Phosphate-methylated DNA Research Articles

Specific and efficient knockdown of intracellular miRNA using partially neutralized phosphate-methylated DNA oligonucleic acid-loaded mesoporous silica nanoparticles.

Antisense oligonucleotides (ASOs) are molecules used to regulate RNA expression by targeting specific RNA sequences. One specific type of ASO, known as neutralized DNA (nDNA), contains site-specific methyl phosphotriester (MPTE) linkages on the phosphate backbone, changing the negatively charged DNA phosphodiester into a neutralized MPTE with designed locations. While nDNA has previously been employed as a sensitive nucleotide sequencing probe for the PCR, the potential of nDNA in intracellular RNA regulation and gene therapy remains underexplored. Our study aims to evaluate the regulatory capacity of nDNA as an ASO probe in cellular gene expression. We demonstrated that by tuning MPTE locations, partially and intermediately methylated nDNA loaded onto mesoporous silica nanoparticles (MSNs) can effectively knock down the intracellular miRNA, subsequently resulting in downstream mRNA regulation in colorectal cancer cell HCT116. Additionally, the nDNA ASO-loaded MSNs exhibit superior efficacy in reducing miR-21 levels over 72 hours compared to the efficacy of canonical DNA ASO-loaded MSNs. The reduction in the miR-21 level subsequently resulted in the enhanced mRNA levels of tumour-suppressing genes PTEN and PDCD4. Our findings underscore the potential of nDNA in gene therapies, especially in cancer treatment via a fine-tuned methylation location.

Phosphate-Methylated Oligonucleotides Past, Present and Future

At the moment, we see a great interest for application of Anti Sense Oligonucleotides (ASOs) in order to regulate the expression of genes related to certain diseases. These nucleotides obtained a number of fascinating properties by means of chemical manipulation of natural DNA and RNA under conservation of Watson-Crick base-pairing. About 35 years ago for our research in this field, we selected synthetically (short) phosphate-methylated DNA and RNA. It was concluded that there is an exclusive selection in hybridization affinity with natural DNA and RNA. These (bio)chemical and physical-chemical properties are extensively published. ASOs have found their way in public health as is clearly shown in the treatment of (progressive) neurological diseases. We focus specifically on the past, present and future of the phosphate-methylated oligonucleotides, illustrated with different research studies in chemistry and biophysics. A new field of application of modified DNAs is based on interactive improvements of sensitivity and specificity of nanowire field effect transistor gene chip by designing phosphate-methylated DNA as probe.

Retracted HIV Study Provides New Information about the Status of the <i>in</i> <i>Vitro</i> Inhibition of DNA Replication by Backbone Methylation

In this publication attention is given to a retracted article in Science at the end of 1990 concerning the HIV-1 inhibition by a modified backbone DNA as the phosphatemethylated DNA. A disproportion in the presented data resulted in a faulty generalization of the (bio)chemical characteristics of the phosphatemethylated DNA (18- and 20-nucleotides). In the confusion and the outside pressure a related study in Nucleic Acids Research on the in vitro dynamics of a regiospecific inhibition of DNA duplication with long (20- and 18-nucleotides) and short (8-nucleotides) phosphatemethylated DNA was completely ignored. A restoration will be given based on a comprehensive view demonstrating the unique molecular and conformational properties of phosphatemethylated DNA in their (bio)chemistry towards natural DNA and RNA (HIV-1 RNA loops).

A conformational B-Z DNA study monitored with phosphatemethylated DNA as a model for epigenetic dynamics focused on 5-(hydroxy)methylcytosine

This study was directed on the B- into Z-DNA isomerization with alternating CG sequences monitored with artificial DNA model-systems based on methylation of the phosphate backbone. The chemical concept for this transition wherein shielding of the oxygen anions of the backbone phosphates plays an essential role, resulted in the preparation of the phosphatemethylated d(CpG). Even on this primitive level of only two base pair long, the B-Z conformational aspects of this self-complementary duplex could be described in solution with nuclear magnetic resonance (NMR) and circular dichroism (CD) measurements. The exclusivity of this choice became clear after synthesizing phosphatemethylated DNA with longer alternating CG fragments. It could be shown that conflicting conformational effects of the CG and GC fragments resulted in an overall B structure of the phosphatemethylated tetramer d(CPGPCPG). From our model considerations, it is clear that the internal stress introduced by the alternating CG sequences will be promoted by a complete shielding of the phosphate backbone. Elimination of this effect may be realized by a site-specific phosphate shielding. The role of the anti-syn isomerization of G in the CG fragments is clarified by methylation of the phosphate group. This anti-syn transition is absent in corresponding methylphosphonates, suggesting an exclusive role for base-backbone coordination via hydrogen bonding. In addition, we propose that the B- into Z-DNA interconversion may offer a mechanistic view for differences in dynamics between cytosine and its epigenetic derivative 5-methylcytosine. This mechanism has been extended to the demethylation of 5-methylcytosine and the exchange of information between the new epigenetic base, 5-hydroxymethylcytosine and the DNA backbone via an intramolecular phosphorylation. The role of 5-hydroxymethylcytosine in Alzheimer disease has been briefly discussed. In our opinion, this study can be considered as a new dynamic concept for epigenetics based on the dynamics of the B-Z transition in natural and phosphatemethylated DNA.

