Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5402_Библиотеки_им_академика_М_И_Перельмана
.pdf
4.3 Screening Methods 55
https://t.me/med1917
ligand, ligands that work by a covalent mechanism will not be detected as they will
be retained on the RNA.
One of the advantages of ALIS is the overall low hit rate combined with a low
false-positive rate. One reason for this is the unambiguous determination of binders
based on their intrinsic mass. More importantly, however, bound ligands must survive separation by SEC. During the separation process, compounds continue to dissociate from the target with a half-life that is determined by the o-rate for the
interaction (Eq. (4.1)):
0.693
t
=
1
/
2
k
o
[RNA]+[ligand]
k
on
−−−−−⇀
↽−−−−−
k
o
[RNA∶ligand]
(4.1)
Only complexes with suciently slow o-rates will still contain enough compound after SEC to be detected by mass spectrometry. The SEC conditions must
be carefully chosen to ensure the success of ALIS. The separation of RNA from
unbound molecules must have sucient resolution so that the unbound fraction
does not bleed into the bound fraction, resulting in false positives. The speed of the
separation is also critical. As an example, for a korate of 0.1 s−1, only 50% of the
complex remains after ∼7 seconds (Figure 4.3). The issue of ligand dissociation on
the column can be dealt with in several ways. First, a higher starting concentration
of RNA, typically 1–5 μM, will mean a higher concentration remaining after decay.
Second, employing rapid SEC separations to decrease column residence time will
help to minimize the amount of decay. Below-ambient temperature of the SEC
column can be used to slow the rate of ligand-receptor dissociation, assisting
detection. Modern HPLC sizing columns allow for small column volumes and
high ow rates which enable rapid separation of complexes. Column resins are
selected so that the RNA runs in the void volume, the earliest fraction on a sizing
column. RNA presence in the void volume can be veried via placement of a UV
spectrophotometer post-SEC in the chromatographic system; detection of RNA via
UV absorption is also useful as a quality control metric during routine analysis.
1.0
0
0.5
N/N
T
0.0
0
Figure 4.3 Simulated decay curves for complexes with varying dissociation rates. The
half-life for a complex with a k
life) is illustrated. The red line shows the point at which only 10% of the complex remains.
0.01 sec
0.03 sec
0.05 sec
1/2
10%
100
4020
Time (s)
8060
rate constant of 1 × 10−1s−1(approximately 7 second half
off
0.10 sec
–1
–1
–1
–1
56 4 Screening and Lead Generation Techniques for RNA Binders
https://t.me/med1917
O’Connel et al. estimated that with a 5-second column separation and the ability to
observe binders for approximately 5 half-lives they were able to capture compounds
with o-rates of 0.7 s−1or slower [18].
Two other considerations in ALIS, both of which can result in false positives, are
compound breakthrough and compound carryover.Compound breakthrough refers
to small molecules that elute in the void volume in the absence of RNA. This can be
caused by nonideal behavior of the small molecule by mechanisms such as aggregation. Another nonideal behavior is carryover, where the compound shows adherent behavior within the system and becomes detectable in immediately subsequent
experimental runs. Both issues can be addressed with the appropriate placement of
controls and blank injections.
In addition to high-throughput screening, ALIS can be used to obtain quantitative
binding measurements. Several approaches can be used. The rst is the calculation
of a direct KDmeasurement using multipoint titrations. Alternatively, if the target
of interest contains a ligand with a known anity, a competition-based method for
anity ranking can be used [19]. In these experiments, a constant concentration of
the ligand of interest is titrated with increasing concentrations of competitor ligand.
The resulting titration curve yields an ACE50, dened as the anity competition
50% inhibitory concentration. In this case, a higher ACE50concentration indicates a
higher anity for the ligand of interest. Rizvi et al. used competition titration experiments to rank order a series of small molecules binding to the FMN riboswitch [17].
