Despite remaining apparently frozen through the millennia, the genetic code is far more flexible than previously believed and can be extended and repurposed with relative ease.
synthetic DNA; recoding; unnatural base pairs (UBPs); nonstandard amino acids (NSAAs)
Herein, we describe the synthesis and spectroscopic properties of five novel pyrrolodeoxycytidine analogs, and the related 5-(1-pyrenylethynyl)-2’-deoxycytidine analog; as well as fluorescence characterization of 5-(p-methoxyphenylethynyl)-2’-deoxyuridine. Within this series of compounds, rigidification of the structure from 6-phenylpyrrolodeoxycytidine to 5,6-benzopyrroldeoxycytidine made remarkable improvement of the fluorescence quantum yield (Φ ~1, EtOH) and substantially increased the Stokes shift. Exchange of the phenyl group of 6-phenylpyrrolodeoxycytidine for other heterocycles (benzofuryl or indolyl) produced an increase in the extinction coefficient at the excitation wavelength while preserving high quantum yields. The steady-state fluorescence response to the environment was determined by sensitivity of Stokes shift to solvent polarity. The effect of solvent polarity on fluorescence emission intensity was concurrently examined and showed that 5,6-benzopyrrolodeoxycytidine is highly sensitive to the presence of water. On the other hand, the previously synthesized 5-(p-methoxyphenylethynyl)-2’-deoxyuridine was found to be sensitive to solvent viscosity indicating molecular rotor behavior.
Fluorescence; nucleobase; deoxycytidine; Sonogashira cross-coupling; solvent polarity; solvent viscosity
Thrombin-binding aptamer (TBA) is a 15-nt DNA oligomer that efficiently inhibits thrombin. It has been shown that TBA folds into an anti-parallel unimolecular G-quadruplex. Its three-dimensional chair-like structure consists of two G-tetrads connected by TT and TGT loops. TBA undergoes fast degradation by nucleases in vivo. To improve the nuclease resistance of TBA, a number of modified analogs have been proposed. Here, we describe anomeric modifications of TBA. Non-natural α anomers were used to replace selected nucleotides in the loops and core. Significant stabilization of the quadruplex was observed for the anomeric modification of TT loops at T4 and T13. Replacement of the core guanines either prevents quadruplex assembly or induces rearrangement in G-tetrads. It was found that the anticoagulant properties of chimeric aptamers could be retained only with intact TT loops. On the contrary, modification of the TGT loop was shown to substantially increase nuclease resistance of the chimeric aptamer without a notable disturbance of its anticoagulant activity.
thombin binding aptamer; anomer; quadruplex; anticoagulant properties; nuclease resistance
Among various Z-form DNA inducers, such as transition metal complexes, polyamines and high ionic concentrations, 8-methylguanine have received attention as efficient chemical modifications. Although it is clear that m8–modified guanine base markedly stabilizes the Z conformation of short oligonucleotides under physiological salt conditions, how sequence composition affects the preference of Z-DNA is still not well established. In this study, various oligomers of d(CG)n or d(GC)n containing either 8-methylguanine in a different position were synthesized and their capacity of stabilizing Z-DNA were evaluated by CD spectra and then compared with each other. It is was found out that the Z-DNA stabilizing effect depend on the order of arrangement of m8G and m8rG in DNA strands and the center position is the most effective to stabilize the Z-DNA and promote the B to Z transition.