Dna Systems for B-Z Transition and Their Significance as Epigenetic Model: The Fundamental Role of the Methyl Group

Epigenetic systems involved in the dynamics of gene expression, which are fundamental to cell determination and function without alteration in DNA sequences, are based on methylation of the N-terminal tails of lysine residues and DNA methylation. We demonstrate the vital importance for genetic transfer by different (hydrogen) networks, suggesting a complex interaction between the two epigenetic modifications. In other words, the methylation of local lysines can prescribe CPG methylation, which requires that methylation of histones and DNA are cooperative in carrying out an epigenetic instruction for integrating gene-silencing networks. To give a bio-organic description of the epigenetic coherence between histone and base methylation, we used the well-known B- into Z-DNA dynamics in combination with the unique properties of phosphatemethylated DNA on different levels of chemistry.

Optimization of the separation of the Rp and Sp diastereomers of phosphate-methylated DNA and RNA dinucleotides

The separation by reversed-phase high-performance liquid chromatography of Rp and Sp diastereomers of phosphate-methylated DNA and RNA dinucleotides was studied with respect to pH, organic modifier type and concentration and reversed-phase packing material. Drylab G was used to deduce optimum conditions. On the basis of the observed discrepancies between the computer predictions and experimental results, the gradient operation procedure with volatile buffers was improved. By repetitive chromatography on a 250 × 22 mm I.D. reversed-phase column, fourteen diastereomeric pairs were obtained in at least 97% purity and 60% yield, in amounts of 10–100 mg.

Inhibition of HIV-1 Infectivity by Phosphate-Methylated DNA: Retraction

Inhibition of HIV-1 Infectivity by Phosphate-Methylated DNA: Retraction

Phosphate-Methylated DNA Fragments Involved in Parallel and Antiparallel Duplex Formation Novel Aspects of Structure Stability and Biological Activity

Abstract Parallel duplexes are encountered for the phosphate-methylated DNA dinucleotides d(CpC) and d(TpC; for the Sp configuration exclusively. since inward location of the methyl group (Rp,) encounters severe steric interactions in the groove. A parallel duplex with T-T base pairs is found for the natural system d(T10) only after complexation of the cationic peptides polylysine and polyornithine with the phosphate groups, which diminishes phosphate-phosphate repulsions. For natural d(C10), parallel duplex formation is seen exclusively after complexation with polylysine, which only uses pro-S phosphate oxygens in the complexation. Polyornithine complexates with both pro-S and pro-R phosphate oxygens, leading to steric hindrance in the groove of the C-C duplex. For antiparallel duplexes of phosphate-methylated DNA (‘antisense’) with natural polynucleotides (‘sense’) it is found that the hybridization has a cooperative character. Furthermore, it is mentioned that long phosphate-methylated DNA fragments ca...

Characterization of the African HIV-1 isolate CBL-4 (RUT) by partial sequence analysis and virus neutralization with peptide antibody and antisense phosphate-methylated DNA

Goudsmit, Jaap; Geelen, Jan; Keulen, Wilco; Notermans, Daan; Kuiken, Carla; Ramautarsing, Chitra; Smit, Lia; Koole, Leo; van Genderen, Marcel; Buck, Henk; Sninsky, John; Krone, Willy

Characterization of the African HIV-1 isolate CBL-4 (RUT) by partial sequence analysis and virus neutralization with peptide antibody and antisense phosphate-methylated DNA

The HIV-1 isolate CBL-4 (RUT), originating from Tanzania, was characterized using a comprehensive virus-typing system. This system included sequence analysis of the region coding for the neutralization domain in the third variable region (V3) of the external envelope and of the tat responsive (TAR) region after polymerase chain reaction (PCR) amplification of these sequences from cellular DNA in the CBL-4 (RUT) producer line. Based on independent cluster analysis of TAR and V3 sequences the CBL-4 (RUT) virus was positioned closest to the Z6 (and ELI) African virus family. The V3 amino acid sequence on the surface of the virus particle was confirmed by the inhibition of neutralization of CBL-4 (RUT) by a synthetic peptide derived from the nucleic acid sequence. Using antisense phosphate-methylated DNA covering the TAR loop region of LAV-1/HTLV-IIIB, inhibition of HTLV-IIIB and HTLV-IIIRF infection was seen, whereas no inhibition was observed for CBL-4 (RUT), indicating two or more mismatches in the TAR loop region, a characteristic shared with Z6 virus, but not with ELI. We propose a virus-typing system based on sequence analysis confirmed by virus neutralization with a peptide binding antibody and inhibition by antisense phosphate-methylated DNA to group viruses for laboratory use and vaccine design.

