Crystal structures of DNA:DNA and DNA:RNA duplexes containing 5-(N-aminohexyl)carbamoyl-modified uracils reveal the basis for properties as antigene and antisense molecules

Abstract

Oligonucleotides containing 5-(N-aminohexyl)carbamoyl-modified uracils have promising features for applications as antigene and antisense therapies. Relative to unmodified DNA, oligonucleotides containing 5-(N-aminohexyl)carbamoyl-2′-deoxyuridine (NU) or 5-(N-aminohexyl)carbamoyl-2′-O-methyluridine (NUm), respectively exhibit increased binding affinity for DNA and RNA, and enhanced nuclease resistance. To understand the structural implications of NU and NUm substitutions, we have determined the X-ray crystal structures of DNA:DNA duplexes containing either NU or NUm and of DNA:RNA hybrid duplexes containing NUm. The aminohexyl chains are fixed in the major groove through hydrogen bonds between the carbamoyl amino groups and the uracil O4 atoms. The terminal ammonium cations on these chains could interact with the phosphate oxygen anions of the residues in the target strands. These interactions partly account for the increased target binding affinity and nuclease resistance. In contrast to NU, NUm decreases DNA binding affinity. This could be explained by the drastic changes in sugar puckering and in the minor groove widths and hydration structures seen in the NUm containing DNA:DNA duplex structure. The conformation of NUm, however, is compatible with the preferred conformation in DNA:RNA hybrid duplexes. Furthermore, the ability of NUm to render the duplexes with altered minor grooves may increase nuclease resistance and elicit RNase H activity.

Towards Improved Oligonucleotide Therapeutics Through Faster Target Binding Kinetics

“…Although we have not ruled it out, it is unlikely that the triazole moiety produced this effect:S imple triazoles have been conjugated previously to C5o ft hymidine in DNAs, but have only minor effects on DNA/RNA hybridization affinity,u nless cumulated at consecutive stackedp ositions. [21,37] Our data is consistentw ith evidence that free polyamines bind favorably to A-form helical structures, in line with the stronger effects of free spermine on DNA/RNA heteroduplexes than on DNA/DNA duplexes, [17] and the preference of polyamines for cellular RNA over DNA. [34][35][36] Also, positioning of an aminohexyl group into the major groove of duplex RNA considerably increases T M values.…”

Polyamine–Oligonucleotide Conjugates: A Promising Direction for Nucleic Acid Tools and Therapeutics

“…In the majority of cases, incorporation of 5-aminoethyl and/or aminohexyl-dT, -dU and -dC nucleotides (structures 21, 26 and 28, respectively) into various positions of ODN sequences were shown to increase both ODN-DNA and ODN-RNA duplex stabilities with ΔT M =+2-5°C and +1-3°C per modification, respectively [70][71][72][73][74]78]. Crystal structures of DNA duplexes containing 5-(N-aminohexyl)carbamoyl-dU or 5-(N-aminohexyl) carbamoyl-2´-O-Me-U units provided some insight into these findings ( Table 3; structure 26) [79] as they showed an association with and possible partial charge neutralization of the phosphate group in the ODN backbone. For 5-(N-aminohexyl)carbamoyl-2´-O-Me-U, preorganization of the adjacent sugar into the preferred 3´-endo conformation likely further enhanced duplex formation with complementary RNAs.…”

“…It is, therefore, unsurprising that the C-5 position of pyrimidines has been extensively used for conjugation of linear and branched polyamines ( Table 3; substructures 21-31) [18,[65][66][67][68][69][70][71][72][73][74][75][76][77][78][79][80][81][83][84][85][96][97][98]100], predominately via amide linkers. Interestingly, the genome of the bacteriophage ΦW-14 contains a thymine with a putrescinyl-substitution at its C-5 position, which enhances local DNA duplex stability and provides added resistance against endo-and exonucleases [101][102][103].…”

“…All of the modifications, however, showed increased nuclease resistance, and as a likely consequence, when tested, an increased silencing activity. • Conjugation of single amino groups or branched polyamines to the nucleobases appears most useful at the C-5 position of pyrimidines: 5-(N-aminoethyl)carbamoyl, 5-(N-aminohexyl)carbamoyl or triamine substituents were all found to increase nuclease resistance and most of them stabilized duplex ODN-DNA and ODN-RNA structures [70][71][73][74][76][77][78][79][80][81][82]. Increased thermal stability was observed for other spermine conjugates, particularly at the C-4 position.…”

5‐Substituted Nucleosides in Biochemistry and Biotechnology

“…In compounds 15e-h [8,70], the carbonyl is directly conjugated to the ring, and an electron-withdrawing group would be expected to increase the acidity of the N-3 proton. Recent crystal structures of DNAÁ ÁDNA and DNAÁ ÁRNA containing 15f show the side chain projecting from the major groove, thus placing the protonated amino group in close proximity to a phosphodiester group [72]. Several studies have been conducted to determine why the relatively small aminopropyl (15c) is not stabilizing [73][74][75][76].…”