8-methylguanine; Z-form DNA; B-Z transition; CD spectra
Triplex structures generated by sequence-specific triplex-forming oligonucleotides (TFOs) have proven to be promising tools for gene targeting strategies. In addition, triplex technology has been highly utilized to study the molecular mechanisms of DNA repair, recombination and mutagenesis. However, triplex formation utilizing guanine-rich oligonucleotides as third strands can be inhibited by potassium-induced self-association resulting in G-quadruplex formation. We report here that guanine-rich TFOs partially substituted with 8-aza-7-deaza-guanine (PPG) have improved target site binding in potassium compared with TFOs containing the natural guanine base. We designed PPG-substituted TFOs to bind to a polypurine sequence in the supFG1 reporter gene. The binding efficiency of PPG-substituted TFOs to the target sequence was analyzed using electrophoresis mobility gel shift assays. We have determined that in the presence of potassium, the non-substituted TFO, AG30 did not bind to its target sequence, however binding was observed with the PPG-substituted AG30 under conditions with up to 140 mM KCl. The PPG-TFOs were able to maintain their ability to induce genomic modifications as measured by an assay for gene-targeted mutagenesis. In addition, these compounds were capable of triplex-induced DNA double strand breaks, which resulted in activation of apoptosis.
triplex-forming oligonucleotides; 8-aza-7-deaza-guanine
Modified peptide nucleic acids (PNA) containing one or two thymine PNA monomers derived from phenylalanine were synthesized. Triple helix formation by these modified PNAs with RNA and DNA hairpins having a variable base pair in the middle of the helix were studied using isothermal titration calorimetry and compared with triple helix formation by non-modified PNAs. While unmodified PNA had low sequence selectivity against mismatched hairpins, introduction of one or two phenylalanine-derived monomers significantly increased the mismatch discrimination and sequence selectivity of the modified PNA. Consistent with our previous observations, PNA formed more stable triple helices with RNA than with DNA. Interestingly, the phenylalanine modification further improved the preference of PNA for RNA over DNA hairpin.
PNA backbone modification; PNA-RNA triple helix; isothermal titration calorimetry; peptide nucleic acid; recognition of double-stranded RNA
A new variety on non-coding RNA has been discovered by several groups: circular RNA (circRNA). This discovery raises intriguing questions about the possibility of the existence of knotted RNA molecules and the existence of a new class of enzymes changing RNA topology, RNA topoisomerases.
The application of ex vivo synthetic DNA as a high capacity information storage medium is well documented. Herein, we consider the potential for synthetic DNA to be incorporated as part of the human genome; providing a definitive, accessible, in vivo database of patient history.
synthetic DNA; diagnostics; information; storage; identification
Recently, we achieved the first in vitro selection of 2′-O,4′-C-methylene bridged/locked nucleic acid (2′,4′-BNA/LNA) aptamers. High-affinity thrombin-binding aptamers (TBAs) were obtained from DNA-based libraries containing 2′-O,4′-C-methylene-bridged/linked bicyclic ribonucleotides (B/L nucleotides) in the 5′-primer region, using the method of capillary electrophoresis systematic evolution of ligands by exponential enrichment (CE-SELEX). Furthermore, a similar selection protocol could provide TBAs that contain B/L nucleotides in both primer and random regions. We review technical challenges involved in the generation of various BNA libraries using analogs of B/L nucleoside-5′-triphosphate and polymerase variants and also discuss applications of these libraries to the selection of BNA (LNA) aptamers, as well as future prospects for their therapeutic and diagnostic uses.
2′,4′-BNA/LNA; KODDNA polymerase; Bridged (locked) nucleic acid aptamer; Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX); Xeno-nucleic acid (XNA)
The development of a new class of peptide nucleic acids (PNAs), i.e., gamma PNAs (γPNAs), creates the need for a general and effective method for its delivery into cells for regulating gene expression in mammalian cells. Here we report the antisense activity of a recently developed hydrophilic and biocompatible diethylene glycol (miniPEG)-based gamma peptide nucleic acid called MPγPNAs via its delivery by poly(lactide-co-glycolide) (PLGA)-based nanoparticle system. We show that MPγPNA oligomers designed to bind to the selective region of Chemokine Receptor 5 (CCR5) transcript, induce potent and sequence-specific antisense effects as compared with regular PNA oligomers. In addition, PLGA nanoparticle delivery of MPγPNAs is not toxic to the cells. The findings reported in this study provide a combination of γPNA technology and PLGA-based nanoparticle delivery method for regulating gene expression in live cells via the antisense mechanism.
