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For the best experience on our site, be sure to turn on Javascript in your browser. ZNA are oligonucleotides conjugated to cationic units. Their global charge can be modulated by the number of cationic spermine moieties grafted on each oligonucleotide. ZNA are produced using standard automated nucleic acid synthesis based on phosphoramidite chemistry. A spermine derivative i. However, the global charge of ZNA defines per se the fields of applications. When negatively charged, ZNA conserve their ability to mediate specific hybridization with complementary strands and they are more suitable for PCR applications, amplification techniques, diagnostic applications or purification technologies.

ZNA molecules bearing excess cationic charges were shown to have the property to stick to anionic cell surfaces and to trigger endocytosis, leading to cell transfection without the need of a transfection reagent or a cargo. Hybridization studies demonstrated that spermine-mediated attraction is the main driving force for the initiation of nucleic acid binding. Then, the standard base pairing, according to Watson-Crick rules, between the target strand sequence and the complementary ZNA leads to a duplex formation. As a consequence of the cationic contribution, the thermal stability of the formed duplex is increased.

The stepwise spermine conjugation raises the Tm in a smooth and linear way while preserving mismatch discrimination. According to the initial study from Noir, et al. In this study, the impact of the number of grafted spermines per oligonucleotide on the melting temperature was determined, with 6-, 8-, , , or mer oligonucleotides bound to complementary sequences within mer oligonucleotides see Figure 1. In this initial study, the melting temperatures were measured in physiological ionic strength mM sodium chloride and at pH 7.

Three groups of sequences with various GC contents low, medium, and high and standard lengths of primers and probes 22 to 16 mers were synthesized see Table 1. Each sequence was modified with an increasing number of spermines. The maximum number of grafted spermines was determined taking into consideration the solubility of the molecule i.

EPA4 - Method for solution phase synthesis of oligonucleotides - Google Patents

Finally, the melting temperature was measured. Tm measurements in PCR buffer conditions indicate that the Tm increase is linear and is a function of the number of spermines grafted to the oligonucleotide, as shown in Figure 2, which presents the results obtained with and mers. The linear increase per conjugated spermine was also confirmed for the and mers. This study also demonstrates that the Tm increase induced by the spermine grafting is lower in GC-rich oligonucleotides.

It also provides additional explanation further to prior studies4 about why, when ZNA are used as primers, the maximal improvement effect on the amplification is obtained with AT-rich sequences. Many recent reports have described the use of ZNA as potent primers and probes for PCR applications, which are mostly based on Z4- and Z5-conjugated oligonucleotides with lengths of 16 to 22 nucleotides.

The Tm increase per oligonucleotide remains a function of the number of spermines grafted onto the oligonucleotide, is dependent on the oligonucleotide length, and is slightly dependent on the GC content. However, the Tm impact per spermine can be affected by the pH which depends on the buffer used and on the salt content having a shielding effect on the cationic charge.

A brief history of oligonucleotide synthesis.

The versatile chemistry of ZNA offers various possibilities of primer and probe chemical structures, such as dual-labelled probes. Such stereoisomeric constraints also apply to cationic backbone-modified ON For example, Letsinger has shown that an ON with alternating anionic phosphodiester and cationic stereo-uniform phosphoramidate linkages formed a stable duplex with its DNA target whereas a similar ON containing the other stereoisomer did not bind efficiently Despite this chirality problem, zwitterionic ON, containing several terminal cationic phosphoramidate linkages as diastereoisomeric mixtures and central phosphodiester linkages, have been reported to bind to their mRNA target and show a greater antisense activity than unmodified ON Of special interest, with respect to potential therapeutic and diagnostic purposes, we demonstrate that these cationic ON bind with high affinity to their single-stranded targets.

Despite this, they were capable of specifically inhibiting translation initiated at the internal ribosome entry site IRES of the human hepatitis C virus HCV in vitro and in intact cells. The oxidation of each block was carried out manually by the syringe technique outside the synthesizer In the same way, fully backbone-modified ON 18, 19 and 20 were prepared via H -phosphonate-chemistry.

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The H -phosphonate diester linkages were then manually oxidized by CCl 4 in the presence of 2-methoxyethylamine for ON 19 or N -dimethylaminopropylamine for ON 18 and Scheme 1. Optical measurements were carried out on a Uvikon spectrophotometer Kontron. The cell compartment was continuously flushed with dry nitrogen when the temperature was below room temperature. During the melting experiments the heating rate was 0. Digitised absorbance and temperature values were stored in a computer for subsequent plotting and analysis.

T m values were determined from the maxima of the first derivative plots of absorbance versus temperature.

