Method for the site-specific enzymatic labelling of nucleic acids in vitro by incorporation of unnatural nucleotides

A nucleobase pair comprising a first nucleobase analog having the structure β9b, and a second nucleobase analog having any of the structures α15a or α15b:

• wherein each X is independently carbon or nitrogen,
• wherein each R1 is independently hydrogen, alkyl group, a reactive linker comprising a reactive center a linker coupled to a cargo,
• wherein each R2 is optional and when present is independently hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno, halogen, cyano, azide, nitro group, a reactive linker comprising a reactive center, or a linker coupled to a cargo,
• wherein each Y is independently sulfur, oxygen, selenium, or a secondary amine,
• wherein each E is independently oxygen, sulfur or selenium,
• and the wavy line indicates a point of bonding to a ribosyl, deoxyribosyl, or dideoxyribosyl moiety or an analog thereof, wherein the ribosyl, deoxyribosyl, or dideoxyribosyl moiety, or analog thereof is in free form, is bound to a mono-phosphate, diphosphate, triphosphate, α-thiotriphosphate, β-thiophosphate, or γ-thiophosphate group, or is included in an RNA or a DNA or in an RNA analog or a DNA analog.