CCR5; PEG; PNA; antisense; nanoparticle; γPNA
We have developed an assay for single strand DNA and RNA detection which is based on novel pyrene−perylene FRET pairs attached to short LNA/DNA probes. The assay is based on ratiometric emission upon binding of target DNA/RNA by three combinations of fluorescent LNA/DNA reporter strands. Specific geometry of the pyrene fluorophore attached to the 2′-amino group of 2′-amino-LNA in position 4 allows for the first time to efficiently utilize dipole−dipole orientation parameter for sensing of single-nucleotide polymorphisms (SNPs) in nucleic acid targets by FRET. Using novel probes, SNP detection is achieved with advantages of large Stokes shift (115 nm), high fluorescence quantum yields and low limit of target detection values (< 5 nM). Rapid and accurate genotyping of highly polymorphic HIV Pol cDNA and RNA fragments performed herein proves the possibility for broad application of the novel pyrene−perylene FRET pairs, e.g., in imaging and clinical diagnostics.
oligonucleotide; LNA; pyrene; perylene; fluorescence; FRET; SNP
The affinity-mediated selection of large libraries of DNA-encoded small molecules is increasingly being used to initiate drug discovery programs. We present universal methods for the encoding of such libraries using the chemical ligation of oligonucleotides. These methods may be used to record the chemical history of individual library members during combinatorial synthesis processes. We demonstrate three different chemical ligation methods as examples of information recording processes (writing) for such libraries and two different cDNA-generation methods as examples of information retrieval processes (reading) from such libraries. The example writing methods include uncatalyzed and Cu(I)-catalyzed alkyne-azide cycloadditions and a novel photochemical thymidine-psoralen cycloaddition. The first reading method “relay primer-dependent bypass” utilizes a relay primer that hybridizes across a chemical ligation junction embedded in a fixed-sequence and is extended at its 3′-terminus prior to ligation to adjacent oligonucleotides. The second reading method “repeat-dependent bypass” utilizes chemical ligation junctions that are flanked by repeated sequences. The upstream repeat is copied prior to a rearrangement event during which the 3′-terminus of the cDNA hybridizes to the downstream repeat and polymerization continues. In principle these reading methods may be used with any ligation chemistry and offer universal strategies for the encoding (writing) and interpretation (reading) of DNA-encoded chemical libraries.
chemical ligation; click chemistry; combinatorial chemistry; drug discovery; modified nucleotide; photochemistry; polymerase; template-dependent polymerization; unimolecular information recording and retrieval; in vitro selection
In their recent Science paper, Vafabakhsh and Ha claim that DNA duplexes at the range of 100 bp experience anomalous flexibility, much greater than the flexibility of large DNA molecules.1 However, careful reevaluation of their data leads to the conclusion that the presented data do not warrant the authors’ claim.
DNA bending flexibility; DNA minicircles; DNA sticky ends; DNA extreme bendability; single-molecule data
As a proof-of-principle, two hetero-bimetallic PNA oligomers containing a ruthenium(II) polypyridyl and a cyclopentadienyl manganese tricarbonyl complex have been prepared by serial combination of solid-phase peptide coupling and in-solution thiol chemistry. Solid-phase N-terminus attachment of Ru(II)-polypyridyl carboxylic acid derivative, C1, onto the thiol-functionalized PNA backbone (H-a-a-g-t-c-t-g-c-linker-cys-NH2) has been performed by standard peptide coupling method. As two parallel approaches, the strong affinity of thiols for maleimide and haloacetyl group has been exploited for subsequent post-SPPS addition of cymantrene-based organometallic cores, C2 and C3. Michael-like addition and thioether ligation of thiol functionalized PNA1 (H-gly-a-a-g-t-c-t-g-c-linker-cys-NH2) and PNA2 (C1-a-a-g-t-c-t-g-c-linker-cys-NH2) to cymantrene maleimide and chloroacetyl derivatives, C2 and C3, respectively, has been performed. The synthesized ruthenium(II)-cymantrenyl PNA oligomers have been characterized by mass spectrometry (ESI-MS) and IR spectroscopy. The distinct Mn-CO vibrational IR stretches, between 1,924–2,074 cm−1, have been used as markers to confirm the presence of cymantrenyl units in the PNA sequences and the purity of the HPLC-purified PNA thioethers assessed using LC-MS.
