Intercalating Nucleic Acid Research Articles

Locked Nucleic Acids and Intercalating Nucleic Acids in the Design of Easily Denaturing Nucleic Acids: Thermal Stability Studies

Intercalating nucleic acids (INA(R)s) with insertions of (R)-1-O-(1-pyrenylmethyl)glycerol were hybridized with locked nucleic acids (LNAs). INA/LNA duplexes were found to be less stable than the corresponding DNA/LNA duplexes when the INA monomer was inserted as a bulge close to the LNA monomers in the opposite strand. This property was used to make "quenched" complements that possess LNA in hairpins and in duplexes and are consequently more accessible for targeting native DNA. The duplex between a fully modified 13-mer LNA sequence and a complementary INA with six pyrene residues inserted after every second base as a bulge was found to be very unstable (Tm=30.1 degrees C) in comparison with the unmodified double-stranded DNA (Tm=48.7 degrees C) and the corresponding duplexes of LNA/DNA (Tm=81.6 degrees C) and INA/DNA (Tm=66.4 degrees C). A thermal melting experiment of a mixture of an LNA hairpin, with five LNA nucleotides in the stem, and its complementary DNA sequence gave a transition with an extremely low increase in optical density (hyperchromicity). When two INA monomers were inserted into the stem of the LNA hairpin, the same experiment resulted in a significant hyperchromicity comparable with the one obtained for the corresponding DNA/DNA duplex.

Intercalating nucleic acids: The inversion of the stereocenter in 1- O-(pyren-1-ylmethyl)glycerol from R to S. Thermal stability towards ssDNA, ssRNA and its own type of oligodeoxynucleotides

The synthesis and insertions of ( S)-1- O-(pyren-1-ylmethyl)glycerol into intercalating nucleic acids is described. Insertions of this S-isomer as a bulge lead to reduced binding affinity towards complementary ssDNA compared to intercalating nucleic acids possessing ( R)-1- O-(pyren-1-ylmethyl)glycerol in the same positions. Insertions of both ( R) or ( S) 1- O-(pyren-1-ylmethyl)glycerols as bulges into two complementary strands decreased the stability of the complex compared to dsDNA possessing the pyrene pseudo-nucleotide in one of the strands.

NMR structure determination of a modified DNA oligonucleotide containing a new intercalating nucleic acid.

The intercalating nucleic acid (INA) presented in this paper is a novel 1-O-(1-pyrenylmethyl)glycerol DNA intercalator that induces high thermal affinity for complementary DNA. The duplex examined contained two INA intercalators, denoted X, inserted directly opposite each other: d(C(1)T(2)C(3)A(4)A(5)C(6)X(7)C(8)A(9)A(10)G(11)C(12)T(13)):d(A(14)G(15)C(16)T(17)-T(18)G(19)X(20)G(21)T(22)T(23)G(24)A(25)G(26)). Unlike most other nucleotide analogues, DNA with INA inserted has a lower affinity for hybridizing to complementary DNA with an INA inserted directly opposite than to complementary unmodified DNA. In this study we used two-dimensional (1)H NMR spectroscopy to determine a high-resolution solution structure of the weak INA-INA duplex. A modified ISPA approach was used to obtain interproton distance bounds from NOESY cross-peak intensities. These distance bounds were used as restraints in molecular dynamics (rMD) calculations. Twenty final structures were generated for the duplex from a B-type DNA starting structure. The root-mean-square deviation (RMSD) of the coordinates for the 20 structures of the complex was 1.95 A. This rather large value, together with broad lines in the area of insertion, reflect the high degree of internal motion in the complex. The determination of the structure revealed that both intercalators were situated in the center of the helix, stacking with each other and the neighboring nucleobases. The intercalation of the INAs caused an unwinding of the helix in the insertion area, creating a ladderlike structure. The structural changes observed upon intercalation were mainly of local character; however, a broadening of the minor groove was found throughout the helix.

Intercalating Nucleic Acids: The Influence of Linker Length and Intercalator Type on Their Duplex Stabilities †

Six new examples of intercalating nucleic acids were synthesized in order to evaluate the dependence of the length of the linker between oligo and intercalator on the thermal stability of their corresponding duplexes and triplexes. †In honour and celebration of the 70th birthday of Professor Leroy B. Townsend. ††A research center funded by The National Danish Research Foundation for studies on nucleic acid chemical biology.

