Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

Ligands that stabilize the formation of telomeric DNA G-quadruplexes have potential as cancer treatments, because the G-quadruplex structure cannot be extended by telomerase, an enzyme over-expressed in many cancer cells. Understanding the kinetic, thermodynamic and mechanical properties of small-molecule binding to these structures is therefore important, but classical ensemble assays are unable to measure these simultaneously. Here, we have used a laser tweezers method to investigate such interactions. With a force jump approach, we observe that pyridostatin promotes the folding of telomeric G-quadruplexes. The increased mechanical stability of pyridostatin-bound G-quadruplex permits the determination of a dissociation constant Kd of 490 ± 80 nM. The free-energy change of binding obtained from a Hess-like process provides an identical Kd for pyridostatin and a Kd of 42 ± 3 μM for a weaker ligand RR110. We anticipate that this single-molecule platform can provide detailed insights into the mechanical, kinetic and thermodynamic properties of liganded bio-macromolecules, which have biological relevance.

The insulin linked polymorphism region (ILPR) is known to regulate transcription of the gene coding for insulin. The ILPR has guanine rich segments, suggesting that G quadruplexes may be responsible for this regulatory role. Using mechanical unfolding in a laser tweezers instrument and circular dichroism (CD) spectroscopy, we provide compelling evidence that highly stable parallel and antiparallel G quadruplex structures coexist in the predominant ILPR sequence of (ACAGGGGTGTGGGG)(2) at a physiologically relevant concentration of 100 mM KCl. Experiments at the single molecular level have shown that unfolding forces for parallel and antiparallel structures (F(unfold): 22.6 vs 36.9 pN, respectively) are higher than the stall forces of enzymes having helicase activities. From a mechanical perspective alone, these data support the hypothesis that G quadruplexes may cause replication slippage by blocking replication process. Using the unique combination of the rupture force and the contour length measured by laser tweezers, the simultaneous determination of probable parallel and antiparallel G quadruplex structures in a solution mixture has been achieved. Jarzynski's equality analysis has revealed that the antiparallel G quadruplex is thermodynamically more stable than the parallel conformer (DeltaG (unfold): 23 vs 14 kcal/mol, respectively). On the other hand, kinetic measurements have indicated that both parallel and antiparallel structures fold rather rapidly (k(fold): 0.4 vs 0.3 s(-1), respectively), suggesting that they may be kinetically accessible for gene control. This work provides an unprecedented mechanical perspective on G quadruplex stability, presenting a unique opportunity to predict the functional consequence when motor enzymes encounter such structures.

Investigation of i-motif is of high importance to fully understand the biological functions of G quadruplexes in the context of double stranded DNA. Whereas single molecule approaches have profiled G quadruplexes from a perspective unavailable by bulk techniques, there is a lack of similar literature on the i-motif in the cytosine (C) rich region complementary to G quadruplex forming sequences. Here, we have used laser tweezers to investigate the structures formed in 5′-(TGTCCCCACACCCC)2, a predominate variant in the insulin linked polymorphic region (ILPR). We have observed two species with the change in contour length (ΔL) of 10.4 (±0.1) and 5.1 (±0.5) nm, respectively. Since ΔL of 10.4 nm is located within the expected range for an i-motif structure, we assign this species to the i-motif. The formation of the i-motif in the same sequence has been corroborated by bulk experiments such as Br2 footprinting, circular dichroism, and thermal denaturation. The assignment of the i-motif is further confirmed by decreased formation of this structure (23 % to 1.3 %) with pH 5.5 7.0, which is a well established behavior for i-motifs. In contrast to the i-motif, the formation of the second species with ΔL of 5.1 nm remains unchanged (6.1±1.6 %) in the same pH range, implying that pH sensitive C:CH+ pairs may not contribute to the structure as significantly as those to the i-motif. Compared to the ΔGunfold of i-motif (16.0 ±0.8 kcal/mol), the decreased free energy in the partially folded structure (ΔGunfold 10.4 ± 0.7 kcal/mol) may reflect a weakened structure with reduced C:CH+ pairs. Both ΔL and ΔGunfold argue for the intermediate nature of the partially folded structure in comparison to the i-motif. In line with this argument, we have directly observed the unfolding of i-motif through the partially folded structure. The i-motif and the partially folded structure share similar rupture forces of 22-26 pN, which are higher than those that can stall transcription catalyzed by RNA polymerases. This suggests, from a mechanical perspective alone, that either of the structures can stop RNA transcription.

