To investigate the ICP27 RNA binding specificity , HSV-1 RNA sequences that interact with ICP27 were first identified . A yeast three hybrid analysis identified 31 HSV-1 sense RNAs , mapping to 28 different open reading frames , which interacted specifically with ICP27 in vivo ( 26 ) . ICP27 was also found to specifically associate with seven HSV-1 RNAs in in vivo UV - crosslinking studies . The late RNA for the glycoprotein C ( gC ) gene was one of the RNAs that UV - cross - linked to ICP27 in infected cells ( 3 ) . To further refine the pools of possible RNA binding sequences , a yeast three - hybrid screen was performed with an RNA library generated from nucleotides 95 155 - 98 129 of the KOS HSV-1 genome , which encompasses two late genes , UL43 and UL44 ( gC ) . Sequences corresponding to both the mRNA coding and noncoding strands from UL43 and gC were identified in the yeast three hybrid screen as interacting with ICP27 ( data not shown ) . No obvious consensus sequences were identified in these sequences ; consequently EMSA was used to screen these sequences for those that bound to ICP27 with high affinity . A 294 - nt region of gC , corresponding to nucleotides 96 946 - 97 239 of the coding strand of the KOS HSV-1 gC mRNA , was chosen as an EMSA binding substrate . This region of the gC mRNA was chosen over the other sequences because it was within the coding region of the gC mRNA and was small enough to screen . Nineteen overlapping 30 - mer DNA oligonucleotides corresponding to coding strand of the gC RNA were used in EMSA experiments ( Figure 1C ) . RGG box motifs in other proteins recognize both RNA and DNA quadruplex structures ( 30,31 ) , so gC DNA was used instead of gC RNA for this initial screening to provide a more stable substrate for analysis . The ICP27 N - terminus was incubated with individual radiolabeled gC DNA 30mers at 37 C and resolved by nondenaturing acrylamide gel electrophoresis . Figure 2A shows a gC DNA sequence that the ICP27 N - terminus was able to shift its migration to a more slowly migrating band , gC 11 - 40 , and a second sequence , gC 31 - 60 , whose migration was not affected by addition of protein . Some sequences ' migration were moderately affected ( Figure 2B ; 121 - 150 ) , whereas most gC sequences were shifted well by the ICP27 N - terminus ( Figure 2B ; gC 1 - 30 , 91 - 120 and Figure 2C ; 61 - 90 . ) Of the gC DNA oligonucleotides tested , fourteen were shifted , two were shifted moderately and three showed no discernable shift ( Table 1 ) .