chemical modification. Both supramolecular and covalent modification with small molecules and polymers have been shown to expand or enhance the functions of DNA nanostructures in biological contexts. These alterations include the addition of small molecule, protein, or nucleic acid moieties that enable structural stability under physiological conditions, more efficient cellular uptake and targeting, delivery of various molecular cargos, stimulus-responsive behaviors, or modulation of a host immune response. Herein, various types of DNA nanostructure modifications and their functional consequences are examined, followed by a brief discussion of the future opportunities for functionalized DNA nanostructures as well as the barriers that must be overcome before their translational use. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology Therapeutic Approaches and Drug Discovery > Emerging Technologies Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures.

Trinucleotide Cap Analogue Bearing a Locked Nucleic Acid Moiety: Synthesis, mRNA Modification, and Translation for Therapeutic Applications.

The synthesis of a new trinucleotide cap analogue containing a locked nucleic acid (LNA) moiety such as m7(LNA)G(5')ppp(5')AmpG and its molecular biology applications are described. The most appealing feature is that this new cap analogue outperforms the standard trinucleotide cap m7G(5')ppp(5')AmpG and the anti-reverse cap analogue m27,3'-OG(5')ppp(5')G by a factor of 5 in terms of translational efficiency.

Chemical Synthesis of a Locked Nucleic Acid-Substituted Dinucleotide Cap Analog.

This article describes a reliable and efficient method for synthesis of the dinucleotide cap analog m7(LNA) G[5']ppp[5']G containing a locked nucleic acid moiety. The required LNA intermediate for the final coupling reaction, m7(LNA) GDP, is prepared in six steps starting from 5'-DMTr-N-DMF LNA guanosine. The overall reaction involves removal of DMTr and DMF groups, 5' monophosphorylation, imidazolide formation, diphosphorylation, and regioselective m7 methylation. The final coupling reaction of m7(LNA) GDP with ImGMP in the presence of zinc chloride as a catalyst affords m7(LNA) G[5']ppp[5']G in 59% yield. © 2021 Wiley Periodicals LLC. Basic Protocol: Synthesis of an LNA-substituted dinucleotide cap analog Support Protocol: Preparation of the tris(tributylammonium) phosphate linker.

Fluorescence and Morphology of Self-Assembled Nucleobases and Their Diphenylalanine Hybrid Aggregates.

Studies carried out in recent decades have revealed that the ability to self-assemble is a widespread property among biomolecules. Small nucleic acid moieties or very short peptides are able to generate intricate assemblies endowed with remarkable structural and spectroscopic properties. Herein, the structural/spectroscopic characterization of aggregates formed by nucleobases and peptide nucleic acid (PNA)-peptide conjugates are reported. At high concentration, all studied nucleobases form aggregates characterized by previously unreported fluorescence properties. The conjugation of these bases, as PNA derivatives, to the dipeptide Phe-Phe leads to the formation of novel hybrid assemblies, which are characterized by an amyloid-like association of the monomers. Although these compounds share the same basic cross-β motif, the nature and number of PNA units have an important impact on both the level of structural order and the intrinsic fluorescence of the self-assembled nanostructure.

Original article - Cytoplasmic nucleic acid-based XNAs directly enhance live cardiac cell function by a Ca2+ cycling-independent mechanism via the sarcomere

Nucleic acid - protein interactions are critical for regulating gene activation in the nucleus. In the cytoplasm, however, potential nucleic acid-protein functional interactions are less clear. The emergence of a large and expanding number of non-coding RNAs and DNA fragments raises the possibility that the cytoplasmic nucleic acids may interact with cytoplasmic cellular components to directly alter key biological processes within the cell. We now show that both natural and synthetic nucleic acids, collectively XNAs, when introduced to the cytoplasm of live cell cardiac myocytes, markedly enhance contractile function via a mechanism that is independent of new translation, activation of the TLR-9 pathway or by altered intracellular Ca2+ cycling. Findings show a steep XNA oligo length-dependence, but not sequence dependence or nucleic acid moiety dependence, for cytoplasmic XNAs to hasten myocyte relaxation. XNAs localized to the sarcomere in a striated pattern and bound the cardiac troponin regulatory complex with high affinity in an electrostatic-dependent manner. Mechanistically, XNAs phenocopy PKA-based modified troponin to cause faster relaxation. Collectively, these data support a new role for cytoplasmic nucleic acids in directly modulating live cell cardiac performance and raise the possibility that cytoplasmic nucleic acid - protein interactions may alter functionally relevant pathways in other cell types.

