α-Putrescinylthymine and the sensitivity of bacteriophageφW-14 DNA to restriction endonucleases

Abstract

The modified base alpha-putrescinylthymine (putT) in phi W-14 DNA blocks cleavage of the DNA by 17 of 32 Type II restriction endonucleases. The enzymes cleaving the DNA do so to widely varying extents. The frequencies of cleavage of three altered forms of the DNA show that putT blocks recognition sites either when it occurs within the site or when it is in a sequence flanking the site. The blocking is dependent on both charge and steric factors. The charge effects can be greater than the steric effects for some of the enzymes tested. All the enzymes cleaving phi W-14 DNA release discrete fragments, showing that the distribution of putT is ordered. The cleavage frequencies for different enzymes suggest that the sequence CAputTG occurs frequently in the DNA. Only TaqI of the enzymes tested appeared not to be blocked by putT, but it was slowed down. TaqI generated fragments are joinable by T4 DNA ligase.

Oligonucleotides Containing 7-Deazaadenines: The Influence of the 7-Substituent Chain Length and Charge on the Duplex Stability

“…Also structural modifications have a strong impact on the stability of such duplexes. Therefore, in a first series of experiments, 7-deazaadenine residues carrying alkynyl groups different in length or size of the alkynyl chain were used to replace the adenine base of 5'-d(A-T) 6 (8). The T m values and thermodynamic data of the various oligonucleotide duplexes were determined for buffer solutions containing Mg 2 ions and 1.0m or 0.1m NaCl (see Table 3).…”

“…The bulky estradiol-modified ethynyl residue (see 1i) resulted in significant duplex destabilization when only one modified base was incorporated ( Table 3). For selfcomplementary oligonucleotide duplexes containing a modified dA 6 tract, enhanced duplex stability was observed when a 7-deaza-7-ethynyladenine was introduced (see 5'- Table 3, it can be concluded that alkynyl side chains up to a length of 6 ± 7 , which is about the depth of a B-DNA major groove (8.8 ; [33]), lead to duplex stabilization, while longer residues destabilize the duplex structure.…”

“…One or three dA replacements by 1h in the sequence 8´8 led to a duplex stabilization (see 50´50 and 51´51), whereas, in contrast to the duplex 5'-d[(1g-T ) 6 ] 2 (40´40), no cooperative melting was observed for 5'-d[(1h-T ) 6 ] 2 (52´52). Such an observation has already been made for the corresponding 7-(5-aminohex-1-ynyl) derivative of 7-deaza-2'-deoxyguanosine [3].…”

“…Nature has implemented this type of structure in bacteriophage DNA where half of the thymidine residues are replaced by a-putrescinylthymidine ( a-[(4-aminobutyl)amino]thymidine) [6] [7]. This zwitterionic DNA has been shown to resist a variety of endonucleases and exonucleases [6] [8] [9], and in the case of 5-aminoalkylated pyrimidines, it was found that these oligomers hybridize with natural single-stranded DNA as well or even better than natural DNA itself does [10]. Furthermore, these partially neutralized DNA fragments are membrane-permeable [11 ± 13] and can find application in the transfection of DNA molecules [14] [15].…”

“…Comparable effects were reported when the 7-deazaguanine moiety carries a 5-aminopent-1-ynyl side chain which is positively charged under physiological conditions [3]. Nature has implemented this type of structure in bacteriophage DNA where half of the thymidine residues are replaced by a-putrescinylthymidine ( a-[(4-aminobutyl)amino]thymidine) [6] [7]. This zwitterionic DNA has been shown to resist a variety of endonucleases and exonucleases [6] [8] [9], and in the case of 5-aminoalkylated pyrimidines, it was found that these oligomers hybridize with natural single-stranded DNA as well or even better than natural DNA itself does [10].…”