Development of antiparallel-type triplex-forming oligonucleotides containing quinoline derivatives capable of recognizing a T-A base pair in a DNA duplex.

A triplex-forming oligonucleotide (TFO) can bind to genomic DNA and inhibit the expression of genes with specific sequences. However, to date, there have been a few reports of modified bases in antiparallel TFOs that can recognize and strongly bind to T-A base pairs. In this study, we introduced several quinoline derivatives into antiparallel TFOs to develop modified bases that can recognize the T-A base pair and evaluated their ability to form triplexes and to discriminate between base pairs. The introduction of 2-acetamido-6-aminoquinoline6DAQac) into an antiparallel TFO allowed the selective recognition of a T-A base pair at both low and high salt concentrations.

Oligonucleotide Synthesis on Porous Glass Resins Containing Activators.

For the advancement of nucleic acid-related research, high-efficiency, low-cost synthesis of high-purity oligonucleotides is necessary. Herein, we introduced hydroxybenzotriazole (HOBt) activators on controlled pore glass resins to improve the efficiency of chain elongation (the synthesis efficiency increased from 48% without an activator to 92% with an activator). In particular, the use of the resin containing 6-trifluoromethyl HOBt with a linker of lauric acid and succinic acid significantly improved the synthesis efficiency for both DNA and RNA syntheses.

Chemical Synthesis of Modified Oligonucleotides Containing 5'-Amino-5'-Deoxy-5'-Hydroxymethylthymidine Residues.

Introduction of cationic modifications into an oligonucleotide can increase its nuclease resistance and duplex- or triplex-forming abilities. In a recent study, we found that the nuclease resistance and RNA binding selectivity of an oligonucleotide containing a 5'-(R)-amino-5'-deoxy-5'-(R)-hydroxymethylthymidine residue were greater than those of the unmodified oligonucleotide. In this article, we describe the synthesis of 5'-amino-5'-deoxy-5'-hydroxymethylthymidine via dihydroxylation of the 5'-alkene derivative using either of two commercial AD (asymmetric dehydroxylation) mixes or via epoxidation and ring opening. We also provide detailed protocols for the syntheses of oligonucleotides containing 5'-amino-5'-deoxy-5'-hydroxymethylthymidine residues. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of 5'-amino-5'-deoxy-5'-hydroxymethylthymidine phosphoramidites 9a and 9b Basic Protocol 2: Synthesis of oligonucleotides 1 and 2 containing 5'-amino-5'-deoxy-5'-hydoxymethylthymidine residues (R T and S T).

Chemical synthesis and biochemical characterization of cyclic oligonucleotides containing acyl groups at both 5'- and 3'-terminal positions.

Modified oligonucleotides, whose ON-OFF switch of hybridization can be controlled by an external stimulus, are important to understanding life phenomena and efficient treatment of diseases. The ON-OFF switch can be completely controlled by chemical modification of the oligonucleotide such as cyclization. However, their chemical modifications of the previous cyclic oligonucleotides remain after the addition of an external stimulus. To overcome this problem, we carried out the first synthesis of cyclic oligonucleotides containing acyl groups at both 5'- and 3'-terminal positions, which can be hydrolyzed by intracellular esterase. The cyclic oligonucleotides were successfully synthesized via disulfide bond formation and the phosphoramidite method without base protection on polymer supports containing a silyl linker. Subsequently, we were able to introduce a functional group into the cyclic oligonucleotide using the corresponding isothiocyanate reagent. Additionally, a cyclic oligonucleotide with acyl groups was found to have a much lower binding ability than the corresponding linear oligonucleotide. Moreover, we demonstrated its structural conversion to the corresponding linear oligonucleotide with two thiol groups under reducing conditions using dithiothreitol. It was also confirmed that the two terminal acyl groups of the linear oligonucleotide were hydrolyzed by pig liver esterase. These results indicate that hybridization of cyclic acylated nucleic acid drugs with high nuclease resistance is regulated by intracellular esterase under the reducing conditions in the cell cytoplasm.

The ability of a triplex-forming oligonucleotide to recognize T-A and C-G base pairs in a DNA duplex is enhanced by incorporating N-acetyl-2,7-diaminoquinoline.