Finally, if the goal is to rank order compounds without the need for a quantitative
binding constant, then relative anity ranking can be performed by titrating the target molecule against xed concentrationsof compounds. As the target concentration
decreases, competition among compounds increases with weaker compounds disappearing rst. The advantage of this method is its quickness and the ability to screen
high numbers of compounds in a single experiment.
4.3.1.4 DNA-Encoded Libraries (DELs)
Recently, researchers have used DNA-encoded libraries (DELs) to discover RNA
binders. A DEL is a mixture of small molecules conjugated to unique DNA tags,
wherein the structure information is encoded within DNA sequences. This allows
the screening of billions of compounds simultaneously in a single vessel [20].
Researchers have not typically used DEL screening to discover RNA binders [21],
but Mukherjee, Blain, Petter, and co-workers utilized the Vipergen yoctoReactor
DNA-encoded library (DEL) approach [22] to screen for compounds that bound
Aptamer 21 [23]. Among the primary hits identied from this DEL screen, one compound was selected for photoprobe development based on its binding anity
in conrmatory SPR assays (KD= 130 nM). More recently, Dou and co-authors
used DEL technology on the E. coli avin mononucleotide (FMN) Riboswitch
to nd compounds with mid-nanomolar binding anity [24]. Originally, Dou
and co-authors screened a DEL consisting of 10.38 billion ligands against HIV1
trans-acting responsive region (TAR) RNA and found signicant false-positive
signals due to DNA–RNA base-pairing interactions. To solve this issue, Dou and
co-authors developed an algorithm to dierentiate DNA–RNA-binding signals from
4.3 Screening Methods 57
https://t.me/med1917
small molecule–RNA binding and they realized certain compounds were enriched
because their DNA tags (rather than the small molecules) were interacting with the
TAR RNA from base 21 to 29 through the “GGCAGAGAG” motif. In view of this,
Dou and co-authors were able to reduce false positive signals by preincubating the
DELs with RNA fragments from the RNA target and using competitive elution with
50 μM Pra-tat. Using this protocol, they did not nd any active compounds from
screening the DEL against TAR but found compounds with mid-nanomolar binding
anity against FMN riboswitch after applying what they learned from screening
HIV1 TAR.
Paegel, Disney, and co-authors have recently reported success in screening a
DEL of 73,728 ligands against a library of RNA structures (4096 targets) [25]. They
pooled, amplied, sequenced, and decoded the DEL hits to identify hit structures for
synthesis and subsequent validation. One of the hits bound a 5′GAG/3′CCC internal
loop that is present in primary microRNA-27a (pri-miR-27a), the oncogenic precursor of microRNA-27a with a KDof 40 ± 30 nM as measured by a competitive binding
assay with a constant concentration of compound, a cyanine5 (Cy5)-labeled model
of pri-miR-27a’s Drosha site, and varying concentrations of unlabeled miR-27a
precursor. This compound was cell active, inhibiting pri-miR-27a processing in
MCF-10a cells transfected with a plasmid encoding wild-type pri-miR-27a. Further,
the compound inhibited pri-miR-27a biogenesis in MDA-Mb-231 TNBC cells with
a measured IC50of ∼1μM. Most recently, Paegel, Disney, and co-authors used a
solid phase DEL [26] to nd binders of the RNA repeat expansion r(CUG)
exp
, widely
considered the cause of the most common form of adult-onset muscular dystrophy,
myotonic dystrophy type 1 (DM1) [27].
4.3.1.5 Microarray Screening
SMM is a high-throughput method that can be used to identify ligands for a given
RNA target. SMMs work through the immobilization of a library of compounds in
an array onto a glass surface [28]. Labeled RNA is then exposed to the immobilized
library. Through the detection of the labeled RNA, compounds that bind are
identied.
A clear advantage to the use of SMM is the minute amount of material required
for each assay, as well as the large number of samples that can be evaluated at a
time via high throughput screens. However, SMMs also present a few challenges.
