Characterization of Single Stranded Binding Protein’s Nucleic Acid Binding Requirements


Protein-nucleic acid interactions are critical during all phases of the life of a cell. Among the proteins that bind nucleic acids are those that preferentially bind to single stranded DNA. One such protein, the single stranded DNA binding protein (SSB) from E. coli is involved in cellular reproduction and maintenance of genetic information (Krauss et al. 1981). In this application note we demonstrate the capability of the BiOptix 404Pi™ to analyzing protein-oligonucleotide interactions in real time and without the need of prior molecular labeling (Meyer and Laine, 1990; Kozlov and Lohman, 2002).

Materials and Methods

Biotinylated and non-biotinylated oligonucleotides were purchased from Integrated DNA Technologies. Oligonucleotide sequences are listed in Table 1. SSB was purchased from Epicentre Biotechnologies. BiOptix SA-150™ (Streptavidin) SensorChips were used. All binding studies were performed in PBS at 20˚ C.

Oligonucleotide Name



Comparing SSB binding affinity for double stranded and single stranded oligonucleotides

An experiment was performed to demonstrate the binding specificity of SSB for single stranded DNA (ssDNA) versus double stranded DNA (dsDNA). Biotinylated oligonucleotide 4511 was first immobilized by injection onto a BiOptix SA-150™ SensorChip. dsDNA was generated by injecting the complementary 7434 oligonucleotide over the same SensorChip. As expected, when SSB was injected onto a SensorChip containing dsDNA, no significant binding was observed (<30 RUs). Denaturation of the dsDNA with 20mM NaOH for 20 sec, followed by a subsequent injection of SSB resulted in an instrument response of around 2000 RUs. (Figure 1). The sigmoidal-like curve shape observed at the higher concentrations is consistent with the cooperative binding modality of this protein (Meyer and Laine, 1990).

Download the Full Application Note for Complete Results, Data and Figures