This chapter covers selected papers published in the years 2016 and 2017 on chemical synthesis and biological application of nucleotides and their analogues, including data on related prodrugs (mostly exerting antiviral properties) and specific and highly sensitive probes for detection of biomolecules important for elucidation of biochemical processes. Whereas the commonly known nucleotides (i.e. nucleosides bearing a phosphate group) are mostly the building blocks of DNA or RNA, there is a growing field of less recognised nucleotide and nucleotide-like molecules playing specific signalling or regulatory roles, and their analogues are designed and used for investigation or tuning of biochemical pathways, both in vitro and in vivo. The progress in chemistry of nucleic acids components responds to those needs, and chemists and biochemists provide compounds using either purely chemical or combined chemo-enzymatic approaches. It should be emphasised that all those efforts are greatly supported by enormous development of analytical methods, which allow for fast and precise detection of investigated complexes or metabolites.