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
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.
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
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
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
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
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
Caged Substrates Applied to HCS255
Caged Substrates, Caging Chromophores, and Efficiencies Applied in Biological Studies
Classes of released
and so on
740 (2 hν)
740 (2 hν)
0.33 1 GM
Oligofecamine) and peptide based systems (e.g., Pep1  and MPG ) 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 ).
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.
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)
Classes of released
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.
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).
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-
[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
HPLC and the purity verified by gel electrophoresis and mass spectrometry (see Note 2). The
sense and antisense strands were annealed:
1. Dissolve the oligonucleotide pellet in standard annealing buffer to a concentration of 300 µM.
2. Confirm the concentration through UV absorption and dilute the sample to 100 µM stock solution (see
3. Combine equal volumes of each oligonucleotide in a 500 µL amber vial.
4. Vortex and centrifuge the sample for several seconds, heat the solution at 85oC for 5 min, and allow
the sample to cool to room temperature over 4 h.
5. Vortex and centrifuge the sample for several seconds. The final concentration for the stock solution of
annealed csiRNA or siRNA is 50 µM, which is confirmed using UV absorption.
6. Samples might be aliquoted and diluted for working stock solutions.
7. Annealed samples can be stored at –20°C for up to 6 mo and thawed for desired use.
HPLC analysis of the antisense csiRNA was used to establish a light–dosage working curve
for csiRNA. Concentration curves of pure starting material (GAPDH csiRNA) and photoproduct
(5′-phosphate GAPDH siRNA) were established. Samples of csiRNA were exposed to 365 nm
light using the UCOM while monitoring the amount of caged and released csiRNA through
HPLC analysis (Fig. 1). The energy light flux to uncage 100% of csiRNA at 2 µM was found to
be 5 J/cm2. The initial energy light flux of 1.4 J/cm2released approx 26 pmol of csiRNA, which
is nine times greater than the amount of csiRNA exposed to cells. For in vivo release of csiRNA
cells will be exposed to 1.4 J/cm2of 365 nm light (see Note 5).
3.3. Cell Preparation: Transfection of csiRNAs
HEK 293 or HeLa cells were transfected using Lipofecamine 2000 in a 96-well clear-bottom,
black-wall microtiter plate in accordance with the csiRNA manual (see Note 4). Approximately
5000 cells were plated. The final concentration of csiRNA delivered to the cells was 3 nM. At
t = 4 h post-transfection, the complexes were removed and replaced with 120 µL of fresh com-
plete growth medium (see Note 6).
3.4. Preliminary Studies: GAPDH Expression Knockdown
Through csiRNA Activation at Different Time-Points
A key advantage of caged materials is the ability to keep the substrates silent until it is
required, experimentally, to activate the substrate. Here, this feature is demonstrated with
258Conrad, II et al.
Fig. 1. Light dose–response curve for photorelease of β-actin csiRNA.
csiRNA by transfecting cells as described in Subheading 3.3. and incubating cells for 4, 24, or
48 h posttransfection. Cells were exposed to 365 nm light using UCOM, according to the UCOM
user manual, at t = 4 or 24 h time-points. GAPDH expression levels were analyzed using
Quantigene, according to the user manual (Fig. 2).
Cells that were not transfected with GAPDH siRNA or csiRNA maintained their normal
GAPDH expression levels. Cells that were transfected with csiRNA maintained their normal
GAPDH expression levels until csiRNA was activated with the UCOM. The most important
advantage is allowing substrates to remain dormant in cells until the desired time to activate
them, illustrated with cells irradiated at 24 h. Prior to irradiation, GAPDH expression levels were
normal. However, at t = 24 h, cells were exposed to 365 nm light and GAPDH expression was
knocked down (t = 48 h) to less than 60% below normal GAPDH levels.
3.5. Light Dosable Photo-Activation of csiRNA
The importance of controlling dose release in biological studies cannot be overstated (see
Note 6). Kinetic studies,as well as phenotypic assays,are greatly enhanced when modulators intro-
duced to cells can be activated with a high degree of accuracy and precision. For most phenotypic
assays, especially, it is highly desirable to accurately titrate the amount of material required to elicit
a phenotypic response. In this context, we demonstrate the activity of csiRNA can be tuned by con-
trolling the energy exposed to transfected cells. By exposing the cells to increasing light energy, an
increase in siRNA activity is achieved. The beauty of this system (as with all caged systems) is that
a known number of photons (i.e., energy) will trigger a known quantity of siRNA precisely because
there is a single caging group positioned at the 5′-end per siRNA molecule.
Cells were prepared as previously described in Subheading 3.3.
1. The cells were exposed to 0.0–1.4 J/cm2of 365 nm light using a UCOM Microplate Photo-Activator
(Panomics) according to the UCOM manual.
2. The cells were incubated at 37oC for t = 24 h post-transfection, and lysed using QuantiGene lysis buffer
according to the QuantiGene user manual. Replicates of three wells were run for all conditions tested.
Gene expression levels were measured using QuantiGene according to the user manual.
Caged Substrates Applied to HCS 259
Fig. 2. GAPDH expression knockdown at various time-points. GAPDH csiRNA was transfected into
HeLa cells and exposed to 365 nm light at t = 4 or 24 h posttransfection. Control conditions include cells
exposed to transfection reagent only, GAPDH csiRNA without light activation, and GAPDH siRNA.
Expression levels were measured at t = 4, 24, and 48 h for all conditions. Control experiments (delivery
complex only and csiRNA without 365 nm light exposure) shows GAPDH expression continues unim-
peded. GAPDH expression levels are knocked down for GAPDH siRNA and for csiRNA only after expo-
sure to 365 nm light.
