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 ATPgammaS. 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 ATPgammaS 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.

Figure 1.

(A) Pseudocomplementary pairing of nA and sT. (B) A double D-loop joint, with each strand identified according to its role in RecA-mediated joint formation. (C) Formation of a double D-loop joint with pseudocomplementary ONs (pcONs; in red) creates additional base pairs. Formation of the same joint with regular ONs (rONs; in black) does not alter the number of base pairs. (D) Incoming and annealing oligonucleotides used in this study for strand exchange and double D-loop joint formation. A and T bases in red are replaced by nA and sT analogs in pseudocomplementary versions of the respective ONs. The T/sT bases in annealing ONs with a 2′-OMe RNA backbone were attached to 2′-deoxyribose sugars. DNA duplexes were formed by hybridizing an incoming DNA 30-mer, 45-mer or 70-mer to a complement of the same length. Specificity of joint molecule formation was determined using 70-bp DNA targets that were mismatched to the incoming/annealing ONs.

Figure 2.

Gel mobility shift analysis of hybridization between incoming and annealing ONs. Complementary ONs (80 nM radiolabeled incoming ON and 640 nM annealing ON) were incubated 10 min at 37°C in SEB. In panel A, aliquots were directly analyzed by PAGE in a gel preequilibrated at 37°C. In panel B, reactions were quenched by adding 6.4 μM competitor ON and then placed in an ice bath until analyzed by PAGE at 8°C. The base composition of each ON (r = regular bases; pc = substitution with nA and sT bases) is indicated above each lane.

Figure 3.

RecA-mediated strand exchange of DNA substituted with nA and sT in the presence of ATPγS. Exchange was monitored using an incoming 30-mer DNA and a homologous 30-bp DNA duplex that was radiolabeled in the outgoing strand. The base composition of each strand (r = regular bases; pc = substitution with nA and sT bases) is indicated above each lane.

Figure 4.

RecA-mediated double D-loop formation in the presence of ATPγS. Different combinations of incoming and annealing ONs with regular (r) or pseudocomplementary (pc) bases were tested for joint formation with radiolabeled 70-bp DNA target using a three-step reaction protocol. A sequential hybridization reaction is analyzed in the last lane on the right.

Figure 5.

Comparison of RecA-mediated double D-loop formation using one-, two- and three-step reaction protocols with ATPγS. Pseudocomplementary incoming and annealing ONs were used to target radiolabeled 70-bp DNA duplex. The fast moving band in some of the lanes is a hybrid between the outgoing DNA 70-mer and the indicated annealing ON.

Figure 6.

Specificity of RecA-mediated double D-loop formation. Joints were formed using the three-step reaction protocol with pseudocomplementary incoming and annealing ONs and ATPγS. In panel A, the altered 70-bp DNA targets were mismatched to both the ONs at one, two or three positions. In panel B, the altered 70-bp DNA target contained a mismatched base pair such that the annealing–outgoing arm of the double D-loop joint was perfectly matched, while the incoming–recipient arm contained a single mismatch.

Figure 7.

RecA-mediated pairing in the presence of ATP. Strand exchange and double D-loop formation were conducted in the presence of ATPγS, ATP or an ATP regenerating system. Strand exchange was conducted with regular incoming ONs whereas joint molecules were formed with pseudocomplementary incoming and annealing ONs.

Figure 8.

Stability of double D-loop joints at 37°C in PB buffer. The joints were prepared by sequential hybridization between regular or pseudocomplementary incoming and annealing DNA 30-mers and complementary 70-mer DNA strands.