Caged substrates applied to high content screening: an introduction with an eye to the future.

Abstract

The use of photoremovable protecting groups in biology affords the end user high temporal, spatial, and concentration control of reagents and substrates. High content screening and other large-scale biology applications would benefit greatly from these advantages. Herein, we report progress in this field by highlighting the recent development of controllable siRNA (csiRNA), which is a dormant siRNA that can be activated using 365 nm light. Two different experimental designs are described to highlight the temporal and concentration variables that can be controlled. First, the RNAi process is activated at two timepoints, 24- and 48-h post-transfection, to demonstrate that the action of csiRNA does not begin until activated. Second, increasing light dosage exposure to cells transfected with csiRNA that controls the concentration of active siRNA molecules. All experiments are conducted in a 96-well format with light delivered through the UCOM device.

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Caged Substrates Applied to High Content Screening
An Introduction With an Eye to the Future
Peter G. Conrad, II, Rajesh V. Chavli, and Richard S. Givens
Summary
The use of photoremovable protecting groups in biology affords the end user high temporal, spatial, and
concentration control of reagents and substrates. High content screening and other large-scale biology
applications would benefit greatly from these advantages. Herein, we report progress in this field by high-
lighting the recent development of controllable siRNA (csiRNA™), which is a dormant siRNA that can be
activated using 365 nm light. Two different experimental designs are described to highlight the temporal
and concentration variables that can be controlled. First, the RNAi process is activated at two timepoints,
24- and 48-h post-transfection, to demonstrate that the action of csiRNA does not begin until activated.
Second, increasing light dosage exposure to cells transfected with csiRNA that controls the concentration
of active siRNA molecules. All experiments are conducted in a 96-well format with light delivered through
the UCOM™ device.
Key Words: Caged compounds; high-throughput light delivery; light-activated; photoremovable pro-
tecting group; siRNA; spatial and concentration control; temporal control.
1. Introduction
Sudden activation of chemical, biological, and physical processes has been a goal in funda-
mental studies in biology, chemistry, and physics since the initial flash photolysis studies by
Nobel Laureate Sir George Porter at the end of World War II (1). A most effective means for
rapid activation of such a process has developed through photochemistry by employing photo-
chemical reactions of modified biological substrates or reagents (2), or employing photo acti-
vated fluorescent sensors (3–6). These substrates are attached to photoremoveable protecting
groups, commonly known as “cages,” and are truly effective in altering the chemical and bio-
logical activity of the protected substrates. Cages might provide “protection” or inactivation in a
number of ways including steric hindrance to the substrate’s entrée into tight binding domains
or masking the functional group(s) responsible for binding by H-bonding or electrostatic attrac-
tion. The location of the attachment of the cage on the substrate, therefore, is of critical impor-
tance in that the position of the cage must prevent the normal chemical and biological action of
the substrate. Other practical considerations must also be addressed including the photochemi-
cal efficiency of the release of the substrate, the available wavelength region for activation of the
caging chromophore, and the ease of synthesis or installation of the caged substrate (7).
The advantages of caged substrates are extensive. In addition to control of the temporal
release of the substrate through manipulations of the light source, the precise location and the
253
From: Methods in Molecular Biology, vol. 356:
High Content Screening: A Powerful Approach to Systems Cell Biology and Drug Discovery
Edited by: D. L. Taylor, J. R. Haskins, and K. Giuliano © Humana Press, Inc., Totowa, NJ

