Kinetically selective binding of single stranded RNA over DNA by a pyrrolidine-amide oligonucleotide mimic (POM)

Replacing the sugar-phosphodiester backbone of nucleic acids with a pyrrolidine-amide backbone results in an oligonucleotide mimic POM 1 which binds with high affinity and specificity to complementary DNA and RNA. Unlike other modified oligonucleotides, POM binds much more rapidly to single stranded RNA than DNA.     

Physico-chemical and Biological Properties of Antisense Phosphodiester Oligonucleotides with Various Secondary Structures

Abstract

The influence of the secondary structure of oligonucleotides having a natural phosphodiester backbone on their ability to interact with DNA and RNA targets and on their resistance to the nucleolytic digestion is investigated. Oligonucleotides having hairpin, looped and snail-like structure are found to be much more stable to nuclease degradation in different biological media and inside cells than the linear ones. The structured oligonucleotides can also hybridise with their DNA and RNA targets.     

Evaluating the Effect of Ionic Strength on Duplex Stability for PNA Having Negatively or Positively Charged Side Chains

The enhanced thermodynamic stability of PNA:DNA and PNA:RNA duplexes compared with DNA:DNA and DNA:RNA duplexes has been attributed in part to the lack of electrostatic repulsion between the uncharged PNA backbone and negatively charged DNA or RNA backbone. However, there are no previously reported studies that systematically evaluate the effect of ionic strength on duplex stability for PNA having a charged backbone. Here we investigate the role of charge repulsion in PNA binding by synthesizing PNA strands having negatively or positively charged side chains, then measuring their duplex stability with DNA or RNA at varying salt concentrations. At low salt concentrations, positively charged PNA binds more strongly to DNA and RNA than does negatively charged PNA. However, at medium to high salt concentrations, this trend is reversed, and negatively charged PNA shows higher affinity for DNA and RNA than does positively charged PNA. These results show that charge screening by counterions in solution enables negatively charged side chains to be incorporated into the PNA backbone without reducing duplex stability with DNA and RNA. This research provides new insight into the role of electrostatics in PNA binding, and demonstrates that introduction of negatively charged side chains is not significantly detrimental to PNA binding affinity at physiological ionic strength. The ability to incorporate negative charge without sacrificing binding affinity is anticipated to enable the development of PNA therapeutics that take advantage of both the inherent benefits of PNA and the multitude of charge-based delivery technologies currently being developed for DNA and RNA.     

OLigonucleotide (Cyanomethyl) Phosphonates

Abstract

A dinucleoside (cyanomethyl) phosphonate has been prepared, and its properties have been studied. This compound was converted into an oligonucleotide possessing alternating (cyanomethyl) phosphonate and phosphodiester backbone groups and its hybridization to complementary DNA and RNA sequences was studied versus methylphosphonate and phosphodiester controls.     

Synthesis and Properties of 2′-O-Methylribonucleotide Methylphosphonate Containing Chimeric Oligonucleotides

Abstract

2′-O-methylribonucleoside methylphosphonamidites are synthesized and incorporated into oligonucleotides to obtain chimeric antisense oligonucleotides. The resulting oligonucleotide binds to their target RNA/DNA sequences efficiently and stable in a medium containing bovine serum.     

肽核酸在基因诊断和治疗中的研究进展

肽核酸是一种以多肽为骨架,类似核苷酸的物质。它不带电荷,能抵抗核酸酶和蛋白酶的降解;它与DNA或RNA杂交特异性很强,可与靶基因形成稳定的三螺旋结构。肽核酸能够抑制基因的复制、转录、逆转录和翻译过程,在基因诊断及治疗方面有着广泛的用途。     

Tris (phenanthroline) Metal Complexes: Probes for DNA Helicity

Abstract

The intercalative binding of chiral tris(phenanthroline) metal complexes to DNA is stereo-selective. The enantiomeric selectivity is based upon the differential steric interactions between the two non-intercalating phenanthroline ligands of each isomer with the DNA phosphate backbone. Gel electrophoretic assays of helical unwinding, optical enrichment studies by equilibrium dialysis and luminescence titrations with separated enantiomers of (phen)₃Ru²⁺ all indicate that the delta isomer binds preferentially to the right-handed duplex. The chiral discrimination is governed by the DNA helical asymmetry. Complete stereospecifity is seen with isomers of the bulkier RuDIP (tris-4,7-diphenylphenanthrolineruthenium(II)). While both isomers bind to Z-DNA, a poor template for discrimination, binding of Λ-RuDIP to B-DNA is precluded. These chiral complexes therefore serve as a chemical probe to distinguish left and right-handed DNA helices in solution.     