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  • Radioactive bands were visualised by autoradiography. The plasmid is more fully described by Honda et al. Specificity is demonstrated by the lack of activity on Renilla luciferase translation and the lack of activity of a scrambled ON 20 on firefly luciferase translation.

    No ON were added in control reactions C. Representative gels from three independent experiments are presented. For each concentration, signal from the firefly luciferase band is normalized to signal from the Renilla luciferase band and compared to the control lane. The mean values of three independent experiments are given. The translation reactions were primed with ng of capped RNA in all cases. ON were added, on ice, prior to the addition of the lysate, with no specific prehybridisation step. Vacuum-dried gels were analysed by autoradiography using Kodak Biomax films. Protein bands were quantified using NIH Image software version 1.

    The stability of the double-stranded helix formed between an ON and a complementary ssDNA or RNA target is a compromise between hydrogen bonding and the electrostatic repulsion between the negatively charged phosphate moieties of the two strands. To investigate the extent to which the introduction of cationic internucleoside bonds which reduce the net negative charge into the backbone could stabilize duplexes, we evaluated melting temperatures T m by thermal denaturation and renaturation experiments.

    This stabilisation is proportional to the number of modifications. The average stabilisation was 4. The average stabilisation was greatly increased at low ionic strength 6. The sharpness of the melting curves with the DNA targets was slightly affected with increasing modifications. The higher the number of cationic linkages, the broader the temperature range over which the duplexes melted Fig.

    As with DNA targets, no differences between the thermal association and dissociation curves were observed with RNA targets. In contrast with the DNA targets, all duplexes formed with RNA exhibited the same cooperativity of melting, regardless of the number of modifications and their distribution Fig. As it has been reported that the enhancement of duplex stability by cationic polyamines can be sufficient to counter-balance the instability caused by mismatched bases 38 , we evaluated the influence of a single mismatch AX in the DNA 8 or RNA 9 targets on the stability of the hybrids.

    ON complementary to the IIId loop of the HCV IRES were tested in dose-response in an in vitro translation assay programmed with a bi-cistronic construction that allows the simultaneous monitoring of translation initiated at the HCV IRES firefly luciferase and from a classical, eukaryote, cap-dependent translation initiation site Renilla luciferase Fig.

    Interestingly, this effect was observed without the use of specific transfection agents. Protein extracts were made 24 h later and firefly and Renilla luciferase activities assayed by luminometry. Comparative efficiencies of ON 18 with respect to a scrambled control ON 20 are shown.

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    Representative values from three independent experiments are presented. Although several antisense ON are currently being developed as drugs, their large size usually 20mer , low stability and poor membrane passage reduce their potential as pharmaceutical agents.

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    The increase in T m was positively correlated to the number of cationic modifications. The lower melting cooperativity of modified duplexes with the DNA target 8 compared to PO duplex accounts for the effect of increasing mixtures of diastereoisomers, when the number of modifications increases, due to the chirality of the phosphorus atom. At low salt concentration, the differences in the affinity of the cationic ON 7 and the natural ON 2 is striking. This reflects the charge repulsion between the two negatively charged PO backbones of 2 and its target 1 and in contrast, the charge attraction between the cationic ON 7 and its anionic target 1.

    In comparison, hybrids with ON 5 and 6, with a lower net charge —1 , were less sensitive to a change of ionic strength.

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    A 12mer oligopyrimidine ON with five cationic linkages, for example, has a substantially higher T m when the charged linkages are evenly distributed compared with when they are bunched into the central region. It is clear that, as with virtually all second-generation i.

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    This has a major implication for their use as antisense reagents as, unless they are coupled to an RNase activator 40 or contain an RNase H-competent central window 41 , their activity is restricted to steric blockade. However, RNase H competence, although potentially increasing activity is also a source of decreased specificity, as only a few contiguous nucleotides need to hybridise to provoke target cleavage and this reduces their ability to distinguish between a bona fide target sequence and a partially complementary by-stander sequence The advantage of steric blocking ON is that specificity is a function of affinity.

    We therefore tested their capacity to selectively arrest the formation of a translational pre-initiation complex on the HCV internal ribosome entry site, in a cell-free assay. Tallet-Lopez et al. The poor uptake of anionic or neutral oligonucleotides by cells has long proved a major obstacle to their utilisation. The incorporation of cationic linkages within the backbone of the ON itself could potentially overcome the need to use cationic vectors. A significant, dose-dependent reduction in firefly luciferase activity IRES-mediated translation was observed. Although the antisense effect was only moderate, it does demonstrate the capacity of cationic ON to be taken up by the cells and to bind to target in a physiologically relevant context.