We investigate the elasticity of two types of single-stranded synthetic DNA homopolydeoxynucletides, poly(dA) and poly(dT), by AFM-based single-molecule force spectroscopy. We find that poly(dT) exhibits the expected entropic elasticity behavior, while poly(dA) unexpectedly displays two overstretching transitions in the force-extension relationship. We suggest that these transitions, which occur at $\sim 23\mathrm{pN}$ and $\sim 113\mathrm{pN}$, directly capture, for the first time, the mechanical signature of base-stacking interactions among adenines in DNA, in the absence of base pairing.

CRISPR-associated endonuclease Cas9 cuts DNA at variable target sites designated by a Cas9-bound RNA molecule. Cas9's ability to be directed by single ‘guide RNA’ molecules to target nearly any sequence has been recently exploited for a number of emerging biological and medical applications. Therefore, understanding the nature of Cas9's off-target activity is of paramount importance for its practical use. Using atomic force microscopy (AFM), we directly resolve individual Cas9 and nuclease-inactive dCas9 proteins as they bind along engineered DNA substrates. High-resolution imaging allows us to determine their relative propensities to bind with different guide RNA variants to targeted or off-target sequences. Mapping the structural properties of Cas9 and dCas9 to their respective binding sites reveals a progressive conformational transformation at DNA sites with increasing sequence similarity to its target. With kinetic Monte Carlo (KMC) simulations, these results provide evidence of a ‘conformational gating’ mechanism driven by the interactions between the guide RNA and the 14th–17th nucleotide region of the targeted DNA, the stabilities of which we find correlate significantly with reported off-target cleavage rates. KMC simulations also reveal potential methodologies to engineer guide RNA sequences with improved specificity by considering the invasion of guide RNAs into targeted DNA duplex.

The past years have witnessed remarkable advances in our use of atomic force microscopy (AFM) for stretching single biomolecules, thereby contributing to answering many outstanding questions in biophysics and chemical biology. In these single-molecule force spectroscopy (SMFS) experiments, the AFM tip is continuously approached to and retracted from the biological sample, while monitoring the interaction force. The obtained force–extension curves provide key insight into the molecular elasticity and localization of single molecules, either on isolated systems or on cellular surfaces. In this tutorial review, we describe the principle of such SMFS experiments, and we survey remarkable breakthroughs made in manipulating single polysaccharides and proteins, including understanding the conformational properties of sugars and controlling them by force, measuring the molecular elasticity of mechanical proteins, unfolding and refolding individual proteins, probing protein–ligand interactions, and tuning enzymatic reactions by force. In addition, we show how SMFS with AFM tips bearing specific bioligands has enabled researchers to stretch and localize single molecules on live cells, in relation with cellular functions.