First, careful consideration of label type and location along with proper quality control of RNA structure should be employed to alleviate concerns about labeled RNA
not replicating native RNA folds. Second, a compound needs to contain an appropriate functional group to adhere to the microarray surface. This functional group
cannot be essential to the interaction with the RNA target, as association with the
surface may inhibit the interaction with the RNA. Further concerns exist around
the binding capacity of the immobilized ligands, as this environment varies from a
small molecule binding to RNA completely in solution. Recently, a technique called
AbsorbArray was described illustrating the potential for unmodied compounds to
be screened in a microarray-based approach [29]. Using AbsorbArray, compounds
are noncovalently adhered onto hydrated agarose-coated microarray surfaces and
58 4 Screening and Lead Generation Techniques for RNA Binders
https://t.me/med1917
subsequently dried. In this study, FDA-approved drugs were screened against various RNA motifs, and approved anticancer drugs were identied that target the oncogenic noncoding RNA microRNA-21.
Despite these concerns, SMMs have been used successfully for numerous RNA
targets. Though SMMs have long been used to identify ligands for protein targets,
they were rst used with RNA by Disney and co-authors to look at the interactions
between aminoglycosides and bacterial rRNA [30]. Since then, microarrays for RNA
targets haveevolved to assess binding with drug-like compounds. To our knowledge,
the rst use of an SMM for RNA targeting specically with drug-like compounds
was completed by Schneekloth and co-authors [31]. This work focused on targeting
an HIV TAR hairpin structure and identied two hits from a library of 20,000 compounds. Most notably, one of the hits was found to be structurally distinct from any
ligands previously known to bind the HIV TAR hairpin. Additional SMM screens
completed by Schneekloth and co-authors identied ligands for microRNA-21 [32],
pre-Q1 riboswitches [33], MALAT-1 [34], and the ZTP riboswitch [35]. Additionally,
Disney and co-authors more recently used SMM to screen a newly designed
RNA fragment library against a variety of RNA structures displaying randomized
regions of 3 × 3or3× 2 internal loops. This study illustrated that even compounds
with low molecular weight have the potential to selectively aect RNA-mediated
pathways [36].
4.3.1.6 Fragment-Based Drug Discovery
Fragment-based drug discovery (FBDD) is a high-throughput method that uses fragments to screen potential drug targets. In FBDD, a library of fragments is screened,
often in pools. Once a fragment is identied as a hit, if screened in pools, binding is subsequently veried in singleton form, and then via an orthogonal binding
assay. Binding elucidation may then be used to help guide fragment optimization
to enhance binding anity as part of the fragment-to-lead stage of FBDD. Overall,
FBDD requires careful consideration of a fragment library, screening and validation
of fragment binders by two separate methods, elucidation of a fragment’s mode of
binding, and optimization of fragment(s) to drug-like candidates. FBDD can lead to
the identication of fragments with high ligand eciency to their target, therefore
helping to identify compounds that bind in a more specic fashion.
FBDD as a method has become a crucial part of early-stage drug discovery. One
clear advantage of fragment libraries is that they can cover a greater percentage of
chemical space per molecule when compared to HTS of drug-like molecules. It has
been estimated that there are between 1060and 10
200
possible drug-like compounds
like those used in HTS (between 300 and 500Da), but only 107possible molecules
that meet the “Rule of 3” parameters used for fragment libraries [37–39]. Therefore, a
fragment screen using a carefully selected couple of thousands of compounds can be
more eective at exploring the available chemical space when compared to a library
of hundreds of thousands of HTS compounds.
The number of examples of successful FBDD screening against drug targets, both
protein and RNA-based, is rapidly increasing, as is the number of examples of successful progression from fragment to lead. This indicates that FBDD may be a very
4.3 Screening Methods 59
https://t.me/med1917
useful tool in helping to assess the ligandability of RNA structures as well as helping
to identify and develop lead compounds for these targets. FBDD has been used in
numerous instances to illustrate small molecule binding specically to RNA targets.
A few examples where FBDD has been used for RNA targets include the identication of novel ligands to the TPP riboswitch, TERRA, HIV TAR, and the Inuenza A
virus [40–44].
