Synthesis and high stability of complementary complexes of N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides

Lokhov, S. G.; Podyminogin, Mikhail A.; Sergeev, Dmitry S.; Silnikov, Vladimir N.; Kutyavin, Igor V.; Shishkin, G. V.; Zarytova, Valentina P.

doi:10.1021/bc00017a010

Cited by 49 publications

(29 citation statements)

References 23 publications

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“…34 The studied variant of oligonucleotide modification by the neutral phenazine dye gives duplex stabilizing effects that are comparable to those found upon the 3Ј-end attachment of a cationic phenazine derivative via a polymethylene chain. 9 The decrease in standard free energy at 37°C was found to be 2.06 Ϯ 0.4 kcal/mol in the latter case compared to 1.7-2.5 kcal/mol in our case (see Table I).…”

Section: Discussionsupporting

confidence: 42%

Anchorage of oligonucleotide hybridization by tethered phenazine nucleoside analogue

et al. 2003

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UV absorption and fluorescence techniques with a thermal denaturation procedure were used in studies of the anchorage of an oligonucleotide hybridization by a covalently tethered nucleoside analogue of an intercalating imidazophenazine derivative (Pzn). The formation by the (dT)(10)Pzn conjugate of the duplex complex with (dA)(15) and the triplex complex with (dA)(15) or poly(dA).poly(dT) was studied in buffered solutions with 0.11 and/or 1M sodium ions at the oligomer strand concentration of 10 microM. Because of the Pzn emission sensitivity to the interaction with adenine bases, a fluorescence technique was found to be effective in the detection of melting transitions. The attached Pzn substantially enhanced the thermal stability of complexes formed by (dT)(10) because of the intercalation mechanism, which increased the temperature of half-dissociation of the duplex by 10-12 degrees C and of the triplexes by approximately 13 degrees C. With the assumption of a two-state model of transition, the thermodynamic parameters for duplex formations were derived. The investigated variant of conjugation has a certain advantage over the widely used attachment via a flexible linker, consisting of a predetermined location of the Pzn chromophore in target sequences that makes it useful as a fluorescent reporter of the hybridization correctness. Molecular modeling was used to construct the geometries of the intercalation sites that turned out to be in conformity with the behavior of the Pzn fluorescence.

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Section: Discussionsupporting

confidence: 42%

Anchorage of oligonucleotide hybridization by tethered phenazine nucleoside analogue

et al. 2003

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“…The toolbox of such modifications is actually rich and well represented. [25][26][27][28][29][30][31][32] The Taq DNA polymerase used in the study does not have 3 ¢ ?5 ¢ exonuclease proofreading activity. Consequently, more than half of the PCR products may incorporate an extra nucleotide residue, usually adenosine, at the 3 ¢ end.…”

Section: Discussionmentioning

confidence: 99%

“…The first is to modify the primers and probes. The toolbox includes peptide nucleic acid (PNA), 25 locked nucleic acid (LNA), 26,27 conjugated minor groove binders, 28,29 intercalators, 30,31 and certain base modifications. 32 The second approach is based on modification of the detected target DNAs, and it can be as potent in duplex stabilization as the traditional approaches cited above.…”

Section: Base Modification Of Pcr Products For Specific Stabilizationmentioning

confidence: 99%

Use of Base Modifications in Primers and Amplicons to Improve Nucleic Acids Detection in the Real-Time Snake Polymerase Chain Reaction

Kutyavin¹

2011

ASSAY and Drug Development Technologies

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The addition of relatively short flap sequence at the 5 ¢ -end of one of the polymerase chain reaction (PCR) primers considerably improves performance of real-time assays based on 5 ¢ -nuclease activity. This new technology, called Snake, was shown to supersede the conventional methods like TaqMan, Molecular Beacons, and Scorpions in the signal productivity and discrimination of target polymorphic variations as small as single nucleotides. The present article describes a number of reaction conditions and methods that allow further improvement of the assay performance. One of the identified approaches is the use of duplex-destabilizing modifications such as deoxyinosine and deoxyuridine in the design of the Snake primers. This approach was shown to solve the most serious problem associated with the antisense amplicon folding and cleavage. As a result, the method permits the use of relatively long-in this study-14-mer flap sequences. Investigation also revealed that only the 5 ¢ -segment of the flap requires the deoxyinosine/ deoxyuridine destabilization, whereas the 3 ¢ -segment is preferably left unmodified or even stabilized using 2-amino deoxyadenosine d(2-amA) and 5-propynyl deoxyuridine d(5-PrU) modifications. The basemodification technique is especially effective when applied in combination with asymmetric three-step PCR. The most valuable discovery of the present study is the effective application of modified deoxynucleoside 5 ¢ -triphosphates d(2-amA)TP and d(5-PrU)TP in Snake PCR. This method made possible the use of very short 6-8-mer 5 ¢ -flap sequences in Snake primers.