A dual-targeted nucleic acid moiety decorated SPION nanoparticles for chemo-photodynamic synergistic therapy

Administering a cocktail of different anticancer drugs or combining chemotherapeutic agents with other conventional cancer treatment modalities has attracted great attentions of scientists and pharmaceutical industries in recent years. Herein, a hybridized DNA structure composed of aptamer, i-motif and ds-DNA, was designed and served as an active targeted coat. The hybridized DNA was conjugated with superparamagnetic iron oxide nanoparticles (SPION) and then developed as a multifunctional nanocarrier for co-delivery of a chemotherapeutic agent and a photosensitizer molecule. The i-motif component of the DNA structure endowed the nanocarriers with the property of releasing its payload rapidly and effectively in simulated tumor environment. Moreover, the aptamer@SPION drug delivery nanoplatform displayed active and magnetic target-specific efficiency to cancer cells. The successful cell internalization and sensitive pH-controlled intracellular drug release greatly assisted the nanomedicine to exhibit high chemical and photodynamic anticancer activity. This new nucleic acid conjugated SPION nanocarrier successfully integrates dual-targeted, dual-therapy and pH stimuli responsive drug release, into one single system, and thus maybe an ideal drug delivery vehicle with great potential in the era of precision medicine.

Chemically Caged Nucleic Acids

Chemically caged nucleic acids, which comprise a nucleic acid moiety and a synthetic molecular moiety (or “responsive unit”) that may be removed by an appropriate chemical stimulus, are valuable for the development of nucleic acid-based therapeutic strategies like prodrugs and medicine delivery systems. Light is one such stimulus, and excellent examples of photocaged nucleic acids have been developed. However, external light sources must be used for this methodology, and this presents problems concerning light penetration and off-target cytotoxicity. In contrast, chemically caged nucleic acids, which respond to chemical stimuli and autonomously modulate their functions without the need for external stimuli, present an alternative strategy for bioapplications. Herein, recent examples of chemically caged nucleic acids are presented, and the current limitations and future challenges encountered in this field are discussed.

Balancing the Interactions of Mg2+ in Aqueous Solution and with Nucleic Acid Moieties For a Polarizable Force Field Based on the Classical Drude Oscillator Model.

Mg2+ ions are important in biological systems, particularly in stabilizing compact RNA folds. Mg2+ is strongly polarizing, and representing its interactions in heterogeneous environments is a challenge for empirical force field development. To date, the most commonly used force fields in molecular dynamics simulations utilize a pairwise-additive approximation for electrostatic interactions, which cannot account for the significant polarization response in systems containing Mg2+. In the present work, we refine the interactions of Mg2+ with water, Cl- ions, and nucleic acid moieties using a polarizable force field based on the classical Drude oscillator model. By targeting gas-phase quantum mechanical interaction energies and geometries of hydrated complexes, as well as condensed-phase osmotic pressure calculations, we present a model for Mg2+ that yields quantitative agreement with experimental measurements of water dissociation free energy and osmotic pressure across a broad range of concentrations. Notable is the direct modeling of steric repulsion between the water Drude oscillators and Mg2+ to treat the Pauli exclusion effects associated with overlap of the electron clouds of water molecules in the first hydration shell around Mg2+. Combined with the refined interactions with nucleic acid moieties, the present model represents a significant advancement in simulating nucleic acid systems containing Mg2+.

