Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids

Abstract

In the past few years, several researchers have focused their attention on nucleic acid mimics due to the increasing necessity of developing a more robust recognition of DNA or RNA sequences. Fluorescence in situ hybridization (FISH) is an example of a method where the use of these novel nucleic acid monomers might be crucial to the success of the analysis. To achieve the expected accuracy in detection, FISH probes should have high binding affinity towards their complementary strands and discriminate effectively the noncomplementary strands. In this study, we investigate the effect of different chemical modifications in fluorescent probes on their ability to successfully detect the complementary target and discriminate the mismatched base pairs by FISH. To our knowledge, this paper presents the first study where this analysis is per-formed with different types of FISH probes directly in biological targets, Helicobacter pylori and Helicobacter acinonychis. This is also the first study where unlocked nucleic acids (UNA) were used as chemistry modification in oligonucleotides for FISH methodologies. The effectiveness in detecting the specific target and in mismatch discrimination appears to be improved using locked nucleic acids 2 (LNA)/2′-O-methyl RNA (2′OMe) or peptide nucleic acid (PNA) in comparison to LNA/DNA, LNA/UNA, or DNA probes. Further, the use of LNA modifications together with 2′OMe monomers allowed the use of shorter fluorescent probes and increased the range of hybridization temperatures at which FISH would work.

Modified Nucleic Acids: Expanding the Capabilities of Functional Oligonucleotides

“…Modified oligonucleotides such as LNA have demonstrated their abilities as thermally robust capture agents of natural oligonucleotides present at low copy numbers [ 59 ]. Comparison of probes incorporating different modified nucleotides also indicates better mismatch discrimination capabilities for LNA, 2′-OMe and PNA over pure DNA probes; however, these same studies indicate that probes with unlocked nucleic acids (UNA) perform more poorly as capture agents for mismatched targets [ 60 ]. These thermodynamic studies suggest UNA allows for hydrogen bonding arising from Watson–Crick base-pair matching; however, in contrast to the locked conformation of the cyclic sugar group in LNA, the intrinsic flexibility of the acyclic sugar group in UNA could disrupt pi-pi stacking of base-pairs to cause duplex destabilization.…”

Development and application of Peptide Nucleic Acid Fluorescence in situ Hybridization for the specific detection of Listeria monocytogenes

“…While PNA has been described as highly effective for the discrimination of single-base mismatch sequences (Fontenete et al, 2015;Lefmann et al, 2006), the fact is that effective discrimination might also be dependent on the mismatch position. Those at the center are usually associated with an easier discrimination, while those near the 5′ or 3′ ends, which is the case in here, are reported as less effective for discrimination purposes (Amann, 1995;Lefmann et al, 2006).…”

Guidelines for Rational Cancer Therapeutics

“…Such a level of flexibility can be achieved through sugar modifications. These include, among others: incorporation of 2′ -O- Me units to increase affinity and nuclease stability ( 4 , 5 ); the incorporation of LNA analogs ( 6 , 7 ) to dramatically increase binding affinity, improve nuclease stability, and reduce immunogenicity; and the incorporation of a 2′ -O- MOE [2′ -O- (2-methoxyethyl)] modification to increase affinity and nuclease stability ( 8 ). A thorough review on the subject can be found in Ref.…”