Fragment Library Design Fragment libraries consist of a set of compounds which all
fall under the parameters found within the “Rule of 3” (Ro3). The “Rule of 3” indicates that a fragment is less than or equal to 300 Da, has a cLogP less than or equal
to 3, and contains no more than 3 H-bond donors and acceptors. Fragment library
design should consider the Ro3 guidelines [45], three-dimensional space, and the
ease of chemical elaboration [46]. Since hits coming from fragment-based screening are small, they often need to be grown, linked, or merged. Therefore, molecules
containing favorablefunctionality to serve as growth vectors are preferred. However,
care must also be taken to avoid reactive or unstable scaolds.
The largest RNA-specic fragment library we are aware of was recently developed
by the Disney lab [36]. In addition to this fragment library, there are numerous
studies of small molecule libraries used for RNA that can help design the generation
of a fragment library specically focused around targeting RNA [29, 47, 48].
Additionally, RNA-binding sites can be similar in size and hydrophobicity to
druggable protein-binding sites. For this reason, general purpose fragment libraries
should deliver sucient hits. However, there are some known privileged scaolds
for RNA ligands that could lead to higher hit rates [49]. For example, Giacomo
Padroni and co-authors claim that the H-bonding and hydrophobic eects of
sulfur-mediated interactions are underexplored for RNA-targeted drug discovery
[50]. Additionally, Hamid Nasiri and co-authors ran a study on cMYC illustrating
a higher propensity for binding the target with fragments containing either 5- and
6-membered heterocyclic rings, two fused 6-membered heterocyclic rings, or four
substituted aniline derivatives [51].
Fragment Screening Methods Fragment screening is dierent from traditional
high-throughput screening in various ways. First, fragments tend to bind targets
with low anity. To identify these low-anity compounds, it is necessary to screen
them at high concentrations, typically up to 1 or 2 mM [52]. Unfortunately, these
circumstances create unsuitable conditions for some traditional biophysical or
biochemical assays such as uorescent-based competition assays or cell-based
assays. Additionally, adverse eects of using such high concentrations include
an increased rate of false positives due to aggregation and the identication of
nonspecic binders. For this reason, it is essential to verify the binding results of
the primary screen utilizing at least one orthogonal screening method. Compounds
identied by at least two screening methods can more condently be conrmed
as hits and optimized via medicinal chemistry. There are many screening methods
used for fragment screening but NMR, SPR, and virtual screens are the most utilized
methods. We will focus on these methods later.
60 4 Screening and Lead Generation Techniques for RNA Binders
https://t.me/med1917
NMR for Fragment Screening NMRis a commonly used screening method for FBDD
due to its sensitivity and capability to characterize fragments with a broad range of
RNA and fragment binding anities, from μM to mM. Additionally, NMR screening
data can provide structural information on the binding interface and modes. There
are two main NMR techniques used for FBDD: RNA-observed NMR spectroscopy
and ligand-observed NMR spectroscopy.
1
H NMR-binding assays are widely used as RNA imino protons that arise
from the formation of base pairs are resolved from typical proton peaks from the
fragments. For this reason, the chemical shifts of the imino protons are sensitive,
often with clear shifts upon binding to fragments. RNA target size can vary for
RNA-observed NMR screens, though they typically favor small, well-folded RNA
less than 50 nucleotides long as peak overlap issues may arise for longer RNA
[53]. For this screening method, a pooled set of fragments (typically 5–10) can be
screened simultaneously as long as there is minimal signal overlap among the1H
resonances of each fragment in the pool. Binding is observed through shifts in
imino1H resonances based on interaction with the RNA target. If the initial screen
was carried out in pools, once a binding event is observed, screening of individual
fragments must be carried out from the positive pools to conrm binding. Since
each imino proton corresponds to a base pair, the binding site on the RNA target
can be immediately mapped based on the assignment. Unfortunately, this method
is less sensitive if the fragment only binds with a loop or a bulge residue due to lack
of imino proton signals from these regions [54].
