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G-quadruplex (G4) DNA structures are prevalent secondary DNA structures implicated in fundamental cellular functions, such as replication and transcription. Furthermore, G4 structures are directly correlated to human diseases such as cancer and have been highlighted as promising therapeutic targets for their ability to regulate disease-causing genes, e.g., oncogenes. Small molecules that bind and stabilize these structures are thus valuable from a therapeutic perspective and helpful in studying the biological functions of the G4 structures. However, there are hundreds of thousands of G4 DNA motifs in the human genome, and a long-standing problem in the field is how to achieve specificity among these different G4 structures. Here, we developed a strategy to selectively target an individual G4 DNA structure. The strategy is based on a ligand that binds and stabilizes G4s without selectivity, conjugated to a guide oligonucleotide, that specifically directs the G4-Ligand-conjugated oligo (GL-O) to the single target G4 structure. By employing various biophysical and biochemical techniques, we show that the developed method enables the targeting of a unique, specific G4 structure without impacting other off-target G4 formations. Considering the vast amount of G4s in the human genome, this represents a promising strategy to study the presence and functions of individual G4s but may also hold potential as a future therapeutic modality.

Oligonucleotide (ON) conjugates are increasingly important tools for various molecular diagnostics, nanotechnological applications, and for the development of nucleic acid-based therapies. Multiple labeling of ONs can further equip ON-conjugates and provide improved or additional tailored properties. Typically, the preparation of ON multiconjugates involves additional synthetic steps and/or manipulations in post-ON assembly. This report describes the simplified methodology allowing for multiple labeling of ONs on a solid support and is compatible with phosphodiester as well as phosphorothioate (PS) ONs. The current approach utilizes two novel alkyne- and amino-functionalized linker phosphoramidites that can be readily synthesized from a common aminodiol intermediate in three steps. The combination of new linkers provides orthogonal functionalities, which allow for multiple attachments of similar or varied moieties. The linkers are incorporated into ONs during automated solid-phase ON synthesis, and the conjugation with functional entities is achieved by either amide bond formation or by copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The versatility of the approach is demonstrated by the synthesis of 5'-site ON multiconjugates with small molecules, peptides, and fatty acids as well as in the preparation of an internal peptide-ON conjugate., Competing Interests: The authors declare no competing financial interest., (© 2020 The Authors. Published by American Chemical Society.)

Improving oligonucleotide delivery is critical for the further development of oligonucleotide-based therapeutics. Covalent attachment of reporter molecules is one of the most promising approaches toward efficient oligonucleotide-based therapies. An efficient methods for the attachment of a variety of reporter groups is Cu(I)-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition. However, the majority of potential oligonucleotide (ON) therapeutics in clinical trials are carrying phosphorothioate (PS) linkages, and this robust conjugation method is not yet established for these ONs due to a general concern of Cu-S interaction. Here, we developed a method allowing for efficient conjugation of peptides to PS oligonucleotides. The method utilizes solid supported oligonucleotides that can be readily transformed into "clickable ONs" by simple linker conjugation and further reacted with an azido containing moiety (e.g., a peptide) using the CuBr × Me 2 S complex as a superior catalyst in that reaction. This study opens the way for further development of PS oligonucleotide-conjugates by means of efficient Cu(I)-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition.

A new generation of ligands designed to interact with the α-helix/β-strand discordant region of the amyloid-β peptide (Aβ) and to counteract its oligomerization is presented. These ligands are designed to interact with and stabilize the Aβ central helix (residues 13-26) in an α-helical conformation with increased interaction by combining properties of several first-generation ligands. The new peptide-like ligands aim at extended hydrophobic and polar contacts across the central part of the Aβ, that is, "clamping" the target. Molecular dynamics (MD) simulations of the stability of the Aβ central helix in the presence of a set of second-generation ligands were performed and revealed further stabilization of the Aβ α-helical conformation, with larger number of polar and nonpolar contacts between ligand and Aβ, compared to first-generation ligands. The synthesis of selected novel Aβ-targeting ligands was performed in solution via an active ester coupling approach or on solid-phase using an Fmoc chemistry protocol. This included incorporation of aliphatic hydrocarbon moieties, a branched triamino acid with an aliphatic hydrocarbon tail, and an amino acid with a 4'- N, N-dimethylamino-1,8-naphthalimido group in the side chain. The ability of the ligands to reduce Aβ 1-42 neurotoxicity was evaluated by gamma oscillation experiments in hippocampal slice preparations. The "clamping" second-generation ligands were found to be effective antineurotoxicity agents and strongly prevented the degradation of gamma oscillations by physiological concentration of monomeric Aβ 1-42 at a stoichiometric ratio.