Functional interplay between NTP leaving group and base pair recognition during RNA polymerase II nucleotide incorporation revealed by methylene substitution.

RNA polymerase II (pol II) utilizes a complex interaction network to select and incorporate correct nucleoside triphosphate (NTP) substrates with high efficiency and fidelity. Our previous ‘synthetic nucleic acid substitution’ strategy has been successfully applied in dissecting the function of nucleic acid moieties in pol II transcription. However, how the triphosphate moiety of substrate influences the rate of P-O bond cleavage and formation during nucleotide incorporation is still unclear. Here, by employing β,γ-bridging atom-‘substituted’ NTPs, we elucidate how the methylene substitution in the pyrophosphate leaving group affects cognate and non-cognate nucleotide incorporation. Intriguingly, the effect of the β,γ-methylene substitution on the non-cognate UTP/dT scaffold (∼3-fold decrease in kpol) is significantly different from that of the cognate ATP/dT scaffold (∼130-fold decrease in kpol). Removal of the wobble hydrogen bonds in U:dT recovers a strong response to methylene substitution of UTP. Our kinetic and modeling studies are consistent with a unique altered transition state for bond formation and cleavage for UTP/dT incorporation compared with ATP/dT incorporation. Collectively, our data reveals the functional interplay between NTP triphosphate moiety and base pair hydrogen bonding recognition during nucleotide incorporation.

RNPs and autoimmunity: 20 years later.

RNPs and autoimmunity: 20 years later.

A Polysaccharide from Stigma Maydis

Stigma maydis (corn silk) comprises the stigma and style of Zea mays L. and is a traditional Chinese medicine that has been listed in the Chinese Pharmacopeia (1977). Stigma maydis is a herbacious component of many Chinese herbal medicines used clinically for the treatment of hyperglycemia [1, 2], nephritis gallstone, jaundice, tumor [3], diuresis [4], and other ailments. Stigma maydis contains proteins, carbohydrates, and flavonoids, and there have been many reports on the biological activities of its constituents. Aqueous extracts of Stigma maydis have shown hyperglycemic activity on blood glucose levels in studies using hyperglycemic mice induced by alloxan. Effects on blood glucose, glycohemoglobin (HbA1c), insulin secretion, damaged pancreatic -cells, hepatic glycogen, and gluconeogenesis in hyperglycemic mice have been studied [1]. Here, we have systematically studied, for the first time, the links between the composition of Stigma maydis polysaccharide (SMPS) and its chemistry and pharmacology. Water-soluble polysaccharides from Stigma maydis were extracted and purified and the present paper describes the acid hydrolysis, methylation analysis, and hyperglycemic activity of one of its constituent polysaccharides, SMPS1. Dried Stigma maydis was soaked in water at 90 C for 30 min. SMPS was obtained by precipitation with 70% alcohol for at least 24 h from the aqueous extract of the Stigma maydis after its concentration with a film evaporator, deproteinization by the Sevag enzyme method [5–7], decoloring by hydrogen peroxide, and dialysis with distilled water. A portion of purified SMPS was dissolved in water and applied to a DEAE anion exchange chromatography column (500 25 mm), eluted with a 0–2.0 M NaCl gradient (125 mL gradient volume, about 5 mL per tube). The CH3OH and H2O eluate was purified on a Sephadex LH-20 column (500 20 mm) to yield four fractions: SMPS1–4. We report some of the properties of SMPS1 in the present paper. From the elution curve and HPLC analysis, SMPS1 was determined as a single peak by size-exclusion chromatography, with a molecular weight estimated to be over 10 kDa, and it had specific rotation [ ]20 D +4.18 (c 1.40, 50% CH3OH). There was no characteristic absorption at 260 or 280 nm through ultraviolet scanning in the range from 190 to 400 nm, indicating that SMPS1 is a polysaccharide, without nucleic acid or protein moieties. IR spectroscopy showed no characteristic absorption at 1731 cm–1 (carboxyl groups), indicating that SMPS1 is a neutral polysaccharide. After complete hydrolysis with 2 M TFA, GC analysis of the alditol acetates from SMPS1 showed that it contained glucose, rhamnose, mannose, and galactose at a molar ratio of 1:0.38:0.14:1.65. Methylation analysis of SMPS1 showed that it mostly comprised -1,4-linked-L-rhamnose, -1,4;1,6-D-linked glucose, -1,4-linked-D-galactose, and -4-linked-Dmannose (Table 1). These results require further verification. Using the diabetic model induced by alloxan in Kunming mice [1], the conclusion reached was that the active blood sugar lowering components of Stigma maydis were polysaccharides. We demonstrated that SMPS1 shows effective hyperglycemic activities in a dose-dependent manner (Fig. 1). (P < 0.05).