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254Conrad, II et al.
exact quantity of released substrate are experimental parameters that are controlled by the
researcher through the choice of the optical light source (from pulsed laser and high-intensity
two-photon laser excitation to conventional continuous light sources) as well as the optical and
light transmission pathways employed. Furthermore, because light is both a “traceless” and “ran-
dom access” reagent (8); it leaves no residue that might cause further, deleterious reactions, is
indiscriminate in molecular selection; and it provides great versatility in activating caged sub-
strates. The initiation, detection, and detailed study of a great number of biochemical and physio-
logical processes have been successfully pursued (9,10). As an example, caged fluorescent
probes have been commercially available for the study of molecular processes for at least two
decades (11).
A compilation of caged substrates that illustrate the range and scope of this field would itself
be timely but such a list would necessarily be dated. In this growing field, new chromophores
and additional substrate candidates for caging are constantly being added. Hence, we choose
here to present representative published examples that have stood the test of careful examination
and warrant further consideration for the developing field of high content screening (HCS)
analysis and imaging. Although many reagents have been “caged” and their release rates, effi-
ciencies, and reaction parameters determined, the examples selected for this chapter (listed in
Table 1) are restricted to representatives of the range and scope of known biological agonists,
antagonists, and inhibitors that have been studied.
There is very limited information on applications of caged compounds in high content and
high-throughput screening owing, in large part, to the limits of the instrumentation and tech-
niques necessary for photorelease under the rigorous conditions required for the screening and
imaging methodology. The situation is changing, however, as exploratory, innovative applica-
tions appear. The most recent applications of caging chemistry include the use of caged fluo-
rophores, which penetrate cell membranes, resulting in high loading of fluorescent precursors
within cells for imaging and bioconjugation (9). Other studies, directed toward two-photon
decaging, provide more precise spatial control (10). Although these applications are very useful
for single cell investigations, they do not address the needs of high-throughput systems, which
would greatly benefit from the spatial and temporal control afforded by caged compounds. In
addition, caged compounds would open up the possibility of continuous monitoring of responses
in cells under repetitious stimulation of a cell after a single transfection step.
To bridge the single molecule caging technology to larger scale biological applications, instru-
mentation (UCOM, Panomics, Fremont, CA) has recently been developed that delivers precise
and uniform lumination over the entire area of a 96-well (or larger) microplate that can release
substrates in cells, with a combination of temporal and dose-dependent control and in a format
compatible with simultaneous, multiple processes in required in HCS. In the high-throughput
mode, the UCOM serves as the essential instrument in development of whole cell assays. One
could easily envision an entire series of caged reagents delivered to cells in 96-, 384- or 1536-well
microplate format. The temporal and concentration variations are experimentally controlled
directly through the UCOM.
It is well understood that HCS systems are designed to yield enormous amounts of informa-
tion per well, including kinetic measurements of on-/off-response rates, along with selective acti-
vation within cellular subdomains. The difficulty for most HCS systems arises from the
implementation of substrate application, usually based on pipet introduction of the active reagent
or substrate.
The advent of multiple technologies to delivery macromolecules into cellular compartments has
been crucial in high content and high-throughput screening and other multiplexed screening
systems. Techniques, such as electroporation, are complemented by technologies that involve the
covalent attachment of reagents such as TAT (11) and antennapedia (12) are now commercially
available. Complexation reagents such as lipid-based systems (e.g., Lipofecamine and

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Caged Substrates Applied to HCS255
Table 1
Caged Substrates, Caging Chromophores, and Efficiencies Applied in Biological Studies
Representative
examples of
substrate
Approximately
λexcit(nm)
rangesb
Classes of released
substrates
Caging
Quantum
yieldsc(Φ) References
0.3–0.38
0.08
0.01–0.15
chromophoresa
Phosphates,
nucleotides,
and so on
H3PO4
pHP
DMCM, DMACM
BNZ
(pH dependent)
pHP
oNB
oNP
DMACM
pHP
oNP
oNB, oNP
oNBP
BNZ
ACM; MCM
NV
oNP
BHC
oNB
oNB
300–340
385
300–365
28
29
30
ATP300–340
300–370
320
385
300–330
300–350
365
350
360
340
>300
360
350–365
308
0.3
0.19
0.63
31
32,33
34
35
36
37
38
39
8,40
41
42
43
44
45
46
47,48
49
0.07–0.09
na
na
0.2
na
0.33
0.07
na
na
na
na
GTP
Thymidine
cAMP
NADP
DNA
RNA
siRNA
Phosphopeptides oNP
Glu
300–365
>300
0.26–0.33
0.14 C-terminus
carboxylic acids
Amino acids,
oligopeptides,
and proteins
pHP
HCM-carbamate
MNI
N-sub-6-oNP-7-
coumaryl-3-carboxyl
pHP, m-substituted
pHP analogs
oNP-carbamate
MNI
oNB
β-DCNB
740 (2 hν)
350
300–400
740 (2 hν)
300–390
1 GM
0.085
0.33 1 GM
50
51
52
GABA0.03–0.38
53
Serine
Aspartate
3500.65
0.09
na
0.14
54,55
56
57
58
59
60
61
62
63
64
38
65
66
334–364
315
308
Alanine
NMDA
Capsaicin
Ala-Ala
Leu-leu-Me
Acetate
Bradykinin
Fluorescein
Phenylephrine
DNBH
oNB, NV
pHP
4-gluco-oNB
DMBNZ
pHP
oNB
oNB, NV
345na
na
0.26
N/A
0.64
0.22
na
0.1–0.4
300–375
313
375
>300
>300
350
300–400 N-terminus
amines, and
so on
Amino acids,
oligo-peptides,
and proteins
Epinephrine
Isoproterenol
oNB, NV
oNB, NV
300–400
300–400
0.1–0.4
0.1–0.4
42
42
NADPCNB>3200.09–0.19
67
(Continued)

