Regulation of 2D DNA Nanostructures by the Coupling of Intrinsic Tile Curvature and Arm Twist

The overwinding and underwinding of duplex segments between junctions have been used in designing both left-handed and right-handed DNA origami nanostructures. For a variety of DNA tubes obtained from self-assembled tiles, only a theoretical approach of the intrinsic curvature of the DNA tile (specified as the intrinsic tile curvature) has been previously used to explain their formation. Details regarding the quantitative and structural descriptions of the tile curvature and its evolution in DNA tubes by the coupling of the twist of the inter-tile arm (specified as the arm twist) have never been addressed. In this work, we designed three types of tile cores built around a circular 128 nucleotide scaffold by using longitudinal weaving (LW), bridged longitudinal weaving (bLW) and transverse weaving (TW). Joining the tiles with inter-tile arms having the length of an odd number of DNA half-turns (termed O-tiling) almost resulted into planar 2D lattices, whereas joining the tiles with the arms having the length of an even number of DNA half-turns (termed E-tiling) nearly generated tubes. Streptavidin bound to biotin was used as a labeling technique to characterize the inside and outside surfaces of the E-tiling tubes and thereby the conformations of their component tiles with addressable concave and convex curvatures. When the arms have the normal winding at the relaxed B-form of DNA, the intrinsic tile curvature deter-mines the chirality of the E-tiling tubes. By regulating the arm length and the sticky end length of the bLW-Ep/q (E-tiling of the bLW cores with the arm length of p-bp and the sticky end length of q-nt) assemblies, the arm can be overwound, resulting in a left-handed twist, and can also be underwound, resulting in a right-handed twist. Chiral bLW-Ep/q tubes with either a right-handed curvature or a left-handed curvature can also be formed by the coupling of the intrinsic tile curvature and the arm twist. We were able to assign the chiral indices (n,m) to each tube using high-resolution AFM images, and therefore were able to estimate the tile curvature using a regular polygon model that approximated the transverse section of the tube. A deeper understanding of the integrated actions of dif-ferent types of twisting forces on the DNA tubes will be extremely helpful in engineering more elaborate DNA nanostructures in the future.

Restriction Enzyme Double Digestion v1

Enzyme digestion is to cut the DNA molecule and the carrier molecule at the sticky end to obtain the corresponding sticky end connection.

Regulation of 2D DNA Nanostructures by the Coupling of Tile Curvatures and Arm Twists

DNA overwinding and underwinding between adjacent Holliday junctions have been applied in DNA origami constructs to design both left-handed and right-handed nanostructures. For a variety of DNA tubes assembled from small tiles, only a theoretical approach of the intrinsic tile curvature was previously used to explain their formation. Details regarding the quantitative and structural descriptions of the intrinsic tile curvature and its evolution in DNA tubes by coupling with arm twists were missing. In this work, we designed three types of tile cores from a circular 128 nucleotide scaffold by longitudinal weaving (LW), bridging longitudinal weaving (bLW), and transverse weaving (TW) and assembled their 2D planar or tubular nanostructures via inter-tile arms with a distance of an odd or even number of DNA half-turns. The biotin/streptavidin (SA) labeling technique was applied to define the tube configuration with addressable inside and outside surfaces and thus their component tile conformation with addressable concave and convex curvatures. Both chiral tubes possessing left-handed and right-handed curvatures could be generated by finely tuning p and q in bLW-E_p/q designs (bLW tile cores joined together by inter-tile arms of an even number of half-turns with the arm length of p base pairs (bp) and the sticky end length of q nucleotides (nt)). We were able to assign the chiral indices (n,m) to each specific tube from the high-resolution AFM images, and thus estimated the tile curvature angle with a regular polygon model that approximates each tube’s transverse section. We attribute the curvature evolution of bLW-E_p/q tubes composed of the same tile core to the coupling of the intrinsic tile curvature and different arm twists. A better understanding of the integrated actions of different types of twisting forces on DNA tubes will be much more helpful in engineering DNA nanostructures in the future.

