X-ray analysis cannot provide quantitative estimates of the relative contribution of non-specific, specific, strong, and weak contacts of extended DNA molecules to their total affinity for enzymes and proteins. The interaction of different enzymes and proteins with long DNA and RNA at the quantitative molecular level can be successfully analyzed using the method of the stepwise increase in ligand complexity (SILC). The present review summarizes the data on stepwise increase in ligand complexity (SILC) analysis of nucleic acid recognition by various enzymes—replication, restriction, integration, topoisomerization, six different repair enzymes (uracil DNA glycosylase, Fpg protein from Escherichia coli, human 8-oxoguanine-DNA glycosylase, human apurinic/apyrim-idinic endonuclease, RecA protein, and DNA-ligase), and five DNA-recognizing proteins (RNA hel-icase, human lactoferrin, alfa-lactalbumin, human blood albumin, and IgGs against DNA). The relative contributions of structural elements of DNA fragments “covered” by globules of enzymes and proteins to the total affinity of DNA have been evaluated. Thermodynamic and catalytic factors providing discrimination of unspecific and specific DNAs by these enzymes on the stages of primary complex formation following changes in enzymes and DNAs or RNAs conformations and direct processing of the catalysis of the reactions were found. General regularities of recognition of nucleic acid by DNA-dependent enzymes, proteins, and antibodies were established. © 2021 by the author. Licensee MDPI, Basel, Switzerland.