Ribosomal frameshifting is a rare but ubiquitous process that is being studied extensively. Meanwhile, frameshifting motifs without any secondary mRNA structures were identified but rarely studied experimentally. We report unambiguous observation of highly efficient “–1” and “–2” frameshiftings on a GA7G slippery mRNA without the downstream secondary structure, using force-induced remnant magnetization spectroscopy combined with unique probing schemes. The result represents the first experimental evidence of multiple frameshifting steps. It is also one of the rare reports of the “–2” frameshifting. Our assay removed the ambiguity of transcriptional slippage involvement in other frameshifting assays. Two significant insights for the frameshifting mechanism were revealed. First, EF-G·GTP is indispensable to frameshifting. Although EFG·GDPCP has been shown to prompt translocation before, we found that it could not induce frameshifting. This implies that the GTP hydrolysis is responsible for the codon–anticodon re-pairing in frameshifting, which corroborates our previous mechanical force measurement of EF-G·GTP. Second, translation in all three reading frames of the slippery sequence can be induced by the corresponding in-frame aminoacyl tRNAs. Although A-site tRNA is known to affect the partition between “0” and “–1” frameshifting, it has not been reported that all three reading frames can be translated by their corresponding tRNAs. The in vitro results were confirmed by toe-printing assay and protein sequencing.

Multivalent interactions remain difficult to be characterized and consequently controlled, particularly on a macroscopic scale. Using force-induced remnant magnetization spectroscopy (FIRMS), we have resolved the single-, double-, and triple-biotin–streptavidin interactions, multivalent DNA interactions and CXCL12–CXCR4 interactions on millimetre-scale surfaces. Our results establish FIRMS as a viable method for systematic resolution and controlled formation of multivalent interactions.

Molecule-specific noncovalent bonding on cell surfaces is the foundation for cellular recognition and functioning. A major challenge in probing these bonds is to resolve the specific bonds quantitatively and efficiently from the nonspecific interactions in a complex environment. Using force-induced remnant magnetization spectroscopy (FIRMS), we were able to resolve quantitatively three different interactions for magnetic beads bearing anti-CD4 antibodies with CD4+ T cell surfaces based upon their binding forces. The binding force of the CD4 antibody–antigen bonds was determined to be 75 ± 3 pN. For comparison, the same bonds were also studied on a functionalized substrate surface, and the binding force was determined to be 90 ± 6 pN. The 15 pN difference revealed by high-resolution FIRMS illustrates the significant impact of the bonding environment. Because the force difference was unaffected by the cell number or the receptor density on the substrate, we attributed it to the possible conformational or local environmental differences of the CD4 antigens between the cell surface and substrate surface. Our results show that the high force resolution and detection efficiency afforded by FIRMS are valuable for studying protein–protein interactions on cell surfaces.

The resolution of molecular bonds and subsequent selective control of their binding are of great significance in chemistry and biology. We have developed a method based on the use of acoustic radiation force to precisely dissociate noncovalent molecular bonds. The acoustic radiation force is produced by extremely low-power ultrasound waves and is mediated by magnetic particles. We successfully distinguished the binding of antibodies of different subclasses and the binding of DNA duplexes with a single-base-pair difference. In contrast to most ultrasound applications in chemistry, the sonication probe is noninvasive and requires a sample volume of only a few microliters. Our method is thus viable for noninvasive and accurate control of molecular bonds that are widely encountered in biochemistry.

We report a technique that is based on exchange-induced remnant magnetization for microRNA (miRNA) detection. In sequence-specific exchange reactions between label-free miRNA and magnetically labelled RNA with one mismatched base, the decrease in magnetization quantitatively represents the target miRNA. The detection limit reaches the zeptomole regime, with no amplification or washing procedures. Therefore, our technique will be suitable for precise miRNA profiling to aid in early diagnosis of cancers.

The specific binding between the two DNA strands in a double helix is one of the most fundamental and influential molecular interactions in biochemistry. Using force-induced remnant magnetization spectroscopy (FIRMS), we obtained well-defined binding forces of DNA oligomers, with a narrow force distribution of 1.8 pN. The narrow force distribution allows for directly resolving two DNA duplexes with a single base-pair difference in the same sample. Therefore, binding force can serve as a discriminating parameter for probing different DNA interactions. Furthermore, we observed that the binding forces depend on the position of the mismatching base pair. Our results show that FIRMS is capable of high-precision mechanical measurements of biochemical processes involving multiple DNA interactions and has the potential for characterizing the binding strength of materials based on DNA origami.

Specific noncovalent binding between antibody and antigen molecules is the basis for molecular recognition in biochemical processes. Quantitative investigation of the binding forces could lead to molecular specific analysis and potentially mechanical manipulation of these processes. Using our force-induced remnant magnetization spectroscopy, we revealed a well-defined binding force for the bonds between mouse immunoglobulin G and magnetically labeled α-mouse immunoglobulin G. The force was calibrated to be 120 ± 15 pN. In comparison, the binding force was only 17 ± 3 pN for physisorption and much higher than 120 pN for biotin–streptavidin bonds. A unique rebinding method was used to confirm the dissociation of the antibody–antigen bonds. A well-defined and molecule-specific binding force opens a new avenue for distinguishing different noncovalent bonds in biochemical processes.

We report a technique that is based on exchange-induced remnant magnetization for microRNA (miRNA) detection. In sequence-specific exchange reactions between label-free miRNA and magnetically labelled RNA with one mismatched base, the decrease in magnetization quantitatively represents the target miRNA. The detection limit reaches the zeptomole regime, with no amplification or washing procedures. Therefore, our technique will be suitable for precise miRNA profiling to aid in early diagnosis of cancers.

The resolution of molecular bonds and subsequent selective control of their binding are of great significance in chemistry and biology. We have developed a method based on the use of acoustic radiation force to precisely dissociate noncovalent molecular bonds. The acoustic radiation force is produced by extremely low-power ultrasound waves and is mediated by magnetic particles. We successfully distinguished the binding of antibodies of different subclasses and the binding of DNA duplexes with a single-base-pair difference. In contrast to most ultrasound applications in chemistry, the sonication probe is noninvasive and requires a sample volume of only a few microliters. Our method is thus viable for noninvasive and accurate control of molecular bonds that are widely encountered in biochemistry.

Multivalent interactions remain difficult to be characterized and consequently controlled, particularly on a macroscopic scale. Using force-induced remnant magnetization spectroscopy (FIRMS), we have resolved the single-, double-, and triple-biotin–streptavidin interactions, multivalent DNA interactions and CXCL12–CXCR4 interactions on millimetre-scale surfaces. Our results establish FIRMS as a viable method for systematic resolution and controlled formation of multivalent interactions.