Particle Display: A Quantitative Screening Method for Generating High‐Affinity Aptamers

Abstract

We report an aptamer discovery technology that reproducibly yields higher affinity aptamers in fewer rounds compared to conventional selection. Our method (termed particle display) transforms libraries of solution-phase aptamers into “aptamer particles”, each displaying many copies of a single sequence on its surface. We then use fluorescence-activated cell sorting (FACS) to individually measure the relative affinities of >108 aptamer particles and sort them in a high-throughput manner. Through mathematical analysis, we identified experimental parameters that enable optimal screening, and demonstrate enrichment performance that exceeds the theoretical maximum achievable with conventional selection by many orders of magnitude. We used particle display to obtain high-affinity DNA aptamers for four different protein targets in three rounds, including proteins for which previous DNA aptamer selection efforts have been unsuccessful. We believe particle display offers an extraordinarily efficient mechanism for generating high quality aptamers in a rapid and economic manner, towards accelerated exploration of the human proteome.

Precision metabolic engineering: The design of responsive, selective, and controllable metabolic systems

“…To date, the effort and difficulty associated with creating aptamers that bind specifically to small molecules of interest has limited their wide-spread use. However, recent improvements on high-throughput technologies to develop sensitive and specific aptamers (Cho et al, 2013; Wang et al, 2014) will likely hasten creation of novel aptamers, potentially broadening the scope of who can reasonably develop new aptamers and increasing the use of aptamer-based regulation. Other significant challenges to be addressed for precision metabolic engineering include reducing the experimental burden of tuning the system to respond in a switch-like way at a specified signal threshold.…”

Characterization of the Neisseria gonorrhoeae Iron and Fur Regulatory Network

“…The bacterial pellets were collected 1 h after the addition of iron or desferal. For quantitative real-time PCR (qRT-PCR), RNA was purified from these pellets using an RNeasy kit (Qiagen, Hilden, Germany) and treated with DNase I (38). qRT-PCR was performed using the QuantiTect SYBR green RT-PCR kit (Qiagen) and analyzed using an ABI Prism 7700 sequence detection system (Applied Biosystems, Foster City, CA) with the primers listed in Table 1.…”

“…For electrophoretic mobility shift assay (EMSA) with either protein, the reaction mixture was separated on a native 6% polyacrylamide gel (acrylamide/bisacrylamide ratio, 37.5:1 [wt/wt]) (Bio-Rad, Hercules, CA) and stained with SYBR green (Invitrogen) following the manufacturer's instructions. For Fur competition assays, 50-bp double-stranded DNA probes were incubated with the reaction mixtures (38). The probes containing a Fur box (fur positive) from the promoter region of the fur gene and the probes without a Fur box (rmp negative) from the promoter region of the rmp gene are listed in Table 1.…”

Devices and approaches for generating specific high-affinity nucleic acid aptamers

“…Recently, a true single sequence/bead selection called particle display utilizing the sorting capabilities of FACS was fully demonstrated. 90 Currently, FACS technologies are restricted to detect and sort about 10 8 total beads/cells which significantly reduces the number of nucleic acids that can be screened in this format. Particle display resolves this sampling limitation by performing the first selection cycle without FACS so that a full size library can be screened, generating a partially enriched pool which can be much more efficiency utilized via FACS.…”