In most models of DNA replication, Watson-Crick hydrogen bonding drives the incorporation of nucleotides into the new strand of DNA and maintains the complementarity of bases with the template strand. Studies with nonpolar analogues of thymine and adenine, however, have shown that replication is still efficient in the absence of hydrogen bonds. The replication of base pairs might also be influenced by steric exclusion, whereby inserted nucleotides need to be the correct size and shape to fit the active site against a template base. A simple steric-exclusion model may not require Watson-Crick hydrogen bonding to explain the fidelity of replication, nor should canonical purine and pyrimidine shapes be necessary for enzymatic synthesis of a base pair if each can fit into the DNA double helix without steric strain. Here we test this idea by using a pyrene nucleoside triphosphate (dPTP) in which the fluorescent 'base' is nearly as large as an entire Watson-Crick base pair. We show that the non-hydrogen-bonding dPTP is efficiently and specifically inserted by DNA polymerases opposite sites that lack DNA bases. The efficiency of this process approaches that of a natural base pair and the specificity is 10(2)-10(4)-fold. We use these properties to sequence abasic lesions in DNA, which are a common form of DNA damage in vivo. In addition to their application in identifying such genetic lesions, our results show that neither hydrogen bonds nor purine and pyrimidine structures are required to form a base pair with high efficiency and selectivity. These findings confirm that steric complementarity is an important factor in the fidelity of DNA synthesis.
Noncovalent interactions between aromatic nucleobases are the major stabilizing forces which contribute to the structural integrity of duplex DNA and RNA.1 -3 However, base pairing in nucleic acids is a consequence of more than just hydrogen bonding alone. The predictive success of nearest neighbor analysis,4 along with "dangling base" measurements which show duplex stabilization in the absence of pairing, 5 points out the importance of stacking interactions to duplex integrity. Despite the existing work on nucleic acid stability, however, it is not yet known whether hydrogen bonds are absolutely required for stabilization of a base pair (bp). To test this question, we have designed and synthesized a series of non-hydrogenbonding nucleoside analogues to probe these fundamental interactions. 6 We report herein the finding of the first stable non-hydrogen-bonded base pair, and its selective formation relative to mismatched pairs.Prior studies on the base pairing properties of nonpolar nucleoside analogues have almost invariably shown that pairs with natural DNA bases are strongly destabilizing. 7 When both members of a base pair are nonpolar, the destabilization is lessened somewhat; 7a however, one study found that a nonpolar-nonpolar pair still resulted in net destabilization of a 12-bp duplex by 2.9-4.0 kcal mol −1 (9-14 °C in T m ) relative to an A-T base pair. 7a Possible reasons for this are the lack of hydrogen bonds between the bases or the imperfect steric fit of the specific pairs studied in the context of duplex B-form DNA. We hypothesized that optimization of the steric fit of the nonpolar base pair within the duplex, combined with the use of strongly stacking groups, might enable selective pairing without compromising duplex stability.Models of B-form DNA suggested that a recently described pyrene nucleoside analogue (1) 8 is sterically large enough to fit well against model abasic site 9 2 (also denoted φ) (Figure 1). This combination yields a single contiguous π-system spanning nearly the entire distance between strands that would normally be occupied by two nucleobases. We synthesized 12mer duplexes containing 1 and 2 and measured their thermodynamic stabilities in aqueous buffer (Table 1) with thermal denaturation studies. To evaluate pairing selectivity, 1 was also paired against the four natural bases as well as itself, and abasic nucleoside 2 was paired against each of the four natural bases.Supporting Information Available: Experimental details, sample melting curves, plots of thermodynamic data, Jobs plot, and CD spectra (5 pages, print/PDF). See any current masthead page for ordering information and Web access instructions. Thermodynamic parameters (25 °C) were determined from van't Hoff plots using at least five different concentrations for each duplex. Under the conditions described above, control duplex 3, containing an A-T pair at the central position, has a T m (5 μM) of 43.2 °C with a corresponding free energy of −12.3 kcal mol −1 . When A is paired with the abasic site, the result i...
5,6-Dihydrothymid-5-yl (4) is generated via Norrish type I cleavage of isopropyl ketone 7. Ketone 7 was site specifically incorporated into chemically synthesized polythymidylates and an oligonucleotide containing all four native deoxyribonucleotides. No damage is induced in oligonucleotides containing 7 upon photolysis under anaerobic conditions. In the presence of O2, strand breaks and alkaline labile lesions are formed at the original site of 7, and at nucleotides adjacent to the 5‘-phosphate of 7. Kinetic isotope effect experiments reveal that direct strand scission at the thymidine adjacent to the 5‘-phosphate of 4 arises from C1‘ hydrogen atom abstraction. The observed KIE (∼3.9) is attributed to hydrogen atom abstraction from C1‘ by the peroxyl radical 35 derived from 4. Enzymatic end group analysis and measurement of free base release are consistent with a process involving C1‘ hydrogen atom abstraction. Cleavage experiments carried out in the presence of t-BuOH (1.05 M) and NaN3 (10 mM) indicate that damage does not result from hydroxyl radical, but that 1O2 is responsible for a significant amount of the observed strand damage.
We have designed, synthesized, and evaluated using physical, chemical and biochemical assays various oligonucleotide N3'-->P5' phosphoramidates, as potential telomerase inhibitors. Among the prepared compounds were 2'-deoxy, 2'-hydroxy, 2'-methoxy, 2'-ribo-fluoro, and 2'-arabino-fluoro oligonucleotide phosphoramidates, as well as novel N3'-->P5' thio-phosphoramidates. The compounds demonstrated sequence specific and dose dependent activity with IC50 values in the sub-nM to pM concentration range.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.