Dual Recognition of Double-Stranded DNA by 2′-Aminoethoxy-Modified Oligonucleotides

Abstract

Molecular recognition of duplex DNA by proteins plays a central role in biology. Although there are no clear and simple rules or codes to describe sequence-specific recognition, hydrogen bonds with the nucleic bases as well as with the phosphodiester backbone are of critical importance.[1] Efforts to mimic such recognition with smaller synthetic biopolymers have been described. [2] However, in all these cases, specific contacts with the DNA duplex are only provided by hydrogen bonds with the bases, and potential interactions with the DNA backbone are left unexploited. Here we report that 2'-aminoethoxy-modified oligonucleotides can interact simultaneously with the bases and the phosphodiester backbone at each base-pair step of the DNA target, which provides for a dramatic increase in the binding affinity as well as in the association rate constant.The sequence-specific recognition of duplex DNA by pyrimidine oligonucleotides involves the formation of triplehelical structures, which are stabilized by Hoogsteen hydrogen bonds between the bases on the DNA target and the pyrimidine third strand.[3] Examination of a molecular model of a triple helix with a RNA third strand indicates that the 2'-hydroxyl groups of the RNA and the phosphate groups of the DNA second strand are in close proximity.[4] Thus, it appeared conceivable that the attachment of a short amino alkyl group at the 2' position of the ribose of the third strand would allow the protonated amino group to form a specific intermolecular contact with a proximal phosphate group of the DNA duplex. Such charge ± charge interactions should strongly enhance the affinity of the modified oligonucleotide for double-strand DNA.To test this hypothesis, we first examined the properties of a 2'-aminoethoxy-modified oligonucleotide with regard to triplex formation. The synthesis of the 2'-aminoethyl-modified monomeric tymidine and C5-methylcytidine building blocks is summarized in Scheme 1. Alkylation of protected ribothymidine 1[5] with methyl bromoacetate followed by reduction of the ester group and tosylation gave the tosylate 2. Hydrogenolytic removal of the BOM group and subsequent replacement of the tosyloxy group by an azide substituent yielded the azidoethyl derivative 3, which was further transformed into the desired protected 2'-aminoethoxy-thymidine Scheme 1. Synthesis of protected 2'-aminoethoxy phosphoramidites thymidine (5) and C5-methylcytidine (8): a) BrCH 2 CO 2 Me (5 equiv), NaH (2.2 equiv), DMF, 1.5 h, À 5 8C, 98 %; b) LiBH 4 (4 equiv), MeOH/THF (2/ 8), 1.5 h, 5 8C, 84 %; c) TsCl (1.5 equiv), NEt 3 (1.6 equiv), DMAP (10 wt %), CH 2 Cl 2 , 8 h, 22 8C, 89 %; d) H 2 , Pd/C (20 wt %), HCl (0.02 equiv), THF/ MeOH (1/1), 7 h, 22 8C, 88 %; e) NaN 3 (3 equiv), DMF, 3 h, 65 8C, 92 %; f) SnCl 2 (3.5 equiv), MeOH, 1 d, 22 8C, 70 %; g) (CF 3 CO) 2 O (1.2 equiv), pyridine, 2 h, 22 8C, 57 %; h) TBAF (2.1 equiv), THF, 15 min, 22 8C, 100 %; i) DMTrCl (1.2 equiv), pyridine, 16 h, 22 8C, 85 %; j) (iPr 2 N) 2 -POCH 2 CH 2 CN (2.2 equiv), diisopropylammonium tetrazolide (...

Inhibition of Interleukin-4- and CD40-induced IgE Germline Gene Promoter Activity by 2′-Aminoethoxy-modified Triplex-forming Oligonucleotides

“…The binding affinity of the specific TFOs was more than 800-fold higher than that of an unrelated TFO (3595). These numbers are in good agreement with previous results obtained in surface plasmon resonance experiments (36). In addition, the binding reaction in the EMSA assays were carried out at pH Ͼ 7, which does not favor the formation of protonated cytidines, even considering that 5Ј-methylcytidine was used in the synthesis of the TFOs.…”

“…In addition, the binding reaction in the EMSA assays were carried out at pH Ͼ 7, which does not favor the formation of protonated cytidines, even considering that 5Ј-methylcytidine was used in the synthesis of the TFOs. These results support previous findings showing that the interaction of the 2Ј-aminoethoxy side chains of the TFO with phosphate groups of the duplex DNA is a very important contribution to the Hoogsteen hydrogen bonds for the total affinity of the observed DNA triplexes (36,37). Although the shorter 3651 TFO does not hybridize directly with the NF-B site, protein binding was inhibited to a similar degree as PU.1 or STAT6.…”

“…The fusion protein composed of soluble mouse CD40 ligand and soluble mouse CD8 (sCD40L-sCD8) (38), a kind gift of Dr. Peter Lane, was purified by immuno-affinity chromatography. The three TFOs were synthesized as described (36) and dissolved in 8% ammonium hydroxide. The MeC-3654 oligonucleotide was synthesized using 5Ј-methylcytidine (MeC) as building block like in the other three TFOs.…”

“…A number of studies have demonstrated DNA triplex structures with critical polypurine:polypyrimidine cisacting DNA elements found in the regulatory region of genes and the prevention of DNA binding of regulatory proteins (30 -35). Recently, the synthesis and physicochemical properties of chemically modified oligonucleotides containing 2Ј-aminoethoxy-substituted riboses was reported (36). Such triplex-forming oligonucleotides (TFO) assemble into very stable DNAtriplexes and display a Ͼ 1000-fold higher association rate constant compared with their unmodified counterparts (37).…”

Fluorescent Labeling of Plasmid DNA and mRNA: Gains and Losses of Current Labeling Strategies

“…The translation to a broad 12 audience will however benefit from the development of a straight-forward labeling kit, ruling 13 out the need for a thorough understanding of the enzymatic reaction before it can be employed. 14 Nucleic acid-based hybridization probes 83,84 are some examples of modified 6 oligonucleotides used to create a strong and sequence-specific binding to complementary 7 sequences (see Figure 3). 8 9 2'-C, 4'-C-oxymethylene linkage that effects conformational fixation of the furanose ring in a 28 C3'-endo conformation (see Figure 3) was also developed more recently to bind to supercoiled DNA in a fashion similar to bis-PNA 1 (see Figure 2 B) 92 .…”