9 research outputs found
Poisson Statistics of Combinatorial Library Sampling Predict False Discovery Rates of Screening
Microfluidic droplet-based
screening of DNA-encoded one-bead-one-compound
combinatorial libraries is a miniaturized, potentially widely distributable
approach to small molecule discovery. In these screens, a microfluidic
circuit distributes library beads into droplets of activity assay
reagent, photochemically cleaves the compound from the bead, then
incubates and sorts the droplets based on assay result for subsequent
DNA sequencing-based hit compound structure elucidation. Pilot experimental
studies revealed that Poisson statistics describe nearly all aspects
of such screens, prompting the development of simulations to understand
system behavior. Monte Carlo screening simulation data showed that
increasing mean library sampling (ε), mean droplet occupancy,
or library hit rate all increase the false discovery rate (FDR). Compounds
identified as hits on <i>k</i> > 1 beads (the replicate <i>k</i> class) were <i>much</i> more likely to be authentic
hits than singletons (<i>k</i> = 1), in agreement with previous
findings. Here, we explain this observation by deriving an equation
for authenticity, which reduces to the product of a library sampling
bias term (exponential in <i>k</i>) and a sampling saturation
term (exponential in ε) setting a threshold that the <i>k</i>-dependent bias must overcome. The equation thus quantitatively
describes why each hit structure’s FDR is based on its <i>k</i> class, and further predicts the feasibility of intentionally
populating droplets with multiple library beads, assaying the micromixtures
for function, and identifying the active members by statistical deconvolution
<i><i>h</i>ν</i>SABR: Photochemical Dose–Response Bead Screening in Droplets
With
the potential for each droplet to act as a unique reaction
vessel, droplet microfluidics is a powerful tool for high-throughput
discovery. Any attempt at compound screening miniaturization must
address the significant scaling inefficiencies associated with library
handling and distribution. Eschewing microplate-based compound collections
for one-bead-one-compound (OBOC) combinatorial libraries, we have
developed <i><i>h</i>ν</i>SABR (Light-Induced
and -Graduated High-Throughput Screening After Bead Release), a microfluidic
architecture that integrates a suspension hopper for compound library
bead introduction, droplet generation, microfabricated waveguides
to deliver UV light to the droplet flow for photochemical compound
dosing, incubation, and laser-induced fluorescence for assay readout.
Avobenzone-doped PDMS (0.6% w/w) patterning confines UV exposure to
the desired illumination region, generating intradroplet compound
concentrations (>10 μM) that are reproducible between devices.
Beads displaying photochemically cleavable pepstatin A were distributed
into droplets and exposed with five different UV intensities to demonstrate
dose–response screening in an HIV-1 protease activity assay.
This microfluidic architecture introduces a new analytical approach
for OBOC library screening, and represents a key component of a next-generation
distributed small molecule discovery platform
Microfluidic Bead Suspension Hopper
Many
high-throughput analytical platforms, from next-generation
DNA sequencing to drug discovery, rely on beads as carriers of molecular
diversity. Microfluidic systems are ideally suited to handle and analyze
such bead libraries with high precision and at minute volume scales;
however, the challenge of introducing bead suspensions into devices
before they sediment usually confounds microfluidic handling and analysis.
