RecA-mediated strand invasion of DNA by oligonucleotides substituted with 2-aminoadenine and 2-thiothymine

Abstract

Sequence-specific recognition of DNA is a critical step in gene targeting. Here we describe unique oligonucleotide (ON) hybrids that can stably pair to both strands of a linear DNA target in a RecA-dependent reaction with ATP or ATPγS. One strand of the hybrids is a 30-mer DNA ON that contains a 15-nt-long A/T-rich central core. The core sequence, which is substituted with 2-aminoadenine and 2-thiothymine, is weakly hybridized to complementary locked nucleic acid or 2′-OMe RNA ONs that are also substituted with the same base analogs. Robust targeting reactions took place in the presence of ATPγS and generated metastable double D-loop joints. Since the hybrids had pseudocomplementary character, the component ONs hybridized less strongly to each other than to complementary target DNA sequences composed of regular bases. This difference in pairing strength promoted the formation of joints capable of accommodating a single mismatch. If similar joints can form in vivo, virtually any A/T-rich site in genomic DNA could be selectively targeted. By designing the constructs so that the DNA ON is mismatched to its complementary sequence in DNA, joint formation might allow the ON to function as a template for targeted point mutation and gene correction.

Role of the Heat Capacity Change in Understanding and Modeling Melting Thermodynamics of Complementary Duplexes Containing Standard and Nucleobase-Modified LNA

“…Duplexes containing D-H base pairs have a higher T max than their reference duplex (a-t) but still exhibit net lower stability compared to the stabilities of isosequences containing their three LNA-LNA base pair analogues (A-T, D-T, and A-H). The D-H base pair (as well as the d-h, d-H, and D-h base pairs) is therefore pseudocomplementary because of steric hindrance (Figure ) between the 2-amino group in d or D and the 2-thioketo group in h or H that serves to reduce base pair stability. ,,, This could prove to be useful in certain applications, as D-T and H-A base pairs are very stable (Table ) because of the absence of these steric hindrance effects. As a result, oligonucleotides containing A and T, A and H, and/or D and T must be designed carefully, especially in regions of self-complementary, to ensure that highly stable secondary products are not formed.…”

“…We also report DSC-derived melting thermodynamics data and SBT model parameters for the modified nucleotides LNA-2-aminoadenine (D) and LNA-2-thiothymine (H) − and show that duplexes substituted with D and/or H bases are more stable than their isosequential duplexes bearing A and/or T, respectively. Finally, we demonstrate that the D-H base pair (Figure ) is pseudocomplementary, a property that can be exploited in the functional design of oligonucleotides to minimize the formation of undesired stable secondary structures. − …”

Establishing broad generality of DNA catalysts for site-specific hydrolysis of single-stranded DNA

“…Such separation may be possible via suitable strand invasion strategies, whereupon each of the two single-stranded components would then be cleavable by a corresponding deoxyribozyme. However, such strand invasion is experimentally challenging and often requires chemically sophisticated approaches ( 36–39 ). Alternatively, an intact dsDNA substrate can be targeted for cleavage, but achieving this objective will require an entirely different basis for substrate–catalyst interactions that does not rely upon Watson-Crick base pairing of the type shown in Figure 1 (e.g.…”

Nucleotides and Nucleic Acids; Oligo- and Polynucleotides

“…However, when the pcDNA is hybridised to normal DNA it is significantly more thermally stable than even the corresponding DNA duplex. 182 Several different modified nucleotides were also examined to find pcDNA equivalents for the G:C base pair, the most promising being either the 7-nitro-7-deazaguanine and 2-thiocytosine, 308 or 7-alkyl-7deazaguanine and N4-alkylcytosine 309 analogues.…”