MGB oligonucleotide probes
Oligonucleotides conjugated to MGB molecules are known to form stable duplexes with single-stranded DNA targets. The addition of MGBs to DNA probes allows the design of shorter hybridization probes useful for molecular diagnostics, for example for their use in quantitative PCR based assays.
Several designs are possible, including Taqman MGB probes. MGB oligonucleotides form stable hybrid complexes with complementary DNA and RNA target sequences. Several solved 3D structures of MGBs with DNA oligonucleotides as well as a structure of a MGB-oligonucleotide conjugate hybrid provided insight of molecular interactions between minor groove binding moieties and DNA.
What are Minor Groove Binder Molecule?
Minor Groove Binder (MGB) molecules are crescent-shaped molecules that selectively bind non-covalently to the minor groove of DNA, a shallow furrow in the DNA helix. Binding to DNA with specific sequences usually takes place by the combination of directed hydrogen bonding to base pair edges. Tri-imidazole polyamide and the tripeptide of dihydropyrroloindole-carboxylate are good examples of MGBs.
Tri-imidazole polyamides recognize the minor groove of DNA
The design of molecules that can recognize base pairs or mismatched base pairs is desirable for studying mutations leading to mismatched base pairs. The T:G mismatch has been shown to be responsible for most common mutations in human ras oncogenes. Spontaneous deamination of 5-methylcytosine or errors in replication can introduce a T:G mismatch. The combination of pyrrole (Py), hydroxypyrrole (Hp) and imidazole (Im) units of polyamides can confer specific recognition properties for all four different base pairs, A:T, T:A, G:C, and C:G.
Yang et al. in 1999 proposed that an imidazole-imidazole pair containing polyamide may contain a good molecular motif for selective recognition of T:G/G:T base pairs. The researchers reasoned, since the N2 amino group of the G base in a T:G mismatch is not involved in base pairing with T, the free amino group could form two hydrogen bonds, each with one imidazole nitrogen atom of the polyamide. Studying the binding of two polyamides, AR-1-144 and Im-Py-Im, to DNA sequences using NMR, showed that an imidazole-imidazole pair is a good motif for the recognition of a T:G/G:T base pair.
The structures for AR-1-144 and Im-Py-Im are illustrated in figure 1 and the structure of AR-1-144 in complex with a CCGG containing DNA duplex solved by NMR is shown in figure 2.
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Figure 1: Chemical structure of AR-1-144 and Im-Py-Im polyamides. The molecular model of AR-1-144 is shown as well (Yang et al. 1999).
Figure 2: NMR structure of AR-1-144 in complex with a CCGG containing DNA duplex. The tri-imidazole AR-1-144 {N-[2-(dimethylamino)ethyl]-1-methyl-4-[1-methyl-4-[4-formamido-1-methylimidazole-2-carboxamido]imidazole-2-carboxamido]-imidazole-2-carboxamide} is a minor groove binder. AR-1-144 favors the CCGG sequence (Yang et al. 1999).
The dihydropyrroloindole tripeptide CDPI3 is a minor groove binder
Another minor groove binder (MGB) derivative, a dihydropyrroloindole tripeptide (N-3-carbamoyl-1,2-dihydro-3H-pyrrolo[3,2-e]indole-7-carboxylate tripeptide or CDPI3), was shown to arrest sequence-specific primer extension on single-stranded DNA when linked to oligodesoxyribonucleotides.
Afonina et al. in 1996 showed that CDPI3 conjugated to DNA oligonucleotides increased both the specificity and the strength of hybridization using absorption thermal denaturation and slot-blot hybridization studies. Furthermore, a complementary 16-mer conjugated to 5'-CDPI3 when hybridized to phage DNA blocked primer extension by a modified form of phage T7 DNA polymerase (Sequenase).
Kutyavin et al. in 1997 showed that CDPI3 when conjugated to DNA oligonucleotides enhanced the stability of all duplexes studied. The observed stabilization was backbone and sequence-dependent and reached a maximum value of 40-49 °C for d(pT)8: d(pA)8. However, duplexes with a phosphorothioate DNA backbone were less stable than phosphodiesters analogs. Duplexes with a 2'-O-methyl RNA backbone were modestly stable, and the conjugated CDPI3 residue stabilized GC-rich DNA duplexes to a lesser extent than AT-rich duplexes of the same length.
In 1998, Kumar et al. reported the solution structure of a duplex consisting of the oligodeoxyribonucleotide 5'-TGATTATCTG-3' conjugated at the 5'-end to CDPI3 with its complementary strand using NMR. This hybrid duplex was shown to be very stable.
Figure 3: Solution structure of CDPI3-decamer conjugate duplex solved using NMR. (A) The overall shape of the duplex is that of a straight B-type helix and the CDPI3 moiety is bound snugly in the minor groove stabilized by van der Waal’s interactions. (B) Chemical structure of CDPI3. (C) Molecular stick model of CDPI3. The crescent-shape of the molecule is nicely demonstrated here.
In summary,
(i) oligonucleotides conjugated to minor groove binders (MGBs) stabilize DNA
hybrid duplexes,
(ii) conjugation of MGBs to A/T rich oligonucleotides increase the melting
temperature of DNA hybrids by as much as 44 °C,
(iii) conjugates also form stabilized hybrids with complementary RNA with
G/C-rich DNA,
(iv) CDPI3 covalently linked to the 5’-end of oligonucleotides blocks primer
extension by DNA polymerase,
(v) CDPI3 conjugates as short as 8 to 10 mers can function as primers in PCR.
(VI) Adding bridged nucleic acids (BNAs) to selected internal positions of the
probes will stablise the target duplx even further.
Reference
I Afonina, I Kutyavin, E Lukhtanov, R B Meyer, and H Gamper; Sequence-specific arrest of primer extension on single-stranded DNA by an oligonucleotide-minor groove binder conjugate. Proc Natl Acad Sci U S A. 1996 April 16; 93(8): 3199–3204.
Kumar, S., Reed, M. W., Gamper, H. B., Gorn, V. V., Lukhtanov, E. A., Foti, M., … Schweitzer, B. I. (1998). Solution structure of a highly stable DNA duplex conjugated to a minor groove binder. Nucleic Acids Research, 26(3), 831–838.
Kutyavin, I. V., Lukhtanov, E. A., Gamper, H. B., & Meyer, R. B. (1997). Oligonucleotides with conjugated dihydropyrroloindole tripeptides: base composition and backbone effects on hybridization. Nucleic Acids Research, 25(18), 3718–3723.
Yao Y, Nellåker C, Karlsson H.; Evaluation of minor groove binding probe and Taqman probe PCR assays: Influence of mismatches and template complexity on quantification. Mol Cell Probes. 2006 Oct;20(5):311-6. Epub 2006 Apr 21.
Yang XL, Kaenzig C, Lee M, Wang AH.; Binding of AR-1-144, a tri-imidazole DNA minor groove binder, to CCGG sequence analyzed by NMR spectroscopy. Eur J Biochem 1999 Aug; 263(3):646-55.
Xiang-Lei Yang, Richard B. Hubbard, Moses Lee, Zhi-Fu Tao, Hiroshi Sugiyama, Andrew H.-J. Wang; Imidazole-imidazole pair as a minor groove recognition motif for T:G mismatched base pairs, Nucleic Acids Research, Volume 27, Issue 21, 1 November 1999, Pages 4183–4193.
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