An efficient method for the synthesis of multiply functionalized oligonucleotides (ONs) utilizing a novel H-phosphonate alkyne-based linker for multiple functionalization (LMF) is developed. The strategy allows for the conjugation of various active entities to oligonucleotide through the postsynthetic attachment of LMF at the 5'-terminus of ONs using H-phosphonate chemistry followed by conjugation of various entities via [3 + 2] copper(I) catalyzed cycloaddition in a stepwise manner. Each cycle is composed of attachment of the LMF followed by a click reaction with azido-containing units. Sequential solid-phase synthesis of oligonucleotide conjugates containing three attached entities was performed using an acetylated form of MIF peptide conjugated to azido linker, achieving high conversions at each unit addition. In addition, to show the versatility of the method, oligonucleotide conjugates with several different classes of compounds were synthesized. Each conjugate containing three different entities, whose structure and function varied (e.g., sugars, peptides, fluorescent labels, and m 3 G-Caps).

A new peptide nucleic acid (PNA) construct carrying a tris(2-aminobenzimidazole) phosphodiester cleaver is presented. This non-metal-based artificial nuclease hydrolyzes RNA substrates that form a bulge upon binding to the PNA. Reaction rates depend on the bulge sequence. For conjugates of tris(2-aminobenzimidazole), substrate turnover is shown for the first time. Two methods of analysis for the kinetics are compared: IE-HPLC separation of oligonucleotide fragments and analysis of Cy5-labeled oligonucleotide fragments by denaturating PAGE on a DNA sequencer, respectively. The different methods give rates that are in the same range where, in general, the substrates for the sequencer method give slightly lower rates.

DNA-cleaving restriction enzymes are well-known tools in biomedical and biotechnological research. There are, however, no corresponding enzymes known for RNA cleavage. There has been an ongoing development of artificial ribonucleases, including some attempts at sequence selectivity. However, so far these systems have displayed modest rates of cleavage, and in most cases, the cleaver has been used in excess or in stoichiometric amounts. In the current work, we present PNA-based systems (PNAzymes) that carry a Cu(II)-2,9-dimethylphenanthroline group and that act as site and sequence specific RNases. The general basis for the systems is that the target is cleaved at a nonbase paired region (RNA bulge) which is formed in the substrate upon binding of the PNAzyme. With this copper based system, cleavage takes place at virtually only one site and with a half-life of down to 30 min under stoichiometric conditions. Efficient turnover of RNA-substrate is shown with a 100-fold excess of substrate, thus, demonstrating true enzyme behavior. In addition, alteration of the sequence in the RNA bulge or a mismatch in the base-pairing region leads to substantial decreases in rate showing both kinetic resolution and binding discrimination in the substrate selectivity. The selectivity is further demonstrated by the substrates, with two potential cleavage sites differing in only one base, are cleaved only at the site that either does not have a mismatch or is kinetically preferred. We suggest that these systems can serve as a basis for construction of RNA restriction enzymes for in vitro manipulations.

As sugar-modified nucleosides, nucleotides, and oligonucleotides are much used in pharmacology and enzymatic studies, knowledge on the relative pKa values of the secondary hydroxyls can be of great help in design and interpretations. To obtain this, we have determined the acidity constants for ATP, dATP, 2'-F-dATP, 2'-NH2-ATP, and 2'-O-methyl ATP in aqueous solution. The influence of the relative acidities seem to be mainly from inductive effects since a good correlation between pKa values and group electronegativity is found. There is no clear energetic contribution from a much suggested H-bond between the 2'-OH and 3'-oxyanion in adenosine. To clarify if this kind of H-bond would make a more-prominent energetic contribution in less-polar solvents, we also determined the acidity of secondary hydroxyls in adenosine, 2'-O-methyladenosine, and 3'-O-methyladenosine in water, methanol, and DMSO. The relative differences in pKa values were, however, quite similar in all solvents, suggesting that no major energetic contribution is made by an intramolecular H-bond in adenosine.

