Molecular recognition via triplex formation of mixed purine/pyrimidine DNA sequences using oligoTRIPs

Abstract

Stable DNA triple-helical structures are normally restricted to homopurine sequences. We have described a system of four heterocyclic bases (TRIPsides) that, when incorporated into oligomers (oligoTRIPs), can recognize and bind in the major groove to any native sequence of DNA [Li et al., J. Am. Chem. Soc. 2003, 125, 2084]. To date, we have reported on triplex-forming oligomers composed of two of these TRIPsides, i.e., antiTA and antiGC, and their ability to form intramolecular triplexes at mixed purine/pyrimidine sequences. In the present study, we describe the synthesis and characterization of the antiCG TRIPside and its use in conjunction with antiTA and antiGC to form sequence-specific intra- and/or intermolecular triplex structures at mixed purine/pyrimidine sequences that require as many as four major groove crossovers.

Figure 1

UV-visible melting curves for intramolecular triplexes OT’s 1–4 (structures shown in Scheme 4). Absorbance normalized to 1.0 at elevated temperature: (a) OT-1 (■) and OT-2 (○) at 250 nm; (b) OT-1 (■) and OT-2 (○) at 330 nm; (c) OT-3 (■) and OT-4 (○) at 250 nm; (d) OT-3 (■) and OT-4 (○) at 330 nm.

Figure 2

UV melting curves in 10 mM phosphate buffer (pH 7.0) containing 1 M NaCl. Absorbance normalized to 1.0 at elevated temperature: (a) duplex-1 (○) and duplex-1 + OT-6 (●) at 250 nm, (b) duplex-2 ( ) and duplex-2 + OT-7 (■) at 250 nm; (c) curves are constructed by normalization of the absolute values resulting from the subtraction of the triplex (duplex + OT) curves at 250 nm from the corresponding duplex curves shown in (a) and (b); and (d) duplex-1 + OT-6 (●) and duplex-2 + OT-7 (■) at 330 nm.

Figure 3

Fluorescence intensity versus temperature dependence for OT-6 (green), OT-7 (blue), OT-6 + duplex-1 (black) and OT-7 + duplex-2 (red).

Figure 4

Effect of oligoTRIPs on the methylation of DNA by dimethyl sulfate: (a) duplex-1 + OT-6 and (b) duplex-2 + OT-7: number of equivalents of oligoTRIP relative to duplex is indicated.

Figure 5

DSC melting curves of duplex-1 + OT-6 (●) and duplex-1 (○) in 10 mM sodium phosphate buffer (pH 7.0) with 1 M NaCl.

Figure 6

T_M of OT-8 with matched duplex and duplexes with one or more mismatches between the TFO and duplex (see Scheme 6 for putative triplex structures): The UV-vis melting studies at (a) 330 nm and (b) 350 nm for OT-3 with duplex-3 (black), duplex-4 (red), duplex-5 (dark blue), duplex-6 (aqua), duplex-7 (magenta) and duplex-8 (green).

Scheme 1

Structures of the TRIPsides associated with their respective duplex targets.

Scheme 2

Nomenclature used to describe how oligoTRIPs associate with duplex DNA: two unique TFO’s recognize the same sequence in opposite orientation: W, antiAT; X, antiTA; Y, antiGC; Z, antiCG.

Scheme 3

Synthesis of antiCG: a, KHSO₄, N-methylpyrrolidinone, xylene, reflux; b, POCl₃, reflux; c, K₂CO₃, CH₃CONH₂, 200 °C; d, 1,4-anhydro-2-deoxy-3-O-(tert-butyldiphenyl-silyl)-D-pent-1-enitol, Pd(dba)₂, (t-Bu)₃P, dicyclohexylmethylamine, dioxane, reflux; e, TBAF/HOAc; f, NaHB(OAc)₃, −22 °C; g, TMS-Cl, (iPrCO)₂O; h, DMTrCl, pyridine; i, 2-cyano-N,N, diisopropylphosphoramidite, Hunig base, CH₂Cl₂.

Scheme 4

Potential intramolecular structures for OT’s 1–5.

Scheme 5

Potential intermolecular structures for OT-6 + duplex-1 and OT-7 + duplex-2.

Scheme 6

Potential intermolecular structures for OT-8 with duplexes 3–8: X, antiTA; Y, antiGC; bold red X indicates a mismatch site. The color code for each intermolecular triplex structure correlates to the color code used in T_M studies in Figure 6.