We have developed novel nucleic acid probes that recognize and report the presence of specific nucleic acids in homogeneous solutions. These probes undergo a spontaneous fluorogenic conformational change when they hybridize to their targets. Only perfectly complementary targets elicit this response, as hybridization does not occur when the target contains a mismatched nucleotide or a deletion. The probes are particularly suited for monitoring the synthesis of specific nucleic acids in real time. When used in nucleic acid amplification assays, gene detection is homogeneous and sensitive, and can be carried out in a sealed tube. When introduced into living cells, these probes should enable the origin, movement, and fate of specific mRNAs to be traced.

Molecular Beacons: Probes that Fluoresce upon Hybridization

Thermodynamic parameters for prediction of RNA duplex stability are reported. One parameter for duplex initiation and 10 parameters for helix propagation are derived from enthalpy and free-energy changes for helix formation by 45 RNA oligonucleotide duplexes. The oligomer sequences were chosen to maximize reliability of secondary structure predictions. Each of the 10 nearest-neighbor sequences is well-represented among the 45 oligonucleotides, and the sequences were chosen to minimize experimental errors in delta GO at 37 degrees C. These parameters predict melting temperatures of most oligonucleotide duplexes within 5 degrees C. This is about as good as can be expected from the nearest-neighbor model. Free-energy changes for helix propagation at dangling ends, terminal mismatches, and internal G X U mismatches, and free-energy changes for helix initiation at hairpin loops, internal loops, or internal bulges are also tabulated.

https://www.pnas.org/content/pnas/83/24/9373.full.pdf

Improved free-energy parameters for predictions of RNA duplex stability

An in vitro selection procedure was used to develop a DNA enzyme that can be made to cleave almost any targeted RNA substrate under simulated physiological conditions. The enzyme is comprised of a catalytic domain of 15 deoxynucleotides, flanked by two substrate-recognition domains of seven to eight deoxynucleotides each. The RNA substrate is bound through Watson–Crick base pairing and is cleaved at a particular phosphodiester located between an unpaired purine and a paired pyrimidine residue. Despite its small size, the DNA enzyme has a catalytic efficiency (kcat/Km) of ≈109 M−1⋅min−1 under multiple turnover conditions, exceeding that of any other known nucleic acid enzyme. Its activity is dependent on the presence of Mg2+ ion. By changing the sequence of the substrate-recognition domains, the DNA enzyme can be made to target different RNA substrates. In this study, for example, it was directed to cleave synthetic RNAs corresponding to the start codon region of HIV-1 gag/pol, env, vpr, tat, and nef mRNAs.

https://www.pnas.org/content/pnas/94/9/4262.full.pdf

A General Purpose RNA-Cleaving DNA Enzyme

In this paper, we derive the general forms of the equations required to extract thermodynamic data from equilibrium transition curves on oligomeric and polymeric nucleic acids of any molecularity Significantly, since the equations and protocols are general, they also can be used to characterize thermodynamically equilibrium processes in systems other than nucleic acids We briefly review how the reduced forms of the general equations have been used by many investigators to evaluate mono- and bimolecular transitions, and then explain how these equations can be generalized to calculate thermodynamic parameters from common experimental observables for transitions of higher molecularities We emphasize the strengths and weaknesses of each method of data analysis so that investigators can select the approach most appropriate for their experimental circumstances We also describe how to analyze calorimetric heat capacity curves and noncalorimetric differentiated melting curves so as to extract both model-independent and model-dependent thermodynamic data for transitions of any molecularity The general equations and methods of analysis described in this paper should be of particular interest to laboratories that currently are investigating association and dissociation processes in nucleic acids that exhibit molecularities greater than two

Calculating thermodynamic data for transitions of any molecularity from equilibrium melting curves

A general model is proposed for genetic recombination. Its essential new feature is the hypothesis that recombination is initiated by a single-strand (or asymmetric) transfer, which may, after isomerization, become a two-strand (or symmetric) exchange. The likelihood of this transition from asymmetric to symmetric strand exchange determines certain characteristic features of recombination in any particular organism.

https://www.pnas.org/content/pnas/72/1/358.full.pdf

Donald M. Crothers

Papers

Relaxation kinetics of dimer formation by self complementary oligonucleotides

Kinetic discrimination in the folding of intramolecular triple helices.