Interaction of Sulforaphane with DNA and RNA

Sulforaphane (SFN) is an isothiocyanate found in cruciferous vegetables with anti-inflammatory, anti-oxidant and anti-cancer activities. However, the antioxidant and anticancer mechanism of sulforaphane is not well understood. In the present research, we reported binding modes, binding constants and stability of SFN–DNA and -RNA complexes by Fourier transform infrared (FTIR) and UV–Visible spectroscopic methods. Spectroscopic evidence showed DNA intercalation with some degree of groove binding. SFN binds minor and major grooves of DNA and backbone phosphate (PO₂), while RNA binding is through G, U, A bases with some degree of SFN–phosphate (PO₂) interaction. Overall binding constants were estimated to be K_(SFN–DNA)=3.01 (± 0.035)×10⁴ M^-1 and K_(SFN–RNA)= 6.63 (±0.042)×10³ M^-1. At high SFN concentration (SFN/RNA = 1/1), DNA conformation changed from B to A occurred, while RNA remained in A-family structure.     

Stereoselective 5′-Monodeuterated Ribonucleoside Phosphoramidites as Tools for Conformational Studies of RNA by the NMR Approach

Abstract

The analysis of NMR spectra of DNA and RNA, in particular, homo- and heteronuclear vicinal coupling constants of the nuclei of the sugar-phosphate backbone, can provide important information about the conformation of macromolecules¹. For example, 5′H - P coupling constants allows us to obtain a value of β torsional angle, 5′H-4′H - γ, whereas 3′H-P constant gives the angle ?. Unfortunately, due to the complex structure of H5′, H4′ and H3′ multiples in moderate and large RNA fragments (>15 nucleotides), it is very difficult to assign signals and extract accurate structural data.     

A Remarkable Stabilization of Complexes Formed by 2,6-Diaminopurine Oligonucleotide N3′→P5′ Phosphoramidates

Abstract

2′-Deoxyribo- and ribo-oligonucleotide N3′→P5′phosphoramidates containing 2,6-diaminopurine nucleosides were synthesized. Thermal denaturation experiments demonstrated a significant stabilization of the complexes formed by these compounds with DNA and RNA complementary strands, relative to adenosine-containing phosphoramidate counterparts. The increase in melting temperature of the complexes reached up to 6.9 °C per substitution. The observed stabilization was attributed to the apparent synergistic effects of N-type sugar puckering of the oligonucleotide N3′→5′ phosphoramidate backbone, and the ability of 2,6-diaminopurine bases to form three hydrogen bonds.     

Analysis of nucleic acid binding by a recombinant translin-trax complex

Translin is a highly conserved mammalian RNA and DNA-binding protein involved in DNA recombination and RNA trafficking. Crystal structures of mouse and human translin have been solved, but do not provide information about nucleic acid binding or recognition. Translin has a partner protein, translin-associated factor x (trax), which is believed to regulate translin’s subcellular locale and affinity for certain RNA and DNA sequences. Here we present a comparative study of recombinant translin and translin-trax complex binding to specific RNA and DNA sequences. It was observed that translin preferentially binds to G-rich RNA sequences whereas translin-trax preferentially binds G-rich DNA sequences. Translin can bind mRNA sequences with sub-micromolar K_d values, and the complex with trax can bind G-rich DNA with similar affinity. We conclude that trax acts to regulate translin’s RNA and DNA binding affinities as part of a cellular RNA trafficking mechanism.     

Nucleic acid binding properties of allicin: Spectroscopic analysis and estimation of anti-tumor potential

Background

Allicin has received much attention due to its anti-proliferative activity and not-well elucidated underlying mechanism of action. This work focuses towards determining the cellular toxicity of allicin and understanding its interaction with nucleic acid at molecular level.

Methods

MTT assay was used to assess the cell viability of A549 lung cancer cells against allicin. Fourier transform infrared (FTIR) and UV-visible spectroscopy were used to study the binding parameters of nucleic acid-allicin interaction.

Results

Allicin inhibits the proliferation of cancer cells in a concentration dependent manner. FTIR spectroscopy exhibited that allicin binds preferentially to minor groove of DNA via thymine base. Analysis of tRNA allicin complex has also revealed that allicin binds primarily through nitrogenous bases. Some amount of external binding with phosphate backbone was also observed for both DNA and RNA. UV visible spectra of both DNA allicin and RNA allicin complexes showed hypochromic shift with an estimated binding constant of 1.2 × 10⁴ M^- 1 for DNA and 1.06 × 10³ M^− 1for RNA binding. No major transition from the B-form of DNA and A-form of RNA is observed after their interaction with allicin.

Conclusions

The results demonstrated that allicin treatment inhibited the proliferation of A549 cells in a dose-dependent manner. Biophysical outcomes are suggestive of base binding and helix contraction of nucleic acid structure upon binding with allicin.

General significance

The results describe cytotoxic potential of allicin and its binding properties with cellular nucleic acid, which could be helpful in deciphering the complete mechanism of cell death exerted by allicin.     

