There are many methods for the generation of a hairpin, including inverted repeats, RCR plasmids, and conjugation. These methods require only one strand of DNA, but both strands must be present for conjugation to occur. These methods are also limited by strand selectivity, because they produce the same hairpin on top and bottom strands, as well as bulges and top strands.
Inverted repeats
Inverted repeats are structures in DNA that are capable of forming secondary structures. Many of these repeats are involved in mutagenesis, gross chromosomal rearrangements, and mutation. Inverted repeats are among the most well-studied DNA motifs. These motifs occur in both prokaryotic and eukaryotic genomes and can form DNA hairpins or cruciforms when double-stranded. Studies have shown that inverted repeats can impede DNA replication in vitro and induce genomic instability in a wide range of organisms.
This type of cloning is not always successful, however. One way to select the best repeats is to use multiple cloning sites. Multiple cloning sites make it easier to determine the orientation of inserts, as well as to shuttle inverted repeat cassettes. However, this method requires an additional step called shuttle. This is time-consuming, but can be highly effective if multiple inverted repeats are desired.
Unconventional base pairing
The present study examined the structure of the hairpin structure in DNA by performing cMD simulations on folded and single-stranded DNA sequences. The phosphorus atoms in the hairpin structure were subjected to a quadratic restraining potential, and the reference distance was increased in 0.5 A steps up to a total of 28 A. A total of 3750 snapshots were analyzed in order to find the optimal folding and unfolding parameters.
The protonatable 5′-position of the hairpin appears to function as an encapsidation initiation signal. This feature is shared by the Tymovirus genus. In addition to exhibiting a decreased Tm in an acidic environment, the hairpin sequence is accompanied by a novel type of RNA-protein interaction involving protonatable bases. These findings provide a better understanding of how a hairpin sequence interacts with proteins and RNA.
Transcription bubble
RNA polymerase unwinds the elongation complex by binding the 3′ end of the red strand to the active site and forming a 8-bp RNA-DNA hybrid. The remainder of the strand exits polymerase toward the “back” of the structure. The green non-template strand of DNA cannot reanneal with the blue template strand. In this way, the RNA locks open the transcription bubble and releases the polymerase.
In addition, this study showed that the T7RNAP intercalating the hairpin is essential for stabilizing the opened promoter during initiation. However, this protein does not contribute to the structure or stability of the transcription bubble. Thus, this protein is not required for the formation of transcription bubbles. Hairpin transcription is a crucial step in gene expression and is an essential component in the expression of certain genes. Therefore, this protein plays a critical role in the initiation of RNA synthesis.
Recombination events
Molecular analyses of hairpin recombination events show that the opening and closing of the loop are essential for gene transcription. This process is similar to transpositional recombination and uses the free 3′ hydroxyl of one strand to attack the opposing strand with a nucleophilic attack. The resulting covalently sealed hairpin intermediate is known as the hairpin coding end. It is believed that the opening and closing of this intermediate are critical for generating junctional diversity and have been the subject of intensive analysis in recent years.
Recombination events in hairpin RNAs can result in the synthesis of novel RNA molecules. One mutant of RAG1 shows impaired hairpin formation. Recombination events in hairpin RNAs may have evolved through the exchange of sequence patches between RNA molecules. In vitro reactions have shown that RAG proteins play an important role in the opening of hairpins, as they are involved in the cleavage and ligation of two different RNA molecules. Mutants of RAG1 and RAG2 are active in DNA cleavage and inhibit coding joint formation.