A triplex-forming oligonucleotide (TFO) can recognize the homopurine-homopyrimidine sequence in DNA duplexes and inhibit the transcription of targeted mRNAs. Recently, we reported that N-acetyl-2,7-diamino-1,8-naphthyridine (DANac), incorporated into a TFO, has high binding ability and base recognition selectivity for the pyrimidine bases in the purine-rich chain of the DNA duplex at pH 7.4. However, it was found in this study that the difference in the Tm values between the pyrimidine bases and purine bases decreased by more than 4 °C at pH 6.0-7.0. To improve the low base recognition selectivity of the TFO, we designed a new artificial base, DAQac, with a quinoline skeleton. The Tm values of the triplexes containing DAQac:T-A or DAQac:C-G were more than 13 °C higher than those of the triplexes containing DAQac:A-T or DAQac:G-C at pH 7.4. We also observed that under more acidic conditions (pH 6.0-7.0), the base recognition selectivity of DAQac in a triplex was higher than that of DANac, although the binding ability of DAQac in a triplex was similar to that of DANac. Additionally, we found that DAQac, incorporated into the TFO, could accurately recognize the MeC-G base pair in the hairpin DNA, similar to the C-G base pair.

Chemical synthesis and properties of modified oligonucleotides containing 5'-amino-5'-deoxy-5'-hydroxymethylthymidine residues.

In this study, we designed 5'-amino-5'-deoxy-5'-hydroxymethylthymidine as a new oligonucleotide modification with an amino group directly attached to the 5'-carbon atom. We successfully synthesized two isomers of 5'-amino-5'-deoxy-5'-hydroxymethylthymidine via dihydroxylation of the 5'-vinyl group incorporated into 5'-deoxy-5'-C-methenylthymidine derivative. Moreover, it was found that the nuclease resistance, binding selectivity to single-stranded RNA, and triplex-forming ability of an oligonucleotide containing RT residues of the new compound were higher than those of the unmodified oligonucleotide.

DNA triplex-based fluorescence turn-on sensors for adenosine using a fluorescent molecular rotor 5-(3-methylbenzofuran-2-yl) deoxyuridine.

Fluorescence turn-on sensors for adenosine were developed using DNA triplexes modified with a fluorescent molecular rotor 5-(3-methylbenzofuran-2-yl)deoxyuridine (dUMBF) and abasic sites. Binding of adenosine to the abasic site next to the dUMBF changed the microenvironment and conformation (from the twisted to planar state) of dUMBF and enhanced the fluorescence. Adenosine could be selectively detected over other nucleosides and adenosine phosphates. The binding of adenosine was confirmed by UV-thermal melting experiments. Further, the conformational changes of dUMBF from the twisted to coplanar state upon binding of adenosine was supported by MD simulations.

Synthesis of and triplex formation in oligonucleotides containing 2'-deoxy-6-thioxanthosine.

This study aimed to synthesize triplex-forming oligonucleotides (TFOs) containing 2'-deoxy-6-thioxanthosine (s6X) and 2'-deoxy-6-thioguanosine (s6Gs) residues and examined their triplex-forming ability. Consecutive arrangement of s6X and s6Gs residues increased the triplex-forming ability of the oligonucleotides more than 50 times, compared with the unmodified TFOs. Moreover, the stability of triplex containing a mismatched pair was much lower than that of the full-matched triplex, though s6X could form a s6X-GC mismatched pair via tautomerization of s6X. The present results reveal excellent properties of modified TFOs containing s6Xs and s6Gs residues, which may be harnessed in gene therapy and DNA nanotechnology.

pH-Dependent Switching of Base Pairs Using Artificial Nucleobases with Carboxyl Groups.

In this study, we report the synthesis of modified oligonucleotides consisting of benzoic acid or isophthalic acid residues as new nucleobases. As evaluated by UV thermal denaturation analysis at different pH conditions (5.0, 6.0, 7.0, and 8.0), these modified oligonucleotides exhibited pH-dependent recognition of natural nucleobases and one is first found to be capable of base pair switching in response to a pH change. The isophthalic acid residue incorporated into the oligonucleotide on a d-threoninol backbone could preferentially bind with adenine but with guanine in response to a change in the pH conditions from pH 5 to pH 7 (or 8) without significant difference in duplex stability. These findings would be valuable for further developing pH-responsive DNA-based molecular devices.

Fluorescence enhancement of oligodeoxynucleotides modified with green fluorescent protein chromophore mimics upon triplex formation.

Green fluorescent protein (GFP)-based molecular-rotor chromophores were attached to the 5-positions of deoxyuridines, and subsequently, incorporated into the middle positions of oligodeoxynucleotides. These oligonucleotides were designed to form triplex DNA in order to encapsulate the GFP chromophores, mimicking GFP structures. Upon triplex formation, the embedded GFP chromophores exhibited fluorescence enhancement, suggesting the potential application of these fluorescent probes for the detection of nucleic acids.

Coating lanthanide nanoparticles with carbohydrate ligands elicits affinity for HeLa and RAW264.7 cells, enhancing their photodamaging effect.

