Evolution of Hybridization Probes to DNA Machines and Robots (original) (raw)
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Organic & biomolecular chemistry, 2006
Nucleic acids include substantial information in their base sequence and their hybridization-complexation motifs. Recent research efforts attempt to utilize this biomolecular information to develop DNA nanostructures exhibiting machine-like functions. DNA nano-assemblies revealing tweezers, motor, and walker activities exemplify a few such machines. The DNA-based machines provide new components that act as sensitive sensors, transporters, or drug delivery systems.
High-fidelity DNA hybridization using programmable molecular DNA devices
2011
The hybridization of complementary nucleic acid strands is the most basic of all reactions involving nucleic acids, but has a major limitation: the specificity of hybridization reactions depends critically on the lengths of the complementary pairs of strands and can drop (in the presence of other competing strands whose sequences are close to that of a given target strand) to very low values if the strands have sufficiently long length. This reduction in specificity of hybridization reactions occurs especially in the presence of noise in the form of other competing strands that have sequence segments identical to the target. This limitation in specificity depending on strand length significantly limits the scale and accuracy of biotechnology and nanotechnology applications which depend on hybridization reactions. Our paper develops techniques for ensuring specific high-fidelity DNA hybridization reactions for target strands of arbitrary length. Given an in vitro solution which contains various DNA strands with differing sequences, among them a particular known target DNA sequence s of relatively long length (say at least 60 to hundreds of bases), our goal is to bind to each subsequence segment of s with high specificity and exact complementarity for a significant fraction of strands s in the solution. To do this, we develop a protocol that relies only on hybridization reactions between relatively short length (of at most 15 bases) DNA sequence segments. Our basic approach is to design DNA devices that essentially scan over strands in solution, subsegment by subsegment, and determine if one is indeed an instance of the given target strand s. This scanning is achieved by carefully designed relatively short sequences that effectively perform, in sequential order, successive verifications of subsequence identities on the target sequence, which if successfully completed indicate that the complete target sequence has been verified and completely hybridized. Our protocol is executed autonomously, without external mediation. Our high-fidelity DNA hybridization protocol is driven by a series of conversions of single stranded DNA into duplex DNA that help overcome kinetic energy traps, similar to DNA walkers. In addition to the design of our highfidelity DNA hybridization protocol, we also discuss the kinetics of our hybridization reactions, reducing the overall kinetics to a series of well-understood strand-displacement reactions. Further, we describe a detailed design of an ongoing small-scale experimental demonstration of our protocols for high-fidelity hybridization. We also discuss potential applications for our protocols in molecular detection and DNA computing.
Analytical Chemistry, 2010
Quantitative and reproducible data can be obtained from surface-based DNA sensors if variations in the conformation and surface density of immobilized single-stranded DNA capture probes are minimized. Both the conformation and surface density can be independently and deterministically controlled by taking advantage of the preferential adsorption of adenine nucleotides (dA) on gold, as previously demonstrated using a model system in Opdahl, A.; Petrovykh, D. Y.; Kimura-Suda, H.; Tarlov, M. J.; Whitman, L. J. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 9-14. Here, we describe the immobilization and subsequent hybridization properties of a 15-nucleotide DNA probe sequence that has additional m adenine nucleotides, (dA) m , at the 5′ end. Quantitative analysis of immobilization and hybridization for these probes indicates that the (dA) m block preferentially adsorbs on gold, forcing the probe portion of the strand to adopt an upright conformation suited for efficient hybridization. In addition, a wide range of probe-to-probe lateral spacing can be achieved by coimmobilizing the probe DNA with a lateral spacer, a strand of k adenine nucleotides, (dA) k . Altering either the length or relative concentration of the (dA) k spacers added during probe immobilization controls the average surface density of probes; the density of probes, in turn, systematically modulates their hybridization with solution targets.
