Studying the influence of stem composition in pH-sensitive molecular beacons onto their sensing properties

Intracellular sensing using fluorescent molecular beacons is a potentially useful strategy for real-time, in vivo monitoring of important cellular events. This work is focused on evaluation of pyrene excimer signaling molecular beacons (MBs) for the monitoring of pH changes in vitro as well as inside living cells. The recognition element in our MB called pHSO (pH-sensitive oligonucleotide) is the loop enclosing cytosine-rich fragment that is able to form i-motif structure in a specific pH range. However, alteration of a sequence of the 6 base pairs containing stem of MB allowed the design of pHSO probes that exhibited different dynamic pH range and possessed slightly different transition midpoint between i-motif and open loop configuration. Moreover, this conformational transition was accompanied by spectral changes showing developed probes different pyrene excimer-monomer emission ratio triggered by pH changes. The potential of these MBs for intracellular pH sensing is demonstrated on the example of HeLa cells line.