been used in human gene modulation. Limitations and workarounds The major structural drawback to unmodified SPAs as gene modulators is that their recognition sequence cannot be extended beyond 5 Watson-Crick base pairings. The natural curvature of the DNA minor groove is too tight a turn for the hairpin structure to match. There are several groups with proposed workarounds to this problem. SPAs can be made to better follow the curvature of the minor groove by inserting beta-alanine which relaxes the structure. Another approach to extending the recognition length is to use several short hairpins in succession. This approach has increased the recognition length to up to eleven Watson-Crick base pairs. Direct modulation SPAs may inhibit transcription through binding within a transcribed region of a target gene. This inhibition occurs through blocking of elongation by an RNA polymerase. SPAs may also modulate transcription by targeting a transcription regulator binding site. If the regulator is an activator of transcription, this will decrease transcriptional levels. As an example, SPA targeting to the binding site for the activating transcription factor TFIIIA has been demonstrated to inhibit transcription of the downstream 5S RNA. In contrast, if the regulator is a repressor, this will increase transcriptional levels. As an example, SPA targeting to the host factor LSF, which represses expression of the human immunodeficiency virus (HIV) type 1 long terminal repeat (LTR), blocks binding of LSF and consequently de-represses expression of LTR . Conjugate modulation SPAs have not been shown to directly modify DNA or have activity other than direct blocking of other factors or processes. However, modifying agents can be bound to the tail ends of the hairpin structure. The specific binding of the SPA to DNA allows for site-specific targeting of the conjugated modifying agent. SPAs have been paired with the DNA-alkylating moieties cyclopropylpyrroloindole and chlorambucil that were able to damage and crosslink SV40 DNA. This effect inhibited cell cycling and growth. Chlorambucil, a chemotherapeutic agent, was more effective when conjugated to an SPA than without. In 2012, SPAs were conjugated to SAHA, a potent histone deacetylase (HDAC) inhibitor. SPAs with conjugated SAHA were targeted to Oct-3/4 and Nanog which induced epigenetic remodeling and consequently increased expression of multiple pluripotency related genes in mouse embryonic fibroblasts. Designer zinc-finger proteins What they are/structure Designer zinc-finger proteins are engineered proteins used to target specific areas of DNA. These proteins capitalize on the DNA-binding capacity of natural