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

Abstract

The sensitivity and specificity of clinical diagnostic assays using DNA hybridization techniques are limited by the dissociation of double-stranded DNA (dsDNA) antiparallel duplex helices. This situation can be improved by addition of DNA stabilizing molecules such as nucleic acid intercalators. Here, we report the synthesis of a novel ortho-Twisted Intercalating Nucleic Acid (TINA) amidite utilizing the phosphoramidite approach, and examine the stabilizing effect of ortho- and para-TINA molecules in antiparallel DNA duplex formation. In a thermal stability assay, ortho- and para-TINA molecules increased the melting point (Tm) of Watson-Crick based antiparallel DNA duplexes. The increase in Tm was greatest when the intercalators were placed at the 5′ and 3′ termini (preferable) or, if placed internally, for each half or whole helix turn. Terminally positioned TINA molecules improved analytical sensitivity in a DNA hybridization capture assay targeting the Escherichia coli rrs gene. The corresponding sequence from the Pseudomonas aeruginosa rrs gene was used as cross-reactivity control. At 150 mM ionic strength, analytical sensitivity was improved 27-fold by addition of ortho-TINA molecules and 7-fold by addition of para-TINA molecules (versus the unmodified DNA oligonucleotide), with a 4-fold increase retained at 1 M ionic strength. Both intercalators sustained the discrimination of mismatches in the dsDNA (indicated by ΔTm), unless placed directly adjacent to the mismatch – in which case they partly concealed ΔTm (most pronounced for para-TINA molecules). We anticipate that the presented rules for placement of TINA molecules will be broadly applicable in hybridization capture assays and target amplification systems.

MAZ-binding G4-decoy with locked nucleic acid and twisted intercalating nucleic acid modifications suppresses KRAS in pancreatic cancer cells and delays tumor growth in mice

“…Indeed, although quadruplex 32R-3n or 32R-3n with P insertions is degraded in serum, their analogs with two LNA at the 3′ show a fairly good resistance. Previous studies demonstrated that the introduction of P at the ends of duplex or quadruplex DNA strongly increases the T M (10,75). An interesting effect of TINA ( P or O ) is its capacity to favor an intramolecular folding of the strand in which they have been inserted.…”

Chemical Maturation of a Bivalent Aptamer by Single Domain Variation

“…We synthesized aptamer variants with either insertions or replacements of nucleotides by using three different PAHs, namely ortho ‐TINA (O) (( R )‐3‐((2‐(1‐pyrenylethynyl)benzyl)oxy)propane‐1,2‐diol),8 TINA (P) (( R )‐3‐((4‐(1‐pyrenylethynyl)benzyl)oxy)propane‐1,2‐diol) and Amany (Y) (( S )‐4‐(4‐(1 H ‐phenanthro[9,10‐d]imidazol‐2‐yl)phenoxy)butane‐1,2‐diol)9 (Scheme ). We then assessed their inhibitory potential by measuring their impact on routine plasma‐based coagulation assays, such as the thrombin time and the activated partial thromboplastin time.…”

Delivery, Effect on Cell Viability, and Plasticity of Modified Aptamer Constructs

“…It stacks with the nucleobases by its aromatic ring and its stabilizing ability is much dependant on where it is placed in an ON sequence. For instance, when placed terminally, both ortho and para-TINA (figure 4E+F) gave higher stability for an antiparallel duplex compared to when placed at other positions (166).…”