We describe the development of a solid-phase technique for the synthesis of 5'-peptide-oligonucleotide conjugates (POCs) with a uniform protection strategy for the nucleic acid and the peptide fragments. On the alpha-amino function, the amino acid building blocks were protected with the 2-(biphenyl-4-yl)propan-2-yloxycarbonyl (Bpoc) group. This protection is removed during the stepwise peptide elongation by the same acidic conditions used for removal of the dimethoxytrityl (DMT) group used in the oligonucleotide assembly (3% trichloroacetic acid, 2 min). The 2-(3,5-dimethoxyphenyl)propan-2-yloxycarbonyl (Ddz) group was also tested. With this somewhat more stable group, a prolonged contact with the acid (at least 16 min) was required for accomplishing complete alpha-amino deprotection, which resulted in some degree of depurination of the acid-sensitive DNA chain. Base-labile acyl protections were adopted for the side-chains of histidine, lysine, and the nucleobase amino functions. These were all removed in the final deblocking step by ammonolysis. This uniform protection scheme for the peptide and the oligonucleotide enabled the total stepwise synthesis of model conjugates in the 3' --> N direction with high efficiency and purity.

Oligoribonucleotide analogues having amide internucleoside linkages (AM1: 3'-CH(2)CONH-5' and AM2: 3'-CH(2)NHCO-5') at selected positions have been synthesized and the thermal stability of duplexes formed by these analogues with complementary RNA fragments has been evaluated by UV melting experiments. Two series of oligomers with either 2'-OH or 2'-OMe vicinal to the amide linkages were studied. Monomeric synthons (3' and 5'-C amines and carboxylic acids) were synthesized as follows: For synthesis of the AM1 analogue, the known sequence of radical allylation followed by the cleavage of the double bond was adopted. For synthesis of the AM2 analogue, novel routes via addition of nitromethane followed by conversion of the nitro function to either amino or carboxyl groups were developed. Coupling of monomeric amines and carboxylic acids followed by protecting group manipulation and phosphonylation gave dimeric 3'-hydrogenphosphonate building blocks for oligonucleotide synthesis. Monomeric model compounds having 3'-amide and 2'-OH or 2'-OMe groups were also prepared and their conformational equilibrium was determined by (1)H NMR. The AM1 and AM2 models showed equal preferences for the North conformers (at 40 degrees C, 88-89% with 2'-OH, and 92-93% with 2'-OMe). At physiological salt concentration (0.1 M NaCl) the duplexes between AM1 modified oligonucleotides and RNA had stability similar to unmodified RNA-RNA duplexes (Delta t(m)= -0.2 to +0.7 degrees C per modification). However, the AM2 modification resulted in substantial stabilization of duplexes: Delta t(m)= +1 to +2.4 degrees C per modification compared to all RNA. A 2'-O-methyl vicinal to the AM2 linkage further increased the duplex stability. Our results suggest that RNA analogues having amide internucleoside bonds are very promising candidates for medicinal applications.

The positioning of the imidazole protecting group of several histidine derivatives was determined by means of (1)H-(15)N heteronuclear multiple-bond correlation NMR experiments. The cross-peak originated from the three-bond correlation between the histidine side-chain H(beta) and the imidazole N(pi) was used for the identification of the N(pi) signal in the (15)N spectrum. Therefore, based on the fact that the signal of the substituted imidazole nitrogen appears always at lower chemical shift (delta) than the unsubstituted one, the position of the blocking group could easily be inferred. The obtained data confirmed previous findings that were accomplished with other less generally applicable spectroscopic or crystallographic techniques.