Phosphate-Methylated DNA Aimed at HIV-1 RNA Loops and Integrated DNA Inhibits Viral Infectivity

Phosphate-methylated DNA hybridizes strongly and specifically to natural DNA and RNA. Hybridization to single-stranded and double-stranded DNA leads to site-selective blocking of replication and transcription. Phosphate-methylated DNA was used to interrupt the life cycle of the human immunodeficiency virus type-1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS). Both antisense and sense phosphate-methylated DNA 20-nucleotide oligomers, targeted at the transactivator responsive region and the primer binding site, caused complete inhibition of viral infectivity at a low concentration. Hybridization of phosphate-methylated DNA with folded and unfolded RNA was studied by ultraviolet and proton nuclear magnetic resonance spectroscopy. The combined results of hybridization studies and biological experiments suggest that the design of effective antisense phosphate-methylated DNA should focus on hairpin loop structures in the viral RNA. For sense systems, the 5' end of the integrated viral genome is considered to be the important target site.

Thermodynamics of polymolecular duplexes between phosphate‐methylated DNA and natural DNA

Phosphate-methylated (P.M.) DNA possesses a very high affinity for complementary natural DNA, as a result of the absence of interstrand electrostatic repulsions. In this study, a model system phosphate-methylated d[Cn] with natural d(Gk) (n less than k) is chosen for an investigation of the thermodynamic properties that determine duplex stability. The enthalpy change of a melting transition is shown to be considerably larger than is observed for corresponding natural DNA duplexes. It is found that delta Hn0 of GG/CC nearest neighbor pairwise interaction equals -15.6 kcal/mol, compared to -11.0 kcal/mol for the natural analog. The entropy change is strongly dependent on the length of the natural DNA strand and the number of phosphate-methylated DNA oligomers hybridized. The results are explained by means of a model in which a cooperative effect for subsequent hybridizations of phosphate-methylated DNA oligomers is assumed, thus giving additional stability.

Conformation of the phosphate-methylated DNA dinucleotides d(CpC) and d(TpC). Formation of a parallel miniduplex exclusively for the S configuration at phosphorus

From our previous work, it is known that phosphate-methylated d(TpT) forms parallel miniduplexes with an equal stability for the sp and Rp diastereoisomers. Therefore, it is concluded that the R P configuration (which is associated with location of the methyl group inside the helix groove) is unfavorable in the case of C-C base pairing. This conclusion was corroborated by AMBER molecular mechanics calculations, which showed that the larger propeller twist angle for C-C base pairing (∼41 o ), in comparison with T-T base pairing (∼25 o ), results in narrowing of the helix groove

Synthesis of well-defined phosphate-methylated DNA fragments: the application of potassium carbonate in methanol as deprotecting reagent

A new deprotection procedure in the synthesis of (partially) phosphate-methylated oligodeoxynucleotides has been developed, involving treatment of fully protected DNA fragments with methanolic potassium carbonate. It is shown that base deprotection can be accomplished in potassium carbonate/methanol without affecting the methyl phosphotriesters. This methodology enables us to synthesize, both in solution and on a solid support, DNA fragments which are phosphate-methylated at defined positions. The solid phase synthesis, however, turns out to be accompanied by considerable demethylation of the phosphotriesters. It is demonstrated that this demethylation does not occur during the deprotection or work-up procedure. Furthermore, it was found that the latter side-reaction is suppressed when the standard capping procedure with acetic anhydride is included.

Phosphate-Methylated Dna Fragments Involved in Parallel and Antiparallel Duplex Formation. Novel Aspects of Structure. Stability and Biological Activity

Phosphate-Methylated Dna Fragments Involved in Parallel and Antiparallel Duplex Formation. Novel Aspects of Structure. Stability and Biological Activity

Synthesis of phosphate-methylated DNA fragments using 9-fluorenylmethoxycarbonyl as transient base protecting group

Synthesis of phosphate-methylated DNA fragments using 9-fluorenylmethoxycarbonyl as transient base protecting group

Regiospecific inhibition of DNA duplication by antisense phosphate-methylated oligodeoxynucleotides.

A new synthesis route for long phosphate-methylated oligodeoxynucleotides is described, which were used as antisense inhibitors of the DNA replication. Phosphate-methylated oligomers hybridize more strongly with natural DNA than their natural analogues, due to the absence of electrostatic interstrand repulsions. Compared with phosphate-ethylated and methyl phosphonate systems, phosphate-methylated systems are preferable as antisense DNA, which was concluded from the high Tm values and sharp melting transitions of duplexes of phosphate-methylated and natural DNA. By using the Sanger dideoxy technique, it was shown that a complementary phosphate-methylated 18-mer can effectively and site-specifically block the DNA replication process at room temperature.