and the anti-parallel fashion of TFO binding, TFOs composed of pyrimidines (C / T) bind to the purine-rich strand of the target double helix via Hoogsteen hydrogen bonds in a parallel fashion. TFOs composed of purines (A / G), or mixed purine and pyrimidine bind to the same purine-rich strand via reverse Hoogsteen bonds in an anti-parallel fashion. TFO's can recognize purine-rich target strands for duplex DNA. Complications and limitations In order for TFO motifs to bind in a parallel fashion and create hydrogen bonds, the nitrogen atom at position 3 on the cytosine residue needs to be protonated, but at physiological pH levels it is not, which could prevent parallel binding. Another limitation is that TFOs can only bind to purine-rich target strands and this would limit the choice of endogenous gene target sites to polypurine-polypyrimidine stretches in duplex DNA. If a method to also allow TFOs to bind to pyrimidine bases was generated, this would enable TFOs to target any part of the genome. Also the human genome is rich in polypurine and polypyrimidine sequences which could affect the specificity of TFO to bind to a target DNA region. An approach to overcome this limitation is to develop TFOs with modified nucleotides that act as locked nucleic acids to increase the affinity of the TFO for specific target sequences. Other limitations include concerns regarding binding affinity and specificity, in vivo stability, and uptake into cells. Researchers are attempting to overcome these limitations by improving TFO characteristics through chemical modifications, such as modifying the TFO backbone to reduce electrostatic repulsions between the TFO and the DNA duplex. Also due to their high molecular weight, uptake into cells is limited and some strategies to overcome this include DNA condensing agents, coupling of the TFO to hydrophobic residues like cholesterol, or cell permeabilization agents. What can they do Scientists are still refining the technology to turn TFOs into a therapeutic product and much of this revolves around their potential applications in antigene therapy. In particular they have been used as inducers of site-specific mutations, reagents that selectively and specifically cleave target DNA, and as modulators of gene expression. One such gene sequence modification method is through the targeting DNA with TFOs to active a target gene. If a target sequence is located between two inactive copies of a gene, DNA ligands, such as TFOs, can bind to the target site and