Lanthanide nanoparticles (LNPs) conjugated with monosaccharides were synthesized as a photon energy-upconverting nanodevice with affinity to cancer cells. The conjugates were designed to selectively damage the cancer cells containing protoporphyrin IX, a photosensitizer endogenously synthesized from priorly administrated 5-aminolevlunic acid (ALA), by a highly tissue-penetrative near-infrared (NIR) irradiation. First of all, the affinities of monosaccharides toward cells (HeLa, RAW264.7, and MKN45) were assessed by a novel cell aggregation assay with trivalent monosaccharide-citric acid conjugates. As a result, HeLa exhibited high affinity for glucose, while RAW264.7 for glucose, galactose, mannose, and fucose. A similar cell-monosaccharide affinity was microscopically observed when the cells were mixed with monosaccharide-LNP conjugates and rinsed, in which the high affinity LNP probes luminesced on the cells. The high affinity monosaccharide-LNPs showed greater photodamaging effects than the unmodified LNP toward the corresponding cells, when the cells were pretreated with ALA and irradiated by NIR. This study demonstrates that carbohydrates can be used as selective ligands for cancer cells in a photodynamic therapy with LNP.

Enzymatic synthesis and reverse transcription of RNAs incorporating 2'-O-carbamoyl uridine triphosphate.

Enzymatic synthesis and the reverse transcription of RNAs containing 2'-O-carbamoyl uridine were evaluated. A mild acidic deprotection procedure allowed the synthesis of 2'-O-carbamoyl uridine triphosphate (UcmTP). UcmTP was incorporated correctly into long RNAs, and its fidelity during reverse transcription using SuperScript III was sufficient for RNA aptamer selection.

Synthesis of 5-[3-(2-aminopyrimidin-4-yl)aminopropyn-1-yl]uracil derivative that recognizes Ade-Thy base pairs in double-stranded DNA.

5-[3-(2-Aminopyrimidin-4-yl)aminopropyn-1-yl]uracil (Ura(Pyr)) was designed as a new nucleobase to recognize Ade-Thy base pair in double-stranded DNA. We successfully synthesized the dexoynucleoside phosphoramidite having Ura(Pyr) and incorporated it into triplex forming oligonucleotides (TFOs). Melting temperature analysis revealed that introduction of Ura(Pyr) into TFOs could effectively stabilize their triplex structures without loss of base recognition capabilities.

The Effect of Coatings on the Affinity of Lanthanide Nanoparticles to MKN45 and HeLa Cancer Cells and Improvement in Photodynamic Therapy Efficiency.

An improvement in photodynamic therapy (PDT) efficiency against a human gastric cancer cell line (MKN45) with 5-aminolevulinic acid (ALA) and lanthanide nanoparticles (LNPs) is described. An endogenous photosensitizer, protoporphyrin IX, biosynthesized from ALA and selectively accumulated in cancer cells, is sensitizable by the visible lights emitted from up-conversion LNPs, which can be excited by a near-infrared light. Ten kinds of surface modifications were performed on LNPs, NaYF4(Sc/Yb/Er) and NaYF4(Yb/Tm), in an aim to distribute these irradiation light sources near cancer cells. Among these LNPs, only the amino-functionalized LNPs showed affinity to MKN45 and HeLa cancer cells. A PDT assay with MKN45 demonstrated that amino-modified NaYF4(Sc/Yb/Er) gave rise to a dramatically enhanced PDT effect, reaching almost perfect lethality, whereas NaYF4(Yb/Tm)-based systems caused little improvement in PDT efficiency. The improvement of PDT effect with the amino-modified NaYF4(Sc/Yb/Er) is promising for a practical PDT against deep cancer cells that are reachable only by near-infrared lights.

Synthesis and triplex-forming properties of oligonucleotides capable of recognizing corresponding DNA duplexes containing four base pairs.

A triplex-forming oligonucleotide (TFO) could be a useful molecular tool for gene therapy and specific gene modification. However, unmodified TFOs have two serious drawbacks: low binding affinities and high sequence-dependencies. In this paper, we propose a new strategy that uses a new set of modified nucleobases for four-base recognition of TFOs, and thereby overcome these two drawbacks. TFOs containing a 2'-deoxy-4N-(2-guanidoethyl)-5-methylcytidine (d(g)C) residue for a C-G base pair have higher binding and base recognition abilities than those containing 2'-OMe-4N-(2-guanidoethyl)-5-methylcytidine (2'-OMe (g)C), 2'-OMe-4N-(2-guanidoethyl)-5-methyl-2-thiocytidine (2'-OMe (g)Cs), d(g)C and 4S-(2-guanidoethyl)-4-thiothymidine ((gs)T). Further, we observed that N-acetyl-2,7-diamino-1,8-naphtyridine ((DA)Nac) has a higher binding and base recognition abilities for a T-A base pair compared with that of dG and the other DNA derivatives. On the basis of this knowledge, we successfully synthesized a fully modified TFO containing (DA)Nac, d(g)C, 2'-OMe-2-thiothymidine (2'-OMe (s)T) and 2'-OMe-8-thioxoadenosine (2'-OMe (s)A) with high binding and base recognition abilities. To the best of our knowledge, this is the first report in which a fully modified TFO accurately recognizes a complementary DNA duplex having a mixed sequence under neutral conditions.