Interdependence of pyrene interactions and tetramolecular G4-DNA assembly.

Controlling the arrangement of organic chromophores in supramolecular architectures is of primary importance for the development of novel functional molecules. Insertion of a twisted intercalating nucleic acid (TINA) moiety, containing phenylethynylpyren-1-yl derivatives, into a G-rich DNA sequence alters G-quadruplex folding, resulting in supramolecular structures with defined pyrene arrangements. Based on CD, NMR and ESI-mass-spectra, as well as TINA excited dimer (excimer) fluorescence emission we propose that insertion of the TINA monomer in the middle of a dTG4T sequence (i.e. dTGGXGGT, where X is TINA) converts a parallel tetramolecular G-quadruplex into an assembly composed of two identical antiparallel G-quadruplex subunits stacked via TINA-TINA interface. Kinetic analysis showed that TINA-TINA association controls complex formation in the presence of Na(+) but barely competes with guanine-mediated association in K(+) or in the sequence with the longer G-run (dTGGGXGGGT). These results demonstrate new perspectives in the design of molecular entities that can kinetically control G-quadruplex formation and show how tetramolecular G-quadruplexes can be used as a tuneable scaffold to control the arrangement of organic chromophores.

ChemInform Abstract: Nucleic Acid Therapeutics: Basic Concepts and Recent Developments

AbstractReview: [147 refs.

Atomistic investigation of the effect of incremental modification of deoxyribose sugars by locked nucleic acid (β-D-LNA and α-L-LNA) moieties on the structures and thermodynamics of DNA-RNA hybrid duplexes.

Chemically modified oligonucleotides offer many possibilities in utilizing their special features for a vast number of applications in nucleic acid based therapies and synthetic molecular biology. Locked nucleic acid analogues (α-/β-LNA) are modifications having an extra ring of 2'-O,4'-C-methylene group in the furanose sugar. LNA strands have been shown to exhibit high binding affinity toward RNA and DNA strands, and the resultant duplexes show significantly high melting temperatures. In the present study, molecular dynamics (MD) simulations were performed on DNA-RNA hybrid duplexes by systematically modifying their deoxyribose sugars with locked nucleic acid analogues. Several geometrical and energetic analyses were performed using principal component (PCA) analysis and binding free energy methods to understand the consequence of incorporated isomeric LNA modifications on the structure, dynamics, and stability of DNA-RNA hybrid duplex. The β-modification systematically changes the conformation of the DNA-RNA hybrid duplex whereas drastic changes are observed for α-modification. The fully modified duplexes have distinct properties compared to partial and unmodified duplexes, and the partly modified duplexes have properties intermediate to full strand and unmodified duplexes. The distribution of BI versus BII populations suggests that backbone rearrangement is minimal for β-LNA modification in order to accommodate it in duplexes whereas extensive backbone rearrangement is necessary in order to incorporate α-LNA modification which subsequently alters the energetic and structural properties of the duplexes. The simulation results also suggest that the alteration of DNA-RNA hybrid properties depends on the position of modification and the gap between the modifications.