The compatibility of modified nucleic acids with enzymatic copying and ligand selection

Résumé 0

Modified nucleic acids have important applications in molecular biology, genetics and genomics. Here I report a series of modified 5-aminomethyl-deoxyuridine (5-AM-dU) analogues with different functional groups on the 5-position of the pyrimidine ring. These modified 5-AM-dU nucleotides were evaluated in primer extension and polymerase chain reaction (PCR) amplification assays and were shown to be compatible with the polymerase enzymes. In total, six 5-AM-dU analogues with either stabilising or destabilising functional groups were prepared and efficiently incorporated by polymerases. Another modified nucleoside, the fluorescent quadracyclic adenine analogue qAN1 (qAN1) is incorporated with low efficiency in PCR. I was able to show that when this highly duplex stabilising modification is paired with the complementary duplex-destabilising 5-AM-dU, PCR amplification becomes possible. This is because the stabilising effects of one analogue offsets the destabilising properties of the other. Two destabilising thymidine analogues with hydrophobic functional groups at the 5′ position of the nucleobase enabled the use of qAN1 in place of deoxyadenosine at full substitution. In combination, these artificial qAN1-5-AM-dU base pairs allow the preparation chemically modified DNA in which all the A and T nucleobases are modified. These chemically modified 5-AM-dU analogues were used into SELEX to generate a series of aptamers through in vitro selection. In parallel, similar approaches were used to construct modified nucleic acid libraries with aminoethyl glycine (AEG) substituents on thymidine.