Synthesis of peptide nucleic acids containing pyridazine derivatives as cytosine and thymine analogs, and their duplexes with complementary oligodeoxynucleotides.

Synthesis of peptide nucleic acids (PNAs) is reported with new pyridazine-type nucleobases: 3-aminopyridazine (aPz) and 1-aminophthalazine (aPh) as cytosine analogs, and pyridazin-3-one (Pz(O)) and phthalazin-1-one (Ph(O)) as thymine analogs. The PNAs having an aPz or a Pz(O) formed duplexes with each complementary oligodeoxynucleotide forming a base pair with G or A, respectively, as evaluated by using UV melting analyses and circular dichroism (CD) spectra.

A ring-flippable sugar as a stimuli-responsive component of liposomes.

For the development of a liposome that takes in and out a drug in response to stimuli, 2,4-diaminoxylose (Xyl), which allows stimuli-responsive conformational switches between (4)C1 and (1)C4, was incorporated into a lipid structure: Xyl derivatives with C8 and C16 methylene chains at the 1,3-positions (C8Xyl and C16Xyl) were synthesized. (1)H NMR spectroscopy indicates that the addition of Zn(2+) and then H(+) induces conformational switches from the chair ((4)C1) to the reverse chair ((1)C4) and (1)C4-to-(4)C1, respectively, at Xyl; this leads to transformation of the lipids between linear and bent structures. Osmotic pressure and electron microscopy studies demonstrate that C8Xyl in water forms spherical solid aggregates (C8Xyl-Zn), which are converted into liposomes (C8Xyl+Zn) upon the addition of Zn(2+), and C16Xyl forms liposomes regardless of the presence of Zn(2+). The aggregates of C8Xyl±Zn incorporated a fluorophore and only C8Xyl+Zn released the content upon the addition of HCl. This study shows that Xyl could be a stimuli-responsive component of a liposome.

Controlling the fluorescence of benzofuran-modified uracil residues in oligonucleotides by triple-helix formation.

We developed fluorescent turn-on probes containing a fluorescent nucleoside, 5-(benzofuran-2-yl)deoxyuridine (dU(BF)) or 5-(3-methylbenzofuran-2-yl)deoxyuridine (dU(MBF)), for the detection of single-stranded DNA or RNA by utilizing DNA triplex formation. Fluorescence measurements revealed that the probe containing dU(MBF) achieved superior fluorescence enhancement than that containing dU(BF). NMR and fluorescence analyses indicated that the fluorescence intensity increased upon triplex formation partly as a consequence of a conformational change at the bond between the 3-methylbenzofuran and uracil rings. In addition, it is suggested that the microenvironment around the 3-methylbenzofuran ring contributed to the fluorescence enhancement. Further, we developed a method for detecting RNA by rolling circular amplification in combination with triplex-induced fluorescence enhancement of the oligonucleotide probe containing dU(MBF).

Synthesis and properties of oligonucleotides modified with 2'-O-(2-carboxyethyl)nucleotides and their carbamoyl derivatives.

2'-O-Methyl oligoribonucleotides with four kinds of 2'-O-modified uridine derivatives were synthesised. Their duplex stability, hydration behavior and exonuclease resistance were studied by spectroscopic analyses and molecular dynamics simulations. Consequently, 2'-O-modification of the uridine residue with 2-carbamoylethyl or 2-(N-methylcarbamoyl)ethyl groups resulted in a significant improvement of the exonuclease resistance without the loss of duplex stability.

A new modified cytosine base capable of base pairing with guanine using four hydrogen bonds.

Oligonucleotides, containing 4-N-(1H-pyrrol-2-ylcarbonyl)deoxycytidine (dC(Pyc)) and related derivatives, were synthesized via deprotection using 1.5 M NaOMe/MeOH. Among them, oligodeoxynucleotides containing dC(Pyc) exhibited a higher hybridization affinity for DNA and RNA than the unmodified oligodeoxynucleotides. Comparative analysis between dC(Pyc) and its derivatives by molecular dynamic simulation indicated that the C(Pyc) residue could form four hydrogen bonds with the opposite G nucleobase keeping a more planar structure than the C(Inc) residue where the Pyc group was replaced with a 1H-indol-2-ylcarbonyl group.