Despite advances in understanding the theory behind RNA folding, ab initio prediction of the folding process has not been achieved yet. Given only the sequence information we still cannot tell the precise three-dimensional structure of neither RNA nor protein. Knowing the kinetics of folding we hope to learn more about the arrangement of secondary and tertiary structure.For that reason we investigated the folding process of RNA pseudoknot VPK with our microfluidic mixing device. VPK (variant pseudoknot) is a variant of the mouse mammary tumor virus (MMTV) pseudoknot and it was specifically designed to prevent the formation of alternative base pairings in the stem regions. Using two differently labeled samples, VPK-2AP and F-VPK, and high and low salt folding conditions, we analyzed the folding process and determined the different folding rates. Our results match very well with recently published findings, but they also raise the possible existence of folding transitions not seen in T-jump experiments. The measured folding times are in the range of 0.5 to several milliseconds.The folding process seems to have different pathways with one of the stems of the pseudoknot forming before, perhaps even initiating the folding of the other.
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