The 2'-OH group in the ribose sugars of an RNA molecule plays an important role in guiding tertiary interactions that stabilize different RNA structural motifs. Deoxyribose, or 2'-OH by 2'-H, substitution in both the single-stranded and the duplex part of an RNA backbone has been routinely used to evaluate what role the 2'-OH plays in different tertiary interactions that guide an RNA-RNA contact. A deoxyribose substitution not only has the effect of removing a hydrogen bond donating group, but also introduces a sugar moiety with a preference for C2'-endo pucker in a backbone of predominantly C3'-endo sugars. This study evaluates the effects of a single deoxyribose substitution in both single-stranded and double-helical forms of RNA oligomers. A single-stranded, nonrepetitive 7-mer oligoribonucleotide (7-mer RNA) and four different variants having the same base sequence but with a single deoxyribose sugar at different positions in the strands have been studied by ultraviolet (UV) absorption, circular dichroism (CD), and Fourier transform infrared (FTIR) spectroscopy. Duplexes were formed by association with the complementary strand of the 7-mer RNA. The results show that both RNA and DNA single strands have preorganized conformations with spectral properties resembling those of A- and B-form helices, respectively, with RNA being more heterogeneous than its DNA counterpart. A single deoxyribose substitution perturbs the structure of the RNA backbone, with the effect being more pronounced in the single-stranded than in the duplex structure. The perturbation depends on the position of the 2'-H substitution in the strand.

Fourier Transform infrared (FTIR) spectroscopy was used to characterize the Mg2+ dependent association of a 23-mer mixed ribo-deoxyribonucleotide (23-mer RNA) and a 7-mer oligoribonucleotide (7-mer RNA) that models the triple-helical domain of a self-splicing group I intron [Sarkar et al. (1996) Biochemistry 35, 4678-4688]. To elucidate the effect of deoxyribose substitution in the entire backbone, as well as at specific positions, in the assembly of the triple-helical domain, parallel studies were carried out on the association of pure deoxyribonucleotides having base sequences corresponding to the oligoribonucleotides and also between 23-mer RNA and two 7-mer RNA variants. In the variants, either the ribose attached to G451 or the ribose attached to U453 was changed to deoxyribose. FTIR-monitored thermal denaturation of the two 23-mer hairpins shows two distinct melting regions in 1 M NaCl, in case of the RNA hairpin but not for the 23-mer DNA. Triple-helix association between the two strands (7-mer and 23-mer) studied by FTIR show that only when both strands are RNA, association takes place with the formation of the P6 helix. Our results also show that the interactions between the two RNA strands involve some participation of the riboses, which could also involve the 2'-OH groups of the RNA backbone. The assembly of the triple-helical domain is not possible with a deoxyribose backbone and is completely perturbed even when only one ribose at either G451 or U453 position is substituted by deoxyribose.

Structural studies were performed on synthetic oligonucleotides with sequences corresponding to the P4/P6 and J3/4, J6/7 regions of the self-splicing group I intron of the bacteriophage T4 nrdB pre-mRNA, which correspond to the proposed triple-helical domain in the Tetrahymena thermophila intron. A 23-mer RNA was synthesized as a mixed ribo-deoxyribo oligonucleotide, modeling an expected base-paired region of P4 along with the J3/4 and P6 (5'-end bases of P6) regions. strand modeling the 3'-end bases of P6 and J6/7 regions, with which a triple helix may form, was synthesized as a pure oligoribonucleotide (7-mer RNA). The interactions of these oligonucleotides have been characterized by UV and circular dichroism (CD) spectroscopy. The results show that the 23-mer RNA forms a stable hairpin modeling the P4 base-paired region. Triple helix association between the 23-mer RNA hairpin and the 7-mer RNA single strand was detected by CD in the presence of Mg2+ (>5mM) but not in presence of a monovalent cation like Na+ (up to 500 mM). Studies on selected variants of both 7-mer and 23-mer RNAs were carried out. The results show that for the association of the two partner strands not only the formation of P6 helix but also triplet interactions between two strands are required. The association of the two strands in general follow a pattern predicted by comparative sequence analysis. Parallel studies on pure oligoribonucleotides having base sequence corresponding to those of the oligoribonucleotides showed no evidence of association under similar conditions, which could indicate that the 2'-hydroxyl groups of the riboses might play an important role in hydrogen bonding to form the required nucleoside triples. Molecular modeling studies on the proposed "plaited triple helix" formed by the association of the 23-mer RNA hairpin and 7-mer RNA single strand showed that the structure is sterically and energetically feasible.