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“…In this case, the reactive antisence oligonucleotides bearing covalently attached 4-[N-(2-chloroethyl)-N-methylamino]benzyl groups (ClRCH 2 NH)-at 5' -terminal phosphate were used as a reagents. The increase in modification yield arising from the presence of both unmodified and modified effectors was supposed to originate from the change in the stability of the target-reagent complex due to cooperative interaction (5). This interaction between oligonucleotides bound to adjacent sites on DNA was suggested to arise largely from stacking between the bases at complex formation (5,11 ).…”

Section: Introductionmentioning

confidence: 99%

“…The increase in modification yield arising from the presence of both unmodified and modified effectors was supposed to originate from the change in the stability of the target-reagent complex due to cooperative interaction (5). This interaction between oligonucleotides bound to adjacent sites on DNA was suggested to arise largely from stacking between the bases at complex formation (5,11 ). The free energy of base stacking which arises from the induced dipole interactions and hydrophobic forces has been shown to range from -0.5 to -3 kcal mol-1 for monomeric nucleosides associated in aqueous solution (6)(7)(8)(9).…”

Section: Introductionmentioning

confidence: 99%

Cooperative Interactions of the Oligodeoxyribonucleotides on the Complementary Template. The Influence of Chemical Groups and Mismatched Nucleotides at the 5′- and 3′-ends of Oligonucleotides on the Parameters of Cooperativity

Adeenah-Zadah

Knorre²,

Fedorova³

1997

Journal of Biomolecular Structure and Dynamics

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Parameters of cooperative interactions of two or three oligodeoxyribonucleotides or their derivatives bound with the adjacent sites of the complementary template were measured using method of "complementary addressed modification titration" (CAMT). Complementary template (target) were modified with the reactive oligonucleotide derivatives (reagents) bearing covalently attached alkylating 4-[N-(2-chloroethyl)-N-methylamino]benzylamino- group (C1RCH2NH)- at 5'-terminal phosphate. The targets had only one binding site for the reagent and either no (T10), or one (T'22 and T22) or two sites (T26) for the oligonucleotides (effectors) cooperatively bound with the adjacent sites on the template. Both unmodified oligonucleotides E1, E2 and their derivatives E1Phn, E2Phn bearing N-(2-hydroxyethyl)-phenazinium residues Phn- both at 5'- and 3'-ends covalently linked via ethylenediamine linker were used as effectors. Effectors E1 and E2 (E1Phn and E2Phn) bind, respectively, upstream or downstream from the reagent. Hexameric (X6) or octameric (X8 or X8m) reagents were used for the target modification. The reagent X8m formed one TT-mismatch with the target at the end opposite to location of the reactive moiety. The cooperativity parameter values characterizing the mutual interactions between the reagents X6, X8, X8m and effectors E1, E2, E1Phn, E2Phn have been found as the ratio of the association constants of the reagents in the presence of effectors. The association constants were calculated from the dependencies of the target modification extent on initial concentrations of the reagents. The use of T26 existing both in linear and hairpin conformations permitted us to estimate additionally the role of indirect cooperativity originating from the induction of the target conformational change by the effectors. The following conclusions were done from the quantitative results. The efficiency of direct cooperativity is independent on the length of oligonucleotide for the same nature of the contact. The cooperativity parameter increases by factor about 3 in the presence of Phn-group covalently attached to oligonucleotides and located at the junctions. The presence of either alkylating group C1RCH2NH- or TT-mismatch at the junctions eliminates cooperative interaction between the bases. In the same time sufficiently effective cooperative interaction takes place in the case of simultaneous presence of both Phn- and either C1RCH2NH- group or TT-mismatch at the junction.

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Synthesis and high stability of complementary complexes of N-(2-hydroxyethyl)phenazinium derivatives of oligonucleotides

Cited by 49 publications

References 23 publications

Anchorage of oligonucleotide hybridization by tethered phenazine nucleoside analogue

Anchorage of oligonucleotide hybridization by tethered phenazine nucleoside analogue

Use of Base Modifications in Primers and Amplicons to Improve Nucleic Acids Detection in the Real-Time Snake Polymerase Chain Reaction

Cooperative Interactions of the Oligodeoxyribonucleotides on the Complementary Template. The Influence of Chemical Groups and Mismatched Nucleotides at the 5′- and 3′-ends of Oligonucleotides on the Parameters of Cooperativity

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