Another NMR screening method is ligand-observed NMR spectroscopy. This
method has the advantage that the target structure is not required, there is no
RNA size limit, less RNA sample is required, and no isotopic labeling is required.
There are several widely used ligand-observed NMR methods, including line broadening, saturation transfer dierence (STD), water-ligand observed via gradient
spectroscopy (WaterLOGSY), and Carr–Purcell–Meiboom–Gill (CPMG). These
NMR methods can not only provide information regarding if the fragment binds
with RNA (Figure 4.4) but can also generate binding information on the fragment
molecules. Due to the high sensitivity of detecting weak binding anity in the mM
range, a combination of multiple methods is recommended during screening to
(a) (b) (d)Fragments STD
Figure 4.41H NMR methods used to identify fragments that bind to a given RNA structure.
WaterLOGSY
Hit
1
H (ppm)
1
H (ppm)
Hit
1
Hit
1
H (ppm)
H (ppm)
(e)(c)
CPMG
4.3 Screening Methods 61
https://t.me/med1917
lower the rate of false positive hits. It is worth mentioning that 19F NMR has also
been widely used for ligand-observed screening due to its higher sensitivity and
throughputs (up to 20 fragments per pool).
Surface Plasmon Resonance (SPR) for Fragment Screening SPR is often utilized for
screening fragments and is especially benecial because it is a sensitive method that
does not require large amounts of RNA. However, SPR is relatively low throughput
and when screening large fragment libraries, higher throughput methods of screening are often more ecient. For this reason, SPR may more commonly be utilized
as a follow-up screen for binding verication. Another utilization of SPR within
fragment screening is for competition experiments [55]. In these competition
experiments, a known ligand is tethered to the chip surface. The RNA is then
added and allowed to form a complex. Addition of a competing fragment will result
in decomplexation and a large signal change can be observed. This can provide
additional information regarding fragment binding and overlap (cf. Section 4.3.2.1).
Fragment Hit Validation/Elucidation of Binding
X-ray Crystallography and SAXS of Fragments X-ray crystallography provides a
unique atomic level structure elucidation that is often crucial for not only validating
a hit but also evolving the hit to a lead compound. Relative to proteins there are
fewer X-ray crystal structures of RNA. This limits the use of X-ray crystallography as
a method of validation. However,once a crystallization condition is established for a
given RNA, obtaining high-resolution structures with dierent fragments becomes
much more feasible. This is illustrated in the successful use of X-ray crystallography
for fragment hit validation achieved in 2014 against the thiM riboswitch [41]. In
addition to X-ray crystallography, the authors used small-angle X-ray scattering
(SAXS) [56]. SAXS provides lower resolution structural determination and can
be especially useful when determining RNA 3D topological structure due to the
stronger X-ray scattering observed in the sugar-phosphate backbone of RNA [57, 58].
Utilizing SAXS, the authors were able to observe the induction of a conformational
change resulting in a structure intermediate between that of the free and that of the
native ligand-bound riboswitch. Recently, Menichelli and coauthors successfully
co-crystallized the theophylline aptamer with theophylline and four unique binders
that have up to 340-fold greater anity compared to theophylline [59]. These results
show that the same approaches to drug discovery that are used for proteins may
also be applied to RNA.
Virtual Screening of Fragments In FBDD of proteins, virtual screening can be a great
tool to identify and elaborate fragments as well as to predict binding modes of ligands from large libraries. Due to many complexities of structure modeling, including
water displacement, protonation states, and accounting for multiple possible binding modes, virtual screening is more useful for later stages in screening such as the
elaboration of fragments rather than initial fragment screening. For RNA-specic
targets, there are only limited examples of virtual screens.