peptide nucleic acids; hetero-metalation; ruthenium; cymantrene; thioether; organometallics
Fluorescent probes for the detection of a double-stranded DNA were prepared by labeling a triplex-forming DNA oligonucleotide with a thiazole orange (TO) dimer unit. They belong to ECHO (exciton-controlled hybridization-sensitive fluorescent oligonucleotide) probes which we have previously reported. The excitonic interaction between the two TO molecules was expected to effectively suppress the background fluorescence of the probes. The applicability of the ECHO probes for the detection of double-stranded DNA was confirmed by examining the thermal stability and photophysical and kinetic properties of the DNA triplexes formed by the ECHO probes.
DNA; duplex; triplex; fluorescence; probe; thiazole orange; excitonic interaction
The synthesis of an azide containing PNA monomer is described. The monomer was incorporated into two PNA sequences for the purpose of synthesizing an intercalating fluorophore-labeled PNA and a metal binding hairpin using a solid phase copper catalyzed azide-alkyne Huisgen cycloaddition (CuAAC). Click chemistry was performed using 2-ethynylfluorene or 1-ethynylpyrene to add a fluorophore to the PNA, which were tested for their ability to recognize an abasic site on a DNA target. A PNA hairpin possessing azide monomers at each termini was synthesized and reacted with 2-ethynylpyridine to form a hairpin that is stabilized by Ni2+.
click chemistry; pyrene; fluorene; metal-binding; hairpin; on-resin; Huisgen cycloaddition
Development of artificial nucleic acids for therapeutic applications warrants that the oligomers be endowed with high specificity, enzymatic stability and with no/reduced off-target effects. The balance between strength of the duplex with target RNA and enzyme stability is therefore the key factor for the designed modification. The chiral serinol derivative combines the attributes of amino- and methoxy- substitution when at 2′- position and at 3′- and 5′- ends, effectively balancing the duplex stability and resistance to hydrolytic enzymes. The biological effect seen is the remarkable improvement in splice correction by the steric blocking antisense oligonucleotide with just 4 modified units, i.e ~20% substitution with R-aminomethoxypropyloxy (R-AMP)-thymidine within the 2′-OMe 18mer sequence.
splice correction; steric-blocking; antisense; oligonucleotides
Here we describe the first example of selective reductive amination in biological fluids using split aptamer proximity ligation (StAPL). Utilizing the cocaine split aptamer, we demonstrate small-molecule-dependent ligation that is dose-dependent over a wide range of target concentrations in buffer, human blood serum and artificial urine medium. We explore the substrate binding preferences of the split aptamer and find that the cinchona alkaloids quinine and quinidine bind to the aptamer with higher affinity than cocaine. This increased affinity leads to improved detection limits for these small-molecule targets. We also demonstrate that linker length and hydrophobicity impact the efficiency of split aptamer ligation. The ability to carry out selective chemical transformations using non-bioorthogonal chemistry in media where competing reactive groups are present highlights the power of the increased effective molarity provided by DNA assembly. Obviating the need for bioorthogonal chemistry would dramatically expand the repertoire of chemical transformations available for use in templated reactions such as proximity ligation assays, in turn enabling the development of novel methods for biomolecule detection.
