The use of fluorescent nucleic acid base analogues is becoming increasingly important in the fields of biology, biochemistry and biophysical chemistry as well as in the field of DNA nanotechnology. The advantage of being able to incorporate a fluorescent probe molecule close to the site of examination in the nucleic acid-containing system of interest with merely a minimal perturbation to the natural structure makes fluorescent base analogues highly attractive. In recent years, there has been a growing interest in developing novel candidates in this group of fluorophores for utilization in various investigations. This review describes the different classes of fluorophores that can be used for studying nucleic acid-containing systems, with an emphasis on choosing the right kind of probe for the system under investigation. It describes the characteristics of the large group of base analogues that has an emission that is sensitive to the surrounding microenvironment and gives examples of investigations in which this group of molecules has been used so far. Furthermore, the characterization and use of fluorescent base analogues that are virtually insensitive to changes in their microenvironment are described in detail. This group of base analogues can be used in several fluorescence investigations of nucleic acids, especially in fluorescence anisotropy and fluorescence resonance energy transfer (FRET) measurements. Finally, the development and characterization of the first nucleic base analogue FRET pair, tCO–tCnitro, and its possible future uses are discussed.

Helicases are a class of nucleic acid (NA) motors that catalyze NTP-dependent unwinding of NA duplexes into single strands, a reaction essential to all areas of NA metabolism. In the last decade, single-molecule (sm) technology has proven to be highly useful in revealing mechanistic insight into helicase activity that is not always detectable via ensemble assays. A combination of methods based on fluorescence, optical and magnetic tweezers, and flow-induced DNA stretching has enabled the study of helicase conformational dynamics, force generation, step size, pausing, reversal and repetitive behaviors during translocation and unwinding by helicases working alone and as part of multiprotein complexes. The contributions of these sm investigations to our understanding of helicase mechanism and function will be discussed.

Some of the rate theories that are most useful for modeling biological processes are reviewed. By delving into some of the details and subtleties in the development of the theories, the review will hopefully help the reader gain a more than superficial perspective. Examples are presented to illustrate how rate theories can be used to generate insight at the microscopic level into biomolecular behaviors. An attempt is made to clear up a number of misconceptions in the literature regarding popular rate theories, including the appearance of Planck's constant in the transition-state theory and the Smoluchowski result as an upper limit for protein–protein and protein–DNA association rate constants. Future work in combining the implementation of rate theories through computer simulations with experimental probes of rate processes, and in modeling effects of intracellular environments so that theories can be used for generating rate constants for systems biology studies is particularly exciting.