62 4 Screening and Lead Generation Techniques for RNA Binders
https://t.me/med1917
Target Engagement of Fragments Target engagement is discussed in more detail
in another section of this chapter (cf. Section 4.4). However, in the context of
RNA-focused fragment screening, Suresh et. al published a technique termed
Chem-CLIP-Frag-Map [60]. This technique utilizes a fragment library where each
fragment contains a photoanity group, which enables covalent attachment of the
fragments to the target. In this technique, each fragment also contains an azide
to bind to streptavidin-coated magnetic beads, allowing for isolation and further
evaluation of the covalently bound fragment and target [60]. While this technique
can be useful to eciently identify fragments that bind to a target, the library is
limited by the requirements to have multiple functionalities for target attachment
and isolation.
Fragment Hit Optimization While fragment screens are widely reported for identi-
fying binders to a variety of RNA targets, little has been reported regarding optimization of these fragments. However,the strategies discussed below are commonly
applied to protein-binding fragments, and the same techniques are anticipated to
also be useful for RNA-binding fragments. Once a set of binding fragments is conrmed via two orthogonal assays, analysis of the binders can help prioritize which
fragments to explore. Analysis of binders includes assessing solubility and ligand
eciency. Many fragments are prioritized because of their good aqueous solubility.
This should lead to a lower rate of false positives from aggregation, which is a common problem in HTS programs [55]. FBDD helps to identify small compounds with
high ligand eciency. However, most fragments due to their small size bind with
low anity.In order to optimize fragment–target interactions, the fragment(s) often
go through an optimization where the fragment is merged, linked, or grown.
Merging starts with compounds with overlapping features; for example, common
binding interactions of functional groups. The compounds can be merged where
they overlap to create a compound with increased binding interactions and higher
anity. Merging has also been shown to be helpful for improving selectivity of
protein ligands [61].
Linking of fragments involves the joining of two fragments that do not bind at
overlapping sites. Finding an ideal linker can be very demanding and requires
exploring multiple parameters. First, a linker may not alter the orientation of each
fragment to the binding site. Doing so could reduce important binding interactions
and favorable geometry thereby reducing binding anity. Second, exibility must
be carefully considered. A rigid linker that reduces the degrees of freedom can serve
to reduce the entropic cost paid upon binding. Alternatively, a exible linker could
cause hydrophobic surfaces to become buried intramolecularly. In this case, the
resulting conformation would create an energetic barrier for binding. An ideal linker
would maintain a careful balance of rigidity and exibility while also incorporating
additional opportunities for favorable interactions with the RNA target.
Growing fragments employs synthesis to capture additional interactions with the
RNA target. Before growing a fragment, close analogs of the fragment hit should be
tested to nd the highest anity starting point. These investigations may lead to the
identication of novel growth vectors, which can include the addition of functional
4.3 Screening Methods 63
https://t.me/med1917
groups or moieties that provide increased opportunities for binding-site interactions. In order to grow the fragment, one should consider ligand eciency, synthetic
tractability, and drug-like properties. It is important to have structural information
on the macromolecular target and the fragment binding mode. Without this information optimization becomes very dicult. Often NMR, X-ray crystallography, and
target engagement assays are used to help guide fragment optimization.
As with other binding rst approaches, FBDD does not account for any potential biological impacts the binding may have. Given the complexity and exibility of
RNA structures, binding does not necessarily indicate function. Care must be taken
to consider biological targets and verify functional activity upon binding conrmation. Despite this distinction, FBDD can still be extremely useful in the design and
development of RNA-binding small molecules, and it has become an accepted part
of early-stage drug discovery.
4.3.1.7 Phage Display
Phage display uses bacteriophages to link proteins with the genetic information that
encodes them [62]. It has been used to discover protein and peptide binders to proteins, DNA, and RNA. In this technique, a gene encoding a protein is inserted into
a phage coat protein gene, causing the phage to “display” the protein on its surface
while containing the gene that codes the protein genome, thus linking genotype and
phenotype. These displaying phages can be screened against an immobilized RNA
sequence to enrich for those displaying protein sequences that bind the RNA. Phage
display can be performed iteratively by using the enriched phage from one round as
input for the next. After screening, the coat protein gene from the enriched phage is
sequenced to determine the protein or peptide sequences of the binders.
