Solution structure of a highly stable DNA duplex conjugated to a minor groove binder

Abstract

The tripeptide 1,2-dihydro-(3 H )-pyrrolo[3,2- e ]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder is conjugated to the 5'-end of short oligonucleotides the conjugates form unusually stable hybrids with complementary DNA and thus may have useful diagnostic and/or therapeutic applications. In order to gain an understanding of the structural interactions between the CDPI3minor groove binding moiety and the DNA, we have determined and compared the solution structure of a duplex consisting of oligodeoxyribonucleotide 5'-TGATTATCTG-3' conjugated at the 5'-end to CDPI3 and its complementary strand to an unmodified control duplex of the same sequence using nuclear magnetic resonance techniques. Thermal denaturation studies indicated that the hybrid of this conjugate with its complementary strand had a melting temperature that was 30 degrees C higher compared with the unmodified control duplex. Following restrained molecular dynamics and relaxation matrix refinement, the solution structure of the CDPI3-conjugated DNA duplex demonstrated that the overall shape of the duplex was that of a straight B-type helix and that the CDPI3moiety was bound snugly in the minor groove, where it was stabilized by extensive van der Waal's interactions.

Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems

“…When such MGBs are conjugated with oligodeoxynucleotides, the conjugates form very stable hybrids with complementary DNA (Kumar et al 1998). Commercial MGB probes have the MGB at their 3 end, since they are easier to synthesise but the MGB can be placed either at the 5 or 3 ends (Afonina et al 1996).…”

Development of an RT-qPCR assay for the specific detection of a distinct genetic lineage of the infectious bursal disease virus

“…The 289 and 290 amino acids are important because they occur within the hvVP2 hydrophilic peak 2, which is antigenically relevant in IBDV (Berg et al, 1996). As these two nucleotides are not present in any other IBDVs, the designed TaqMan-MGB probe is expected to be highly specific; TaqMan-MGB probes form extremely stable duplexes with complementary DNA, and a single mismatch in the probe-target duplex would result in a high Tm difference that prevents cross-hybridization to non-specific targets (Kumar et al, 1998). There are also mismatches between primers and target sites of non-dIBDVs (Figure 1).…”

Development and validation of a TaqMan-MGB real-time RT-PCR assay for simultaneous detection and characterization of infectious bursal disease virus

“…These types of probes had not been previously used for IBDV diagnosis, but they have been successfully applied to detect and characterize many different viral pathogens (Campsall et al, 2004;Chen et al, 2009;Decaro et al, 2008;Steyer et al, 2010). TaqMan-MGB probes form extremely stable duplexes with complementary DNA (Kumar et al, 1998), permitting the use of shorter probes in the assays. The shorter length is a desirable feature in RNA viruses that have few stretches of conserved regions and provides TaqMan-MGB probes with better sequence specificity and lower fluorescent background (Kutyavin et al, 2000).…”