We developed a bead suspension hopper that exploits sedimentation
to load beads into a microfluidic droplet generator. A suspension
hopper continuously delivered synthesis resin beads (17 μm diameter,
112,000 over 2.67 h) functionalized with a photolabile linker and
pepstatin A into picoliter-scale droplets of an HIV-1 protease activity
assay to model ultraminiaturized compound screening. Likewise, trypsinogen
template DNA-coated magnetic beads (2.8 μm diameter, 176,000
over 5.5 h) were loaded into droplets of an in vitro transcription/translation
system to model a protein evolution experiment. The suspension hopper
should effectively remove any barriers to using suspensions as sample
inputs, paving the way for microfluidic automation to replace robotic
library distribution
An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening
DNA-encoded synthesis
is rekindling interest in combinatorial compound
libraries for drug discovery and in technology for automated and quantitative
library screening. Here, we disclose a microfluidic circuit that enables
functional screens of DNA-encoded compound beads. The device carries
out library bead distribution into picoliter-scale assay reagent droplets,
photochemical cleavage of
compound from the bead, assay incubation, laser-induced fluorescence-based
assay detection, and fluorescence-activated droplet sorting to isolate
hits. DNA-encoded compound beads (10-μm diameter) displaying
a photocleavable positive control inhibitor pepstatin A were mixed
(1920 beads, 729 encoding sequences) with negative control beads (58 000
beads, 1728 encoding sequences) and screened for cathepsin D inhibition
using a biochemical enzyme activity assay. The circuit sorted 1518
hit droplets for collection following 18 min incubation over a 240
min analysis. Visual inspection of a subset of droplets (1188 droplets)
yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative
control beads). Using template barcoding strategies, it was possible
to count hit collection beads (1863) using next-generation sequencing
data. Bead-specific barcodes enabled replicate counting, and the false
discovery rate was reduced to 2.6% by only considering hit-encoding
sequences that were observed on >2 beads. This work represents
a complete
distributable small molecule discovery platform, from microfluidic
miniaturized automation to ultrahigh-throughput hit deconvolution
by sequencing
DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis
The
promise of exploiting combinatorial synthesis for small molecule
discovery remains unfulfilled due primarily to the “structure
elucidation problem”: the back-end mass spectrometric analysis
that significantly restricts one-bead-one-compound (OBOC) library
complexity. The very molecular features that confer binding potency
and specificity, such as stereochemistry, regiochemistry, and scaffold
rigidity, are conspicuously absent from most libraries because isomerism
introduces mass redundancy and diverse scaffolds yield uninterpretable
MS fragmentation. Here we present DNA-encoded solid-phase synthesis
(DESPS), comprising parallel compound synthesis in organic solvent
and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides.
Computational encoding language design yielded 148 thermodynamically
optimized sequences with Hamming string distance ≥ 3 and total
read length <100 bases for facile sequencing. Ligation is efficient
(70% yield), specific, and directional over 6 encoding positions.
A series of isomers served as a testbed for DESPS’s utility
in split-and-pool diversification. Single-bead quantitative PCR detected
9 × 10<sup>4</sup> molecules/bead and sequencing allowed for
elucidation of each compound’s synthetic history. We applied
DESPS to the combinatorial synthesis of a 75 645-member OBOC
library containing scaffold, stereochemical and regiochemical diversity
using mixed-scale resin (160-μm quality control beads and 10-μm
screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19
quality control beads showed excellent agreement (<1 ppt) between
DNA sequence-predicted mass and the observed mass. DESPS synergistically
unites the advantages of solid-phase synthesis and DNA encoding, enabling
single-bead structural elucidation of complex compounds and synthesis
using reactions normally considered incompatible with unprotected
DNA. The widespread availability of inexpensive oligonucleotide synthesis,
enzymes, DNA sequencing, and PCR make implementation of DESPS straightforward,
and may prompt the chemistry community to revisit the synthesis of
more complex and diverse libraries
High-throughput identification of DNA-encoded IgG ligands that distinguish active and latent Mycobacterium tuberculosis infections
The circulating antibody repertoire encodes a patient's health status and pathogen exposure history, but identifying antibodies with diagnostic potential usually requires knowledge of the antigen(s). We previously circumvented this problem by screening libraries of bead-displayed small molecules against case and control serum samples to discover "epitope surrogates" (Ligands of IgGs enriched in the case sample). Here, we describe an improved version of this technology that employs DNA-encoded libraries and high-throughput FACS-based screening to discover epitope surrogates that differentiate noninfectious/latent (LTB) patients from infectious/active TB (ATB) patients, which is imperative for proper treatment selection and antibiotic stewardship. Normal control/LTB (10 patients each, NCL) and ATB (10 patients) serum pools were screened against a library (5 × 10 beads, 448 000 unique compounds) using fluorescent antihuman IgG to label hit compound beads for FACS. Deep sequencing decoded all hit structures and each hit's occurrence frequencies. ATB hits were pruned of NCL hits and prioritized for resynthesis based on occurrence and homology. Several structurally homologous families were identified and 16/21 resynthesized representative hits validated as selective ligands of ATB serum IgGs (p < 0.005). The native secreted TB protein Ag85B (though not the E. coli recombinant form) competed with one of the validated ligands for binding to antibodies, suggesting that it mimics a native Ag85B epitope. The use of DNA-encoded libraries and FACS-based screening in epitope surrogate discovery reveals thousands of potential hit structures. Distilling this list down to several consensus chemical structures yielded a diagnostic panel for ATB composed of thermally stable and economically produced small molecule ligands in place of protein antigens