Quantitative analysis of receptors for adenosine nucleotides obtained via in vitro selection from a library incorporating a cationic nucleotide analog.
5-(3"-Aminopropynyl)-2'-deoxyuridine (dJ), a modified nucleoside with a side chain carrying a cationic functional group, was incorporated into an oligonucleotide library, which was amplified using the Vent DNA polymerase in a polymerase chain reaction (PCR). When coupled to an in vitro selection procedure, PCR amplification generated receptors that bind ATP. This is the first example of an in vitro selection generating oligonucleotide receptors where the oligonucleotide library has incorporated a cationic nucleotide functionality. The selection yielded functionalized receptors having sequences differing from a motif known to arise in a standard selection experiment using only natural nucleotides. Surprisingly, both the natural and the functionalized motifs convergently evolved to bind not one, but two ATP molecules cooperatively. Likewise, the affinity of the receptors for ATP had converged; in both cases, the receptors are half saturated at the 3 mM concentrations of ATP presented during the selection. The convergence of phenotype suggests that the outcome of this selection experiment was determined by features of the environment during which selection occurs, in particular, a highly loaded affinity resin used in the selection step. Further, the convergence of phenotype suggests that the optimal molecular phenotype has been achieved by both selections for the selection conditions. This interplay between environmental conditions demanding a function of a biopolymer and the ability of the biopolymer to deliver that function is strictly analogous to that observed during natural selection, illustrating the nature of life as a self-sustaining chemical system capable of Darwinian evolution.
Battersby
T R
TR
Department of Chemistry, Department of Anatomy and Cell Biology, the Florida Center for Heterocyclic Compounds, University of Florida, Gainesville 32611, USA.
Ang
D N
DN
Burgstaller
P
P
Jurczyk
S C
SC
Bowser
M T
MT
Buchanan
D D
DD
Kennedy
R T
RT
Benner
S A
SA
D000227:Adenine Nucleotides / Q000737:chemistry*; D000241:Adenosine / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D001483:Base Sequence; D001704:Biopolymers / Q000737:chemistry; D002412:Cations; D004247:DNA / Q000737:chemistry; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000737:chemistry; D019143:Evolution, Molecular; D007065:Idoxuridine / Q000031:analogs & derivatives / Q000737:chemistry; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D018047:Receptors, Purinergic P1 / Q000737:chemistry*; D018048:Receptors, Purinergic P2 / Q000737:chemistry*; D017421:Sequence Analysis
D000227:Adenine Nucleotides; D001704:Biopolymers; D002412:Cations; D009841:Oligonucleotides; D018047:Receptors, Purinergic P1; D018048:Receptors, Purinergic P2; D000255:Adenosine Triphosphate; D004247:DNA; D000241:Adenosine; D007065:Idoxuridine; D003857:Deoxyuridine