With the help of magnetic tweezers we will unwind a large palindromic DNA, generating a cruciform structure. We will then pull on that molecule with optical tweezers and measure the force required to break successive bonds (between 20 and 30 pN). Because the extent of the resulting denaturation bubble, i.e. the length of the single strands, is controlled by amount of coiling, we will be able to control the amount of elastic energy they store and should in principle be able to achieve the sensitivity required for the detection of the breaking of a single base-pair.  Preparation of proteins and DNA substrates.  The attachment of these substrates to suitably derivatised AFM tips, or surfaces, will be confirmed using local facilities.  Force measurement of the same phenomena using the same palindromic DNA, but with a calibrated AFM system (see Workpackage 2), will allow confirmation of the accuracy of the force measurements

Outcomes:

A number of palindromic constructs have been engineered. Some were used in the study of the RuvAB motor that displaces Holliday junctions. Others are characterized for their formation of tertiary DNA structures, in particular, in conjunction with certain genomic instabilities and diseases.  We, however, dropped the project of using these constructs to probe the possibility of mechanically sequencing DNA, since recent theoretical and experimental results (see below) suggest that this method maybe plagued by serious artefacts (i.e. metastable states).

Deliverable terminated.