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Oligofecamine) and peptide based systems (e.g., Pep1 [13] and MPG [14]) are also available to
deliver substrates such as oligonucleotides, peptides, and proteins through the cell membrane.
Among these, the most effective delivery agents are those that transport the cargo into the cell and
avoid endosomal pathways. The Express™ reagent (Panomics) is such a delivery reagent system,
which is MPG-based and thus successfully evades endosomal pathways (15).
Despite the numerous advantages these delivery reagents offer over microinjection or stan-
dard pipet techniques, spatial and temporal control of cell activation frequently remains elusive
to those implementing in commercially available HCS system. In this aspect, HCS would bene-
fit greatly from a photoactivated caged initiation process.
Batch transfection of caged molecules offers the advantage that equal amounts of silent or
inactive antagonists, agonists or substrates can be delivered to all cells and thereby makes the
transfection independent of the assay outcome. Thereafter, the uniform illumination to multiple
cell arrays with transfected caged reagents under prescribed conditions enables initial null (t = 0)
measurements followed by precise regulation of the substrate release for HCS. In this way, caged
compounds will yield far greater information than simple batch experiments with a group of cells
that produce repeatable responses after a period of recovery. This is illustrated with the recent
development of controllable siRNA (csiRNA [16]).
Caged siRNA is a timely example because RNAi has quickly become one of the most excit-
ing arenas (17), owing in large part to its potential in drug discovery and therapeutics (18). In
addition, recent studies have incorporated RNAi into HCS assays (19). csiRNA is therefore at
the forefront of application of caged substrates to large-scale biology.
csiRNA is a caged siRNA that is incapable of catalyzing the normal gene expression knock-
down process. The biologically benign caged substrate remains dormant and inactive until
absorption of 365 nm light in which siRNA is released. The uncaged siRNA is capable of par-
ticipating in the normal RNAi process. The following sections will highlight two of the control-
lable features of caged reagents, temporal, and dosage control, to illustrate the potential use of
caged reagents in the high content arena.
256Conrad, II et al.
Nitrous oxide5,8-dimethoxy-1-
allylnaphthyl, others
oNB
350 0.66
68,69
Amino acid
side chains
C-Kemptide
(cysteine)
Cysteine,
tyrosine
Aspartate
Arginine
Tamoxifen
EGTA
300–365 0.62
70
oNB 300–350 na
71,72
DNBH
DMoNB
NV
oNB
300–400
300–400
365
347
0.6
73
74
75
11
0.1–0.4
Ca2+
aAbbreviations for the chromophores are: pHP = p-hydroxyphenacyl; oNB = o-nitrobenzyl; oNP = O-nitro-
phenethyl; NV = 4,5-dimethoxy-O-nitrobenzyl; CNB = α-carboxy-O-nitrobenzyl; BNZ = benzoin; DMBNZ =
3′,5′-dimethoxybenzoyl; HCM = 7-hydroxycoumarylmethyl; ACM = 7-acetoxyCM; MCM = 7-methoxyCM;
DMACM = 7-dimethylaminoCM; DMCM = 6,7-dimethoxycoumarylmethyl; MNI = 4-methoxy-7-nitroindoline;
DNBH = o,o′-dinitrobenzhydryl; BHC = 6-bromo-7-hydroxycoumarin-4-ylmethyl.
bThe wavelength or wavelength range is based on data provided from known UV-vis spectra reported in the
references or is estimated based on available data from other sources.
cEfficiencies vary with substituents on the chromophore and with changes in the reaction media and condi-
tions. GM = Goppert-Meyer units for two photon (2 hν) excitation.
Table 1 (Continued)
Representative
examples of
substrate
Approximately
λexcit(nm)
rangesb
Classes of released
substrates
Caging
Quantum
yieldsc(Φ) References chromophoresa