Previously, Chow and co-authors have used a heptapeptide M13 phage-display
library to nd ligands for the tRNA-binding site of bacterial 16S ribosomal RNA
[63, 64]. Galleni, Vandevenne, and co-authors used a phage display selection of a
synthetic nanobody gene library (dedicated for nucleic acid binding) to nd one
nanobody (a camelid heavy-chain antibody named cAb
3) that binds structured
BC1rib
RNA φBC1 with nM anity as measured by biolayer interferometry (BLI) [65].
4.3.2 Orthogonal Methods
4.3.2.1 Surface Plasmon Resonance
Following the identication of small-molecule binders, the next step in drug discovery is to characterize the anity, stoichiometry, and specicity of the binding
interaction. Unlike many protein targets, which may have an intrinsic function like
enzymatic activity or receptor binding, many of the RNAs being targeted lack an
inherent function in isolation. This limits the options to assays with the ability to
measure direct binding. One method that can address all these factors is surface
plasmon resonance (SPR). The technique measures the interaction of molecules to
a target in real time and can determine both equilibrium binding and kinetic constants. The signals observed in an SPR experiment are proportional to the molecular
weights of the binding partners, which can be used to determine the stoichiometry of
64 4 Screening and Lead Generation Techniques for RNA Binders
https://t.me/med1917
Incident light
Glass slide
Gold layer
Flow cell
Prism
Absorbed light: Resonance condition
Detector
SPR angle
Reflected light
Ligand: RNA
Analyte: Compounds
Association
Analyte
Response (RU)
injection
Steady state
Dissociation
Time
Figure 4.5 (a) SPR configuration. Polarized light is focused onto a biosensor surface
through a glass prism, resulting in the creation of surface plasmons. Absorption of light
occurs at the resonance condition. The location of the absorbed light can be defined in
terms of an SPR angle with the location being in part determined by the refractive index
close to the biosensor surface. Binding interactions occurring near the biosensor surface
will change the refractive index, which results in a shift of the absorbed light, thus
changing the SPR angle. (b) SPR sensorgram. The SPR angle is monitored in real time and
represented on the y-axis as response units (RU). Following injection of analyte, the
sensorgram can be divided into three phases: association, steady state, and dissociation.
the interaction based on the observed signal. In addition, inspection of sensorgrams
can be informative, often detecting the presence of nonspecic binding interactions.
A Brief Overview of SPR Theory SPR is a microuidic system in which an analyte (A)
is owed over a ligand (L) that is immobilized to the surface of a bio-sensor chip
(Figure 4.5a). The biosensor detects molecular interactions through the generation
of a SPR, which is highly sensitive to changes in the refractive index close to
the surface of the biosensor chip [66]. Analyte molecules that interact with the
immobilized ligand alter the refractive index at the chip surface, resulting in a
change in the SPR angle, which is measured in real time and represented in the
form of a sensorgram (Figure 4.5b). Analysis of the sensorgram steady-state phase
will provide information on equilibrium binding, and analysis of the association
and dissociation phases can provide kinetic parameters. The determination of
kinetic constants requires sucient curvature in the association and dissociation
phases of the sensorgram (cf. Figure 4.5b).
The ability to match the KDs determined from both kinetic and equilibrium tting
can increase condence in the values being measured. Early-stage compounds typically exhibit fast kinetics with very steep association and dissociation phases which
precludes the robust measurement of kinetic parameters.
In addition to measuring equilibrium binding and rate constants, sensorgrams can
also provide information about the stoichiometry of the interaction. The signals in
an SPR binding experiment are related to the molecular weights of the ligand and
analyte according to Eq. (4.2):
Mr
R
analyte =
max
R
refers to the immobilization level for the ligand in response units, valency is
ligand
analyteRligand
the number of binding sites, and Mr
Mr
Valency
ligand
analyte
ligand
and Mr
refer to the molecular weights
ligand
(4.2)
Соседние файлы в папке Библиотека им академика М.И. Перельмана