Optimal design of parallel triplex forming oligonucleotides containing Twisted Intercalating Nucleic Acids—TINA

Abstract

Twisted intercalating nucleic acid (TINA) is a novel intercalator and stabilizer of Hoogsteen type parallel triplex formations (PT). Specific design rules for position of TINA in triplex forming oligonucleotides (TFOs) have not previously been presented. We describe a complete collection of easy and robust design rules based upon more than 2500 melting points (Tm) determined by FRET. To increase the sensitivity of PT, multiple TINAs should be placed with at least 3 nt in-between or preferable one TINA for each half helixturn and/or whole helixturn. We find that ΔTm of base mismatches on PT is remarkably high (between 7.4 and 15.2°C) compared to antiparallel duplexes (between 3.8 and 9.4°C). The specificity of PT by ΔTm increases when shorter TFOs and higher pH are chosen. To increase ΔTms, base mismatches should be placed in the center of the TFO and when feasible, A, C or T to G base mismatches should be avoided. Base mismatches can be neutralized by intercalation of a TINA on each side of the base mismatch and masked by a TINA intercalating direct 3′ (preferable) or 5′ of it. We predict that TINA stabilized PT will improve the sensitivity and specificity of DNA based clinical diagnostic assays.

Increasing the Analytical Sensitivity by Oligonucleotides Modified with Para- and Ortho-Twisted Intercalating Nucleic Acids – TINA

“…The validity of the design rules are therefore still to be established, but the design rules suggested in this paper are in concordance with previously published thermal stability data on nucleic acid intercalator molecules in other target sequences [11]–[16], [20]. The design rules identified in this study are also identical to the design rules we established previously for placement of para -TINA molecules into Hoogsteen based parallel DNA triplex formations [18]. Since thermal stability data for a number of different nucleic acid intercalating molecules are in perfect agreement with the herein presented design rules [11]–[16], [20], we speculate whether these design rules might represent general design rules for placement of intercalator molecules into Watson-Crick based antiparallel duplex and Hoogsteen type triplex formations.…”

Triplex‐Forming Twisted Intercalating Nucleic Acids (TINAs): Design Rules, Stabilization of Antiparallel DNA Triplexes and Inhibition of G‐Quartet‐Dependent Self‐Association

“…[23] 6) The insertion of TINAs as bulges in the TFO at least three nucleotides apart [23] or, one TINA for each half helix turn, or whole helix turn of the target duplex can be even more advantageous. [31] For example, conservation of at least one block of three contiguous Gs inside the G-tract is necessary for good affinity (compare ONs 3, 5 and 7 to ONs 1 and 4). With the incorporation of each TINA monomer the backbone of the TFO is elongated.…”

“…Furthermore, it gives also a rise to lipophilic interactions (see below). [31] 7) Preferably, the modified TFO should not be part of in any self-associated structures and should exist as free single-stranded DNA. Thus, the TINA monomer(s) should be inserted inside the G-tract in order to negate possible G-quadruplex formation and push the equilibrium towards formation of the antiparallel triple helical structures.…”

“…The remaining GA TINA-TFOs were able to bind to the DNA duplex in an antiparallel fashion even in the presence of K + . [26,27] We [21][22][23][28][29][30] and others [31] have undertaken several studies on the ability of TINA moiety to stabilize pH-dependent parallel CT-triplexes, while very little is known about the effects of TINA on antiparallel GA or GT-containing TFOs. Herein we synthesized several triplex-forming 16-mer GT oligonucleotides with 2-4 TINA insertions to target the polypurine tract of HIV-1 proviral DNA [32] in different orientations.…”

Triplex Formation by Oligonucleotides Containing Organomercurated Base Moieties

“…Weak hybridization is a bigger problem and considerable effort has been invested to overcome it . Approaches explored to improve the hybridization affinity of TFOs include the introduction of positive charges on the sugar or base moieties; replacement of the negatively charged sugar–phosphate backbone with an uncharged one; conformational restraints; and attachment of triplex‐stabilizing conjugate groups, such as intercalators or aminoglycosides …”