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1.1. Equipment and Materials
This section lists the materials and equipment needed to conduct gene expression knockdown
experiments using csiRNA. Although portions of the experimental design are not described as
high throughput, the technology is quite amendable to this technique as well.
2. Materials
1. HEK 293 and HeLa cells (ATCC, Manassas, VA).
2. Growth medium: 10% FBS, DMEM, nonessential amino acids, sodium pyruvate, prepared fresh.
3. PC Phosphoramidite (Glenn Research, Sterling, VA).
4. csiGAPDH™ (Panomics, Fremont, CA), light sensitive, store at –20oC.
5. 5′-phosphate-GAPDH antisense oligonucleotide (TriLink Biotechnologies, San Diego, CA).
6. GAPDH siRNA negative control (Ambion, Austin, TX).
7. Lipofectamine 2000 (Invitrogen, Carlsbad, CA).
8. Standard annealing solution (Panomics).
9. Clear-bottom, black-wall, 96-well microtiter plates.
10. UCOM Microplate Photoactivator (Panomics).
11. QuantiGene Reagent System (Panomics).
12. QuantiGene Probesets (Panomics).
3. Methods
The application of controllable siRNA (csiRNA) to inhibit gene expression will be described
herein under five separate headings:
1. The design and synthesis of csiRNA strands (Subheading 3.1.).
2. The quantum efficiency to establish a working curve for variable gene knockdown and establish the
maximum energy required for 100% release of the siRNA’s activity (Subheading 3.2.).
3. Delivery of csiRNA into cells cultured in a 96-well format (Subheading 3.3.).
This chapter will describe two different experiments to illustrate two features of csiRNA: tem-
poral control and dosable activation.
1. Light-activation of csiRNA at t = 4 or 24 h post-transfection, followed by gene expression analysis at
t = 4, 24, and 48 h (Subheading 3.4.).
2. Increased activation of csiRNA through increasing energy of light, followed by gene expression analy-
sis at t = 24 h post-transfection (Subheading 3.5.).
Finally, we will close with a few concluding remarks (Subheading 3.6.). Throughout the dis-
cussion, items that require specific care or particular attention will be described in Subheading 4.
3.1. Reagent Preparation
SiRNA oligonucleotides were designed in accordance with guidelines set forth by Tuschl
(20). For this discussion, GAPDH was used as the gene of interest. The general structure of
siRNA molecules is a double-stranded 21-mer ribooligonucleotide with TT-overhangs on each
3′-terminus. The sense and complementary antisense strand syntheses were carried out using
standard phosphoramidite chemistry. The GAPDH negative control siRNAs were obtained from
Ambion. The sequence of the GAPDH siRNA sense strand is 5′-caucaucccugccucuacuTT-3′.
The mode of action of siRNA has been well studied (21), and several reports have noted the
importance of the phosphorylation of the 5′-antisense strand during gene expression knockdown
(22). As such, the 5′-end was targeted for protection with a photoactivatable protecting group
(see Note 1). The photolabile phosphoramidite, [1-N-(4,4′-dimethoxytrityl)-5-(6-biotinamido-
capro-amidomethyl)-1-(2-nitrophenyl)ethyl]-2-cyanoethyl-(N′,N′-diisopropyl)-phosphoramidite
[obtained from Glenn Research, Sterling, Virginia] was coupled to the 5′ terminus of the anti-
sense strand of a 21-mer siRNA using standard phosphoramidite chemistry during the normal
oligonucleotide synthesis. The modified, 21-mer antisense strand was purified using RNase-free
Caged Substrates Applied to HCS 257