Recent experiments provided controversial observations that either parallel or non-parallel G-quadruplex exists in molecularly crowded buffers that mimic cellular environment. Here, we used laser tweezers to mechanically unfold structures in a human telomeric DNA fragment, 5′-(TTAGGG)4TTA, along three different trajectories. After the end-to-end distance of each unfolding geometry was measured, it was compared with PDB structures to identify the best-matching G-quadruplex conformation. This method is well-suited to identify biomolecular structures in complex settings not amenable to conventional approaches, such as in a solution with mixed species or at physiologically significant concentrations. With this approach, we found that parallel G-quadruplex coexists with non-parallel species (1:1 ratio) in crowded buffers with dehydrating cosolutes [40% w/v dimethyl sulfoxide (DMSO) or acetonitrile (ACN)]. In crowded solutions with steric cosolutes [40% w/v bovine serum albumin (BSA)], the parallel G-quadruplex constitutes only 10% of the population. This difference unequivocally supports the notion that dehydration promotes the formation of parallel G-quadruplexes. Compared with DNA hairpins that have decreased unfolding forces in crowded (9 pN) versus diluted (15 pN) buffers, those of G-quadruplexes remain the same (20 pN). Such a result implies that in a cellular environment, DNA G-quadruplexes, instead of hairpins, can stop DNA/RNA polymerases with stall forces often <20 pN.

G-quadruplex has demonstrated its biological functions in vivo. Although G-quadruplex in single-stranded DNA (ssDNA) has been well characterized, investigation of this species in double-stranded DNA (dsDNA) lags behind. Here we use chemical footprinting and laser-tweezers-based single-molecule approaches to demonstrate that a dsDNA fragment found in the insulin-linked polymorphic region (ILPR), 5'-(ACA GGGG TGT GGGG)2 TGT, can fold into a G-quadruplex at pH 7.4 with 100 mM K+, and an i-motif at pH 5.5 with 100 mM Li+. Surprisingly, under a condition that favors the formation of both G-quadruplex and i-motif (pH 5.5, 100 mM K+), a unique determination of change in the free energy of unfolding (ΔGunfold) by laser-tweezers experiments provides compelling evidence that only one species is present in each dsDNA. Under this condition, molecules containing G-quadruplex are more stable than those with i-motif. These two species have mechanical stabilities (rupture force≥17 pN) comparable to the stall force of RNA polymerases, which, from a mechanical perspective alone, could justify a regulatory mechanism for tetraplex structures in the expression of human insulin.

In nature, the catalytic efficiency of multienzyme complexes highly depends on their spatial organization. The positions and orientations of the composite enzymes are often precisely controlled to facilitate substrate transport between them. Self-assembled DNA nanostructures hold great promise for organizing biomolecules at the nanoscale. Here, we present detailed protocols for exploiting DNA nanostructures as assembly scaffolds that organize the spatial arrangements of multienzyme cascades with control over their relative distance, compartmentalization and substrate diffusion paths. The protocol describes the preparation and purification of DNA-conjugated enzymes and cofactors, along with the assembly of these prepared complexes on DNA nanostructures. The architecture of assembled enzyme complexes is then readily characterized using a broad selection of techniques from routine gel electrophoresis to advanced single-molecule imaging. We also describe methods of purifying these nano-assemblies and testing them with functional assays based on either bulk or single-molecule fluorescence measurements. The entire assembly and characterization of a multienzyme complex can be completed within 1-2 weeks.

Biological motors are highly complex protein assemblies that generate linear or rotary motion, powered by chemical energy. Synthetic motors based on DNA nanostructures, bio-hybrid designs, or synthetic organic chemistry have been assembled. However, unidirectionally rotating biomimetic wheel motors with rotor-stator units that consume chemical energy are elusive. Here we report a bio-hybrid nanoengine consisting of a catalytic stator that unidirectionally rotates an interlocked DNA wheel, powered by NTP hydrolysis. The engine consists of an engineered T7 RNA polymerase (T7RNAP-ZIF) attached to a double-stranded (ds)DNA nanoring that is catenated to a rigid rotating dsDNA wheel. The wheel motor produces long, repetitive RNA transcripts that remain attached to the engine and are used to guide its movement along predefined single-stranded (ss)DNA tracks arranged on a DNA nanotube. The simplicity of the design renders this walking nanoengine adaptable to other biological nanoarchitectures, facilitating the construction of complex bio-hybrid structures that achieve NTP-driven locomotion.