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

Date Published: June 3, 2011

Publisher: Public Library of Science

Author(s): Uffe V. Schneider, Imrich Géci, Nina Jøhnk, Nikolaj D. Mikkelsen, Erik B. Pedersen, Gorm Lisby, Meni Wanunu.

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.

Partial Text: The stability of double-stranded DNA (dsDNA) is naturally limited to allow cellular processes that require helix dissociation such as gene transcription, gene regulation and cell division. However, the sensitivity of DNA diagnostic assays depends upon the stability of dsDNA helices. The analytical sensitivity of an assay can be improved by decreasing stringency, but at the risk of cross-reactivity to other targets.

In the current paper, we have characterized the stabilizing effect and established design rules for placement of ortho- and para-TINA molecules into Watson-Crick based antiparallel DNA duplexes. According to thermal stability analyses, both para- and ortho-TINA molecules should be placed terminally in the nucleotide sequence, and preferably on both the 5′ and 3′ terminal positions to achieve a maximum increase in Tm. Placement of para-TINA molecules at the 5′ and 3′ termini gave the most pronounced increase in Tm compared to ortho-TINA molecules. The stabilizing effect of para- and ortho-TINA molecules changes when they are placed internally in the oligonucleotide sequence. Ortho-TINA molecules have either a positive effect or no effect on Tm, whereas para-TINA molecules decrease Tm when placed internally. However, neither para- nor ortho-TINA molecules interfere with mismatch-induced ΔTm, unless they are placed internally directly adjacent to the mismatch. Overall, when several TINA molecules are placed in an oligonucleotide, the highest increase in Tm is observed if they are placed at the 5′ and 3′ terminal positions (preferable) or, if placed internally as well, with the modifications separated by a half or whole helix turn.