Methods for generating epitopes for binding to recognition molecules by templated assembly
A composition and biotin technology, applied in chemical instruments and methods, biochemical equipment and methods, medical preparations of non-active ingredients, etc., can solve the problems of finding non-nucleic acid templates, destroying spatial proximity, etc.
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Embodiment 1
[0236] Example 1: Demonstration of co-binding selection on solid-phase targets and sequence confirmation of binary aptamer candidates after co-binding selection
[0237] After 4 separate rounds of selection of the left and right aptamer libraries, the eluted subpopulation was incubated with a biotinylated immunoglobulin Fab target (against the BRD7 protein; ThermoFisher), which was then activated by binding to streptavidin prime magnetic beads to convert to a solid phase. After washing 3 times with 0.5 ml PBSM and once with 1× T4 DNA ligase buffer (NEB, containing 1 mMATP), the preparation was subdivided into two equal parts and splinted with DNA (5′-TCCAGATGTCTTTGCTTTCTTCAGGACACAG (SEQ ID NO: 119); 100 μl, 1 pmol / μl) were + / − annealed by heating at 37°C for 5 min followed by 1 h at room temperature. Thereafter, the preparation was washed an additional 2 times with ligase buffer. Then, the tubes were divided again into two equal parts and treated with + / - T4 DNA ligase. A s...
Embodiment 2
[0240] Example 2: Analysis of single-morphic aptamers and generation of binary aptamers after 10 cycles of selection on Fab fragments (co-binding process)
[0241] After 10 separate selection cycles of monomorphic left and right aptamer subpopulations on biotinylated Fab targets, the resulting subpopulations were cloned and sequenced. Here (compared to the results from the 4th cycle), multiple repetitions of specific aptamer sequences were observed. Three repeats of the specific right aptamer (designated 288 / 10AptR1 ) were seen from 14 sequenced specific single-morphic aptamers (7 each from the left and right clonal populations).
[0242] Then, the 10th cycle subpopulation of left and right aptamers from the Fab target was subjected to a co-binding procedure on the Fab target. The amplified binary products are then sequenced and characterized. The right aptamer clone 228 / 0AptR1 (previously observed as a recurring monomorphic clone) was also found independently in 5 independe...
Embodiment 3
[0245] Example 3: Direct Demonstration of Binding of Specific Generation 10 Singlemorphic Aptamers to Fab Targets
[0246] To assess the binding of the 10th cycle single state aptamer to the selection agent (Fab target), a direct binding assay was performed. Here, single-chain aptamer preparations (usually self-annealing) were incubated in PBSM with or without biotinylated Fab fragments and then adsorbed to streptavidin magnetic beads. After this incubation period, the beads were magnetically separated and the supernatant retained. After 3 washes of the beads, bound material was eluted by a second incubation with 100 μl 0.1 M NaOH 2×20 and the eluate was immediately precipitated with 0.3 M sodium acetate and 3 volumes of ethanol. The precipitate was washed with 70% ethanol and dried. After reconstitution in 5 μl TE, 1 μl samples were denatured in formamide and run on a 10% urea (denaturing) gel. Candidate single state aptamers 228 / 10AptR1 and 229 / 10AptL3 and specific arbitr...
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