DNA; reductive amination; split aptamer; templated reaction; bioorthogonal
We have developed an assay for single strand DNA or RNA detection which is based on the homo-DNA templated Staudinger reduction of the profluorophore rhodamine-azide. The assay is based on a three component system, consisting of a homo-DNA/DNA hybrid probe, a set of homo-DNA reporter strands and the target DNA or RNA. We present two different formats of the assay (Omega probe and linear probe) in which the linear probe was found to perform best with catalytic turnover of the reporter strands (TON: 8) and a match/mismatch discrimination of up to 19. The advantage of this system is that the reporting (homo-DNA) and sensing (DNA) domain are decoupled from each other since the two pairing systems are bioorthogonal. This allows independent optimization of either domain which may lead to higher selectivity in in vivo imaging.
homo-DNA; Staudinger reaction; RNA diagnostics; fluorescence sensing; oligonucleotides
Over the past decade, several technologies have emerged to access nucleic acid-tagged libraries and select the fittest compound within such libraries. This perspective focuses on recent development with PNA-tagged small molecules displayed on DNA templates for screening purposes and to probe the optimal geometry in multivalent interactions.
DNA display; PNA-encoded synthesis; cooperative binding; inhibitor; multivalent interaction; screen
Information storage capabilities are key in most aspects of society and the requirement for storage capacity is rapidly expanding. In principle, DNA could be a high-density medium for information storage. Church and coworkers recently demonstrated how binary data can be encoded, stored in, and retrieved from a library of oligonucleotides, increasing by several orders of magnitude the amount and density of manmade information stored in DNA to date. The technology remains in its infancy and important hurdles have yet to be overcome in order to realize its potential. However, DNA may be particularly useful as a storage-medium over long time-scales (centuries), because data-access is compatible with any large-scale DNA-sequencing and -synthesis technology.
DNA; information storage in DNA; bit; byte; binary encoding
This review highlights the recent methods to prepare PNA-based materials through a combination of self-assembly and self-organization processes. The use of these methods allows easy and versatile preparation of structured hybrid materials showing specific recognition properties and unique physicochemical properties at the nano- and micro-scale levels displaying potential applications in several directions, ranging from sensors and microarrays to nanostructured devices for biochips.
PNA; monolayers; nanoparticles; self-assembly; self-organisation; materials; surfaces; sensors; microarrays; biochips; DNA-PNA duplexes; hybridization
We are happy to publish this special issue dedicated to Prof. Rosangela Marchelli. This issue not only celebrates her long-standing scientific activity on occasion of her significant anniversary, but it is meant to recognize her contribution to Bioorganic Chemistry in the field of Artificial DNA, and in particular of Peptide Nucleic Acids.
The helix is a critical conformation exhibited by biological macromolecules and plays a key role in fundamental biological processes. Biological helical polymers exist in a single helical sense arising from the chiral effect of their primary units—for example, DNA and proteins adopt predominantly a right-handed helix conformation in response to the asymmetric conformational propensity of D-sugars and L-amino acids, respectively. In using these homochiral systems, nature blocks our observations of some fascinating aspects of the cooperativity in helical systems, although when useful for a specific purpose, “wrong” enantiomers may be incorporated in specific places. In synthetic helical systems, on the contrary, incorporation of non-racemic chirality is an additional burden, and the findings discussed in this review show that this burden may be considerably alleviated by taking advantage of the amplification of chirality, in which small chiral influences lead to large consequences. Peptide nucleic acid (PNA), which is a non-chiral synthetic DNA mimic, shows a cooperative response to a small chiral effect induced by a chiral amino acid, which is limited, however, due to the highly flexible nature of this oligomeric chimera. The lack of internal stereochemical bias is an important factor which makes PNA an ideal system to understand some cooperative features that are not directly accessible from DNA.
helix control; chiral amplification; cooperativity; helical polymers; PNA
The combined use of surface plasmon resonance (SPR) and modified or mimic oligonucleotides have expanded diagnostic capabilities of SPR-based biosensors and have allowed detailed studies of molecular recognition processes. This review summarizes the most significant advances made in this area over the past 15 years.
Functional and conformationally restricted DNA analogs (e.g., aptamers and PNAs) when used as components of SPR biosensors contribute to enhance the biosensor sensitivity and selectivity. At the same time, the SPR technology brings advantages that allows forbetter exploration of underlying properties of non-natural nucleic acid structures such us DNAzymes, LNA and HNA.
DNAzyme; LNA; PNA; SPR; aptamer; biosensors