The biophysics of DNA hybridization with immobilized oligonucleotide probes
Biophysical Journal, 1995
A mathematical model based on receptor-ligand interactions at a cell surface has been modified and further developed to represent heterogeneous DNA-DNA hybridization on a solid surface. The immobilized DNA molecules with known sequences are called probes, and the DNA molecules in solution with unknown sequences are called targets in this model. Capture of the perfectly complementary target is modeled as a combined reaction-diffusion limited irreversible reaction. In the model, there are two different mechanisms by which targets can hybridize with the complementary probes: direct hybridization from the solution and hybridization by molecules that adsorb nonspecifically and then surface diffuse to the probe. The results indicate that nonspecific adsorption of single-stranded DNA on the surface and subsequent twodimensional diffusion can significantly enhance the overall reaction rate. Heterogeneous hybridization depends strongly on the rate constants for DNA adsorption/desorption in the non-probe-covered regions of the surface, the two-dimensional (2D) diffusion coefficient, and the size of probes and targets. The model shows that the overall kinetics of DNA hybridization to DNA on a solid support may be an extremely efficient process for physically realistic 2D diffusion coefficients, target concentrations, and surface probe densities. The implication for design and operation of a DNA hybridization surface is that there is an optimal surface probe density when 2D diffusion occurs; values above that optimum do not increase the capture rate. Our model predicts capture rates in agreement with those from recent experimental literature. The results of our analysis predict that several things can be done to improve heterogeneous hybridization: 1) the solution phase target molecules should be about 100 bases or less in size to speed solution-phase and surface diffusion; 2) conditions should be created such that reversible adsorption and two-dimensional diffusion occur in the surface regions between DNA probe molecules; 3) provided that 2) is satisfied, one can achieve results with a sparse probe coverage that are equal to or better than those obtained with a surface totally covered with DNA probes.
Nucleic Acids Research, 2010
One of the main problems in nucleic acid-based techniques for detection of infectious agents, such as influenza viruses, is that of nucleic acid sequence variation. DNA probes, 70-nt long, some including the nucleotide analog deoxyribose-Inosine (dInosine), were analyzed for hybridization tolerance to different amounts and distributions of mismatching bases, e.g. synonymous mutations, in target DNA. Microsphere-linked 70-mer probes were hybridized in 3M TMAC buffer to biotinylated single-stranded (ss) DNA for subsequent analysis in a Luminex Õ system. When mismatches interrupted contiguous matching stretches of 6 nt or longer, it had a strong impact on hybridization. Contiguous matching stretches are more important than the same number of matching nucleotides separated by mismatches into several regions. dInosine, but not 5-nitroindole, substitutions at mismatching positions stabilized hybridization remarkably well, comparable to N (4-fold) wobbles in the same positions. In contrast to shorter probes, 70-nt probes with judiciously placed dInosine substitutions and/or wobble positions were remarkably mismatch tolerant, with preserved specificity. An algorithm, NucZip, was constructed to model the nucleation and zipping phases of hybridization, integrating both local and distant binding contributions. It predicted hybridization more exactly than previous algorithms, and has the potential to guide the design of variation-tolerant yet specific probes.
A probe is a nucleic acid molecule (single-stranded DNA or RNA) with a strong affinity with a specific target (DNA or RNA sequence). Probe and target base sequences must be complementary to each other, but depending on conditions, they do not necessarily have to be exactly complementary. The hybrid (probe–target combination) can be revealed when appropriate labeling and detection systems are used. Gene probes are used in various blotting and in situ techniques for the detection of nucleic acid sequences. In medicine, they can help in the identification of microorganisms and the diagnosis of infectious, inherited, and other diseases.
Strategies for optimizing DNA hybridization on surfaces
Specific and predictable hybridization of the polynucleotide sequences to their complementary counterparts plays a fundamental role in the rational design of new nucleic acid nanodevices. Generally, nucleic acid hybridization can be performed using two major strategies, namely hybridization of DNA or RNA targets to surface-tethered oligonucleotide probes (solid-phase hybridization) and hybridization of the target nucleic acids to randomly distributed probes in solution (solution-phase hybridization). Investigations into thermodynamic and kinetic parameters of these two strategies showed that hybridization on surfaces is less favorable than that of the same sequence in solution. Indeed, the efficiency of DNA hybridization on surfaces suffers from three constraints: (1) electrostatic repulsion between DNA strands on the surface, (2) steric hindrance between tethered DNA probes, and (3) nonspecific adsorption of the attached oligonucleotides to the solid surface. During recent years, several strategies have been developed to overcome the problems associated with DNA hybridization on surfaces. Optimizing the probe surface density, application of a linker between the solid surface and the DNA-recognizing sequence, optimizing the pH of DNA hybridization solutions, application of thiol reagents, and incorporation of a polyadenine block into the terminal end of the recognizing sequence are among the most important strategies for enhancing DNA hybridization on surfaces.