Intercalating Nucleic Acids with Pyrene Nucleotide Analogues as Next‐Nearest Neighbors for Excimer Fluorescence Detection of Single‐Point Mutations under Nonstringent Hybridization Conditions

AbstractFluorescence and hybridization specificity is reported for intercalating nucleic acids (INAs), which are here oligodeoxynucleotides (ODNs) synthesized with insertions using (S)‐1‐[bis(4‐methoxyphenyl)(phenyl)methoxy]‐3‐[(pyren‐1‐yl)methoxy]propan‐2‐ol phosphoramidites. It is shown that an INA with two insertions placed as next‐nearest neighbors can be used for discrimination between nucleic acids and their single‐point mutants. Quenching of an excimer band at 480 nm is observed upon hybridizing to a complementary sequence, whereas the excimer band is present when the nucleobase pair between the two pyrene moieties is mismatched. It is the first example of a solution based on fluorescence detection of single‐point mutants that uses excimer formation and does not rely on stringent hybridization conditions. Furthermore, it is shown that INAs with pyrene insertions retain their sequence specificity in thermal melting.

Intercalating nucleic acids (INAs) with insertion of N-(pyren-1-ylmethyl)-(3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol. DNA (RNA) duplex and DNA three-way junction stabilities.

N-(Pyren-1-ylmethyl)-(3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol was synthesised from (3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol and (3R,4S)-4-[(1S)-1,2-dihydroxyethyl] pyrrolidin-3-ol using alkylation with 1-(chloromethyl)pyrene or reductive amination with pyrene-1-carbaldehyde and NaCNBH3. The incorporation of N-(pyren-1-ylmethyl)azasugar moiety into oligodeoxynucleotides (ODN) as a bulge to form an intercalating nucleic acid (INA) induced a slight destabilization of INA-DNA duplex, whereas the INA-RNA duplex was strongly destabilized and 9 degrees C difference per modification in thermal stability between INA-DNA over INA-RNA duplexes was observed. The stabilization of a DNA three way junction (TWJ) was improved when the intercalator moiety was inserted into the junction region as a bulge.

Intercalating nucleic acids containing insertions of 1-O-(1-pyrenylmethyl)glycerol: stabilisation of dsDNA and discrimination of DNA over RNA.

We have studied hybridisation affinities and fluorescence behaviour of intercalator-modified oligonucleotides. The phosphoramidite of (S)-1-O-(4, 4'-dimethoxytriphenylmethyl)-3-O-(1-pyrenylmethyl)glycerol, an intercalating pseudo-nucleotide (IPN), was synthesised and by standard methods inserted into 7mer and 13mer oligodeoxyribonucleotides (ODNs) to generate intercalating nucleic acids (INAs). INAs showed greatly increased affinity for complementary single-stranded DNA (ssDNA), as determined by a thermal stabilisation of the formed DNA/INA duplex of up to 10.9 degrees C per modification when the IPN was added as a dangling end and up to 6.7 degrees C per modification when the IPN was inserted as a bulge. There was a positive stabilisation effect of the formed DNA/INA duplex on introducing a second IPN in the INA strand, when the two IPNs were separated by at least 1 bp. The effect is more pronounced the larger the separation of the two IPNs. Contrary to the enhanced affinity for ssDNA, the IPNs lower the affinity for complementary single-stranded RNA (ssRNA), giving rise to a difference in melting temperature of up to 25.8 degrees C for two IPN insertions in an RNA/INA duplex when compared with the corresponding DNA/INA duplex. In this way INA is able to discriminate ssDNA over ssRNA with identical sequences. Fluorescence measurements show a stronger interaction of the pyrene moiety with DNA than with RNA, indicating intercalation as the stabilising factor in DNA/INA duplexes.

Application of flow cytometry in toxinology: pathophysiology of human polymorphonuclear leukocytes damaged by a pore-forming toxin from Staphylococcus aureus.

The pore-forming activity of leukocidin (PVL) secreted by Staphylococcus aureus has been investigated on human white cells by flow cytometry techniques. This two-component toxin induced morphological modifications of neutrophils and monocytes as detected by forward light scattering measurements, but was inactive on lymphocytes. These modifications were the consequence of pore formation through the cell membrane leading to its permeabilization. In the absence of calcium, PVL formed pores large enough to allow ethidium ions to penetrate the cells and become fluorescent by intercalating nucleic acids. In the presence of calcium, the pores were too small for ethidium entry but allowed an influx of calcium as shown by the increase in fluorescence of Fluo-3 loaded in the cells. This increase in intracellular calcium concentration induced the activation of neutrophils by PVL as shown by the liberation of their granule content measured by a decrease in side light scattering. Furthermore, ethidium fluorescence was used to discriminate the cells sensitive to PVL, and the analysis of differentiated HL-60 cells and cells obtained from a case of chronic myeloid leukemia led to the conclusion that myeloid cells become sensitive to PVL during differentiation to the metamyelocyte stage.