As Fig. 3 illustrates, increasing light dosage results in more csiRNA uncaged to release active
siRNA effectively reducing residual mRNA levels through the normal RNAi pathway (23–27).
The activity of caged reagents is not simply “on” or “off.” By exposing the appropriate energy
dosage on the UCOM it is possible to tune in the amount of active reagent available in cells.
3.6. Concluding Remarks
There are an infinite number of applications for including cell survival, cell cycle regulation
and cell development. Caging technology offers experimentalists a wide array of control in tem-
poral, spatial, and concentration parameters. And with the advent of tools designed to bring light
control to multiplexed assay systems, caged compounds may now be implemented in high con-
tent and high-throughput screens. We are, in fact, witness to several technologies that have been
available for quite some time, be integrated in complementing fashion. These integrated tech-
nologies will surely help to better understand cellular pathways, off-target and downstream
effects, and substrate effects on these pathways.
1. Attachment of photolabile groups to RNA and DNA using postsynthetic methods has been reported in
literature; however this method does not take siRNA active sites into account. The design described
here requires only a single caging group per siRNA molecule to take full advantage of the caging agent
(vide supra). The postulated methodic placement of the caging group on siRNA molecules is limited
to the following locations: 5′-, 3′-, or 2′-hydroxy groups; on the phosphate backbone; or on an indi-
vidual nucleotide base. It was hypothesized that the 5′-hydroxy group was the most accessible syn-
thetically and would cause the greatest disruption to the RNAi process. To test this, a GAPDH siRNA
was synthesized with derivatives that permanently modified 5′-terminus. 5′-O-methyl siRNA analogs
were shown to have zero activity compared to normal siRNA analogs. In addition, an O-alkyl phos-
phate modified siRNA (5′-C6-amine-GAPDH) also failed to catalyze gene expression knockdown
for GAPDH. These experiments indicated a caging group on the 5′-phosphate would also block siRNA
action. There are reports of other photoactivatable siRNA systems, which do not cage the 5′-end
260Conrad, II et al.
Fig. 3. In vivo light–dosage exposure to HEK 293 cells transfected with GAPDH csiRNA, negative con-
trol siRNA at t = 4 h post-transfection. GAPDH csiRNA that was previously exposed to 1.4 J/cm2365 nm
light was also transfected into HEK 293 cells as a positive control. Cells were incubated at 37oC for t = 24 h
and the GAPDH expression levels were measured.
exclusively (28). These systems are not as potent for a number of reasons. Either there are more caging
groups per siRNA molecule leading to decreased sensitivity to light, and hence less dosable, or siRNA
is caged at random locations. The end result is a system that is not completely silent and might posses
some or all normal activity. In addition, to remove all of the caging groups requires substantially more
light energy, which can lead to cell death (75).
2. Highly purified csiRNA antisense strands are extremely important for successful controllable knock-
down experiments. We investigated the correlation between the purity of csiRNA and the activity of
csiRNA in cells, measured by HPLC chromatograms of the antisense strand. Six different lots of var-
ious purities were transfected into HeLa cells according to the csiRNA manual and incubated for 24 h
at 37oC. The cells were not exposed to 365 nm light in order to keep the csiRNA caged and unreac-
tive. The cells were lysed and GAPDH mRNA expression levels were measured using Quantigene
detection system. Any drop in GAPDH expression levels prior to light activation is viewed as a less
efficient csiRNA. According to Table 2, there is a drop in caging efficiency below 97% purity, and an
even more dramatic drop below 94% purity. This is most likely because of (n–1) residues that make
up the majority of impurities from oligonucleotide syntheses. For csiRNA (n–1) residues are fully
active, complete siRNA molecules.
3. Spectroscopic determination of concentration was carried out for two purposes. First, a 1:1 ratio of
sense to antisense should be used to achieve the highest activity. Second, an accurate measure of
csiRNA concentration is needed to yield optimal delivery to cells. This will result in the highest poten-
tial knockdown activity with the lowest background.
4. It is essential to use clear-bottom microtiter plates, as the UCOM Microplate Photoactivator delivers
light from the bottom. The UCOM has been tested to be compatible with the following microtiter plates:
a. Corning, Costar®(cat. no. 3904).
b. BD, Falcon (cat. no. 353948).
c. Greiner (cat. no. 655090).
d. Nunc, Nalgene (cat. no. 237105).
5. Cytotoxicity experiments for UCOM 365 nm light exposure on HeLa cells showed ED50 values of
21 J/cm2. It is vital to maintain energy doses lower than the ED50 level. Detrimental effects to cells,
including cell death, are evident above the ED50 level. Cells show a very good tolerance to 365 nm
light at energy levels below the ED50 value.
6. It is vital that the media be replaced following the transfection protocol. Although transfection methods
might be highly efficient, it is impossible to have delivered all csiRNA into the cells. As light is com-
pletely unselective toward csiRNA inside or outside of cells, it is necessary to remove the undelivered
extra cellular caged reagents.
We would like to thank Frank Witney, Gary K. McMaster, and Quan Nguyen for helpful
advice and discussions. Support from the National Institutes of Health (Grant no. GM72910
[RSG]) is gratefully acknowledged.
Caged Substrates Applied to HCS 261
Purity of Antisense Csirna™ Compared to Background
Gene Expression Knockdown: The Lower the Purity
of the Antisense Strand, the Higher the siRNA Activity
GAPDH expression level
(normalized to cyclophilin
expression levels [%])
Purity of antisense
strand (%)Lot no.
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