Detection of high-affinity intercalator sites in a ribosomal RNA fragment by the affinity cleavage intercalator methidiumpropyl-EDTA-iron(II)

The affinity cleavage reagent methidiumpropyl-EDTA (MPE) [Hertzberg, R. P., & Dervan, P. B. (1982) J. Am. Chem. Soc. 104, 313-315] intercalates between base pairs in helical DNA and, when complexed with Fe(II), cleaves the DNA by oxidative degradation of the deoxyribose. We find that this reagent is useful for mapping structure in some RNA molecules. The reagent binds to poly(A)-poly(U) with the same or slightly lower affinity as the related ethidium intercalator, selectively binds double-helical in preference to single-stranded RNA, and when complexed with Fe(II) readily cleaves the RNA backbone. The reagent binds to three or four helical locations in tRNAPhe with an affinity of 10(5)-10(6) M-1 (0.1 M Na+, pH 7.6, 37 degrees C). With a 345-base RNA fragment covering the S8/S15 protein binding region of Escherichia coli 16S ribosomal RNA, MPE-Fe(II) intercalates strongly at two helical sites: one is located at or near a single base bulge and the other at the end of a helix. Intense cutting is also seen in a region that is not part of a Watson-Crick helix. Ethidium bromide binds at these sites with high affinity (about 10(7) M-1 at 0.1 M Na+, pH 7.6, 37 degrees C). The sites are all clustered in a region of the RNA thought to bind S15. Tertiary folding of the RNA may distort helices in the molecule to create sites with particularly high affinities for intercalators; such sites may have functional significance in protein recognition or RNA-RNA interactions.     

Silver(I) complexes with DNA and RNA studied by Fourier transform infrared spectroscopy and capillary electrophoresis

Ag(I) is a strong nucleic acids binder and forms several complexes with DNA such as types I, II, and III. However, the details of the binding mode of silver(I) in the Ag-polynucleotides remains unknown. Therefore, it was of interest to examine the binding of Ag(I) with calf-thymus DNA and bakers yeast RNA in aqueous solutions at pH 7.1-6.6 with constant concentration of DNA or RNA and various concentrations of Ag(I). Fourier transform infrared spectroscopy and capillary electrophoresis were used to analyze the Ag(I) binding mode, the binding constant, and the polynucleotides' structural changes in the Ag-DNA and Ag-RNA complexes. The spectroscopic results showed that in the type I complex formed with DNA, Ag(I) binds to guanine N7 at low cation concentration (r = 1/80) and adenine N7 site at higher concentrations (r = 1/20 to 1/10), but not to the backbone phosphate group. At r = 1/2, type II complexes formed with DNA in which Ag(I) binds to the G-C and A-T base pairs. On the other hand, Ag(I) binds to the guanine N7 atom but not to the adenine and the backbone phosphate group in the Ag-RNA complexes. Although a minor alteration of the sugar-phosphate geometry was observed, DNA remained in the B-family structure, whereas RNA retained its A conformation. Scatchard analysis following capillary electrophoresis showed two binding sites for the Ag-DNA complexes with K(1) = 8.3 x 10(4) M(-1) for the guanine and K(2) = 1.5 x 10(4) M(-1) for the adenine bases. On the other hand, Ag-RNA adducts showed one binding site with K = 1.5 x 10(5) M(-1) for the guanine bases.     

Position-dependent effects on stability in tricyclo-DNA modified oligonucleotide duplexes

A series of oligodeoxyribonucleotides and oligoribonucleotides containing single and multiple tricyclo(tc)-nucleosides in various arrangements were prepared and the thermal and thermodynamic transition profiles of duplexes with complementary DNA and RNA evaluated. Tc-residues aligned in a non-continuous fashion in an RNA strand significantly decrease affinity to complementary RNA and DNA, mostly as a consequence of a loss of pairing enthalpy ΔH. Arranging the tc-residues in a continuous fashion rescues T_m and leads to higher DNA and RNA affinity. Substitution of oligodeoxyribonucleotides in the same way causes much less differences in T_m when paired to complementary DNA and leads to substantial increases in T_m when paired to complementary RNA. CD-spectroscopic investigations in combination with molecular dynamics simulations of duplexes with single modifications show that tc-residues in the RNA backbone distinctly influence the conformation of the neighboring nucleotides forcing them into higher energy conformations, while tc-residues in the DNA backbone seem to have negligible influence on the nearest neighbor conformations. These results rationalize the observed affinity differences and are of relevance for the design of tc-DNA containing oligonucleotides for applications in antisense or RNAi therapy.     

An Extended Phosphate Linkage: Synthesis,Hybridization and Modeling Studies of Modified Oligonucleotides

Abstract

Novel stretched oligonucleotides (A-D) containing a 3′-α-C-methylene phosphodiester bridge (5-atoms long) have been synthesized on an automated synthesizer utilizing phosphoramidite chemistry. The key building-block 1-[3′-O-β-cyanoethyldiisopropylaminophosphiryl-2,3-dideoxy-5-O-dimethoxytriphenylmethyl-3-C-(hydroxymethyl)-β-D-erythro-pentofuranosyl]thymine (21) was prepared in a stereoselective manner from thymidine. Hybridization studies indicated a drop (1.8–3.0°C/mod.) in affinity for the complementary RNA and DNA targets. Molecular modeling results indicated that the 5-atom modified backbone had a different geometry around the phosphodiester linkage compared to the natural phosphodiester linkage. The stretched backbone may not be useful for antisense or triplex constructs, however it may find applications in biochemical/enzyme studies.