Regulation of 2D DNA Nanostructures by the Coupling of Tile Cur-Vatures and Arm Twists

DNA overwinding and underwinding between adjacent Holliday junctions have been applied in DNA origami constructs to design both left-handed and right-handed nanostructures. For a variety of DNA tubes assembled from small tiles, only an abstract concept of the intrinsic tile curvature was previously used to explain their formation. Details regarding the quantitative and structural descriptions of the intrinsic tile curvature and its evolution in DNA tubes by coupling with arm twists have been lacking. In this work, we designed three types of tile cores from a circular 128 nucleotide scaffold by longitudinal weaving (LW), bridging longitudinal weaving (bLW), and transverse weaving (TW) and assembled their 2D planar or tubular nanostructures via inter-tile arms with a distance of an odd or even number of DNA half-turns. The biotin/streptavidin (SA) labeling technique was applied to define the tube configuration with addressable inside and outside surfaces and thus their component tile conformation with addressable concave and convex curvatures. Both chiral tubes possessing left-handed and right-handed curvatures could be generated by finely tuning p and q in bLW-E_p/q designs (bLW tile cores joined together by inter-tile arms of even number of half-turns with the arm length of p base pairs (bp) and the sticky end length of q nucleotides (nt)). We were able to assign the chiral indices (n,m) to each specific tube from the high-resolution AFM images, and thus estimated the tile curvature angle with a regular polygon model that approximates each tube’s transverse section. We attribute the curvature evolution of bLW-E_p/q tubes composed of the same tile core to the coupling of the intrinsic tile curvature and different arm twists. A better understanding of integrated actions of different types of twisting forces on DNA tubes will be much more helpful in engineering DNA nanostructures in the future.

Regulation of 2D DNA Nanostructures by the Coupling of Tile Cur-Vatures and Arm Twists

DNA overwinding and underwinding between adjacent Holliday junctions have been applied in DNA origami constructs to design both left-handed and right-handed nanostructures. For a variety of DNA tubes assembled from small tiles, only an abstract concept of the intrinsic tile curvature was previously used to explain their formation. Details regarding the quantitative and structural descriptions of the intrinsic tile curvature and its evolution in DNA tubes by coupling with arm twists have been lacking. In this work, we designed three types of tile cores from a circular 128 nucleotide scaffold by longitudinal weaving (LW), bridging longitudinal weaving (bLW), and transverse weaving (TW) and assembled their 2D planar or tubular nanostructures via inter-tile arms with a distance of an odd or even number of DNA half-turns. The biotin/streptavidin (SA) labeling technique was applied to define the tube configuration with addressable inside and outside surfaces and thus their component tile conformation with addressable concave and convex curvatures. Both chiral tubes possessing left-handed and right-handed curvatures could be generated by finely tuning p and q in bLW-E_p/q designs (bLW tile cores joined together by inter-tile arms of even number of half-turns with the arm length of p base pairs (bp) and the sticky end length of q nucleotides (nt)). We were able to assign the chiral indices (n,m) to each specific tube from the high-resolution AFM images, and thus estimated the tile curvature angle with a regular polygon model that approximates each tube’s transverse section. We attribute the curvature evolution of bLW-E_p/q tubes composed of the same tile core to the coupling of the intrinsic tile curvature and different arm twists. A better understanding of integrated actions of different types of twisting forces on DNA tubes will be much more helpful in engineering DNA nanostructures in the future.

Mitigating the Non-Specific Amplification in RCA: Assessment of the Role of Ligation and Exonuclease Digestion in Circular DNA Preparation

<b>Layman Summary: </b>Rolling circle amplification (RCA) is a popular and extensively used bioanalytical tool. Like any nucleic acid amplifications, non-specific amplification may occur in it and risk generating false positive readouts. The work described in the manuscript investigates non-specific amplification in RCA as a function of ligation and exonuclease digestion assays during the synthesis of circular DNA. In particular, it investigates and compares the role of three different ligation techniques, namely splint-padlock ligation, cohesive end (sticky end ligation), and self-annealing ligation. In addition, it also probes the role of single exonuclease vs dual exonuclease digestions. We employed real time fluorescence to quantify the effect of these factors. Finally, our work hypothesizes the possible origins of non-specific amplification in RCA.

Mitigating the Non-Specific Amplification in RCA: Assessment of the Role of Ligation and Exonuclease Digestion in Circular DNA Preparation

<b>Layman Summary: </b>Rolling circle amplification (RCA) is a popular and extensively used bioanalytical tool. Like any nucleic acid amplifications, non-specific amplification may occur in it and risk generating false positive readouts. The work described in the manuscript investigates non-specific amplification in RCA as a function of ligation and exonuclease digestion assays during the synthesis of circular DNA. In particular, it investigates and compares the role of three different ligation techniques, namely splint-padlock ligation, cohesive end (sticky end ligation), and self-annealing ligation. In addition, it also probes the role of single exonuclease vs dual exonuclease digestions. We employed real time fluorescence to quantify the effect of these factors. Finally, our work hypothesizes the possible origins of non-specific amplification in RCA.