Enhanced Diagnostic Tools
2’,4’-BNA (LNA) with a five-membered bridged structure is insufficiently resistant to nucleases, and fully modified 2’,4’-BNA (LNA) oligonucleotides do not have the flexibility required for efficient triplex formation.
BNA analogues with increased steric bulk and less conformational restriction were developed. 2’,4’-BNACOC has a seven membered bridged structure and exhibits dramatically improved nuclease resistance.
NA-5 = BNACOC: This modification conversely affects duplex stability [i.e., duplexes formed with this nucleic acid analogue are less stable than those formed by 2’,4’-BNA (LNA)].
ENA, with a six-membered bridged structure, has slightly lower duplex-forming ability and significantly higher nuclease resistance than 2’,4’-BNA (LNA).
Triplex formation with ENA provided variable results compared to that of 2’,4’-BNA (LNA).
2’,4’-BNANC, has a six-membered bridged structure with a unique structural feature (N-O bond) in the sugar moiety
The bridged moiety was designed to have a nitrogen atom, which can:
The nitrogen atom on the bridge can be functionalized by hydrophobic and hydrophilic groups, by steric bulk, or by appropriate functional moieties to:
ΔTmvalues in the Table show that any mismatched base in the target RNA strand resulted in a substantial decrease in the Tm of duplexes formed with 2’,4’-BNANC-modified oligonucleotides.
ΔTmvalues of duplexes formed with 2’,4’-BNANC[NH]-modified oligonucleotide having T-U, T-G, and T-C arrangements are -14, -5, -17 °C.
These are lower than those of the corresponding natural DNA-RNA duplexes (ΔTm= -12, -3, and -15 °C, respectively).
Similar to those exhibited by duplexes formed with the 2’,4’-BNA (LNA) oligonucleotide (ΔTm= -13, -5, and -17 °C, respectively).
The mismatch discrimination profiles of 2’,4’-BNANC[NMe] and [NBn] were also similar to that of 2’,4’-BNA (LNA), except in the case of the T-G arrangement for the 2’,4’-BNANC[NMe] derivative, as shown in the Table.
Thus, it appears that 2’,4’-BNANC not only exhibits high-affinity RNA selective hybridization, but is also highly selective in recognizing bases.
The Tm of the triplex formed by the corresponding 2’,4’-BNANC-[NMe]-TFO is 5 °C higher than that of natural-TFO, and that of 2’,4’-BNANC[NBn]-TFO is equal to that of the natural oligonucleotide. In the case of 2’,4’-BNANC[NH], increasing the number of modifications (TFOs) greatly enhanced triplex thermal stability (ΔTm/mod. = +6.2 to +9 °C), which is similar to or even higher than that of the corresponding 2’,4’-BNA (LNA)-TFOs (ΔTm/mod. = +4.9 to +8.7 °C).
The thermal stability of the triplex formed by the corresponding 2’,4’-BNANC[NMe]-TFOs, although significantly higher than that obtained with natural DNA-TFO, is lower than that of 2’,4’-BNANC[NH]- and 2’,4’-BNA (LNA)-TFOs.
2’,4’-BNANC[NH] form more stable TFOs when compared to 2’,4’-BNANC[NMe] and LNAs.
Proper spacing of the monomers appears to make the binding stronger.
Best placement of BNANC[NH] monomers if no Mg2+ ions are present for triplex probes are:
underlined portion indicates the target site for triplex formation. b Conditions: 7 mM sodium phosphate buffer (pH 7.0) containing 140 mM KCl; strand concentration = 1.5 µM. c Data from Rahman et al. 2007.
Oligonucleotides with 2’,4’-BNANC[NH] monomers form more stable TFOs when compared to 2’,4’-BNANC[NMe] and LNAs.
Best placement of BNANC[NH] monomers for triplex probes is:
This design appears to be the best for the design of probes that form stable
triplexes under physiological conditions!
This is similar or close to LNA oligos.
2’,4’-BNANC[NH] oligos form more stable TFOs.
Molecular modeling of a parallel-motif triplex formed by 2’,4’-BNANC[NH]-TFO with dsDNA. BNA: d(TTTTTmCTTTmCTmCTmCT). (Source: Rahman et al., 2008).
(A) The overall view of the triplex is depicted.
The dsDNA is shown as a gray CPK model with the phosphate backbone of the purine strand colored red and purple.
TFO is shown as a colored tube model with three 20,40-BNANC nitrogens represented in green.
(B) Expanded view of the area of the triplex containing three consecutive 20,40-BNANC[NH] residues.
Note that the 20,40-BNANC nitrogens are very close to the phosphate moiety of the purine strand indicating ionic interactions of the compounds.
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