| RNaseAlert® Lab
Test Kit
RNase A Molecules are Used to
Study the Structure of Amyloid-like Fibrils
Ambion’s RNaseAlert® Lab Test Kit is not only used to detect contaminating RNase activity in samples
and reagents but is also useful in biological assays. In just
30 minutes, as little as 3.5 x 10-7 units (~0.5 pg) of RNase
A activity can be detected.
RNaseAlert Tests for Assessing Protein Conformation:
Amyloid-like fibrils that form in vitro from soluble, native
proteins are often studied as models for elucidating disease
processes or for protein folding. Sambashivan and colleagues
[1] recently published data that reconcile the idea that the
native structure of functional proteins has the lowest free energy
of all possible configurations with the observation that many
normally soluble proteins can form amyloid fibrils that are also
very stable (i.e., resistant to degradation and disassembly by
chemicals or proteases). Modified RNase A molecules were used
to show that proteins are not completely refolded to create fibrils,
but instead, retain features of the native protein that have
RNase activity measurable with RNaseAlert technology.
The Significance of Amyloid
Amyloid is a general term for protein deposits
in tissues that share at least three characteristics: (1) extracellular,
nonbranching fibrils, (2) affinity for Congo red, an aromatic
dye, that results in changes in fluorescence intensity, and (3)
cross-beta sheet quaternary structure that creates a characteristic
X-ray diffraction pattern. It is still not known whether amyloid
plaques are a cause or symptom of diseases like systemic amyloidosis;
diabetes mellitus type 2;
Alzheimer’s, Parkinson’s, or Huntington’s disease; and spongiform
encephalopathies (prion diseases). Understanding the structure and formation
of amyloid fibrils may advance disease prevention or treatment options, but
fibrillar features (e.g., large size and variable length) have made the structure
difficult to study. In a carefully designed study, Sambashivan and colleagues
[1] successfully used modified RNase A molecules to examine protein conformation
within fibrils.
Advantages of Using RNase A
Four characteristics of RNase A make it useful
for studying amyloid-like fibril structure. First, each RNase
A monomer contains four disulfide bonds which limit conformational
changes. Second, RNase activity can be easily measured with a
fluorescence-based assay (see box below, More
about the RNaseAlert® Lab
TestKit & QC System). Third, two essential catalytic
histidine residues (H12 and H119) are located on separate domains
(core domain and carboxy-terminal b-strand) that are linked by
a hinge loop. Fourth, RNase A can be manipulated in vitro (freeze-dried
from acetic acid) to form dimers, in which the carboxy-terminal
b-strand interacts with the core domain of its partner [2].
Detecting RNase Activity in Amyloid-like
Fibrils
The last two characteristics described above
were exploited to create two inactive mutants (histidine to alanine
substitutions: H12A and H119A) that each had amyloidogenic sequences
(ten glutamine residues flanked by glycine residues: Q10) in
place of the hinge loop. Each mutant was shown to form amyloid-like
fibrils by Congo red and X-ray diffraction analyses, and the
resulting fibrils did not have RNase activity. When mutants were
mixed and allowed to form fibrils together, RNase activity was
detectable. This indicated that the histidine in the nonmutated
carboxy-terminal b-strand of the Q10-H12A mutant can interact
with the histidine in the nonmutated core domain of the Q10-H119A
mutant to create an RNase A active site.
The Proposed Model
Based on crystal structure and biological activity,
Sambashivan and colleagues propose a model of domain-swapped
functional units for RNase A amyloid-like fibrils. The native
structure of the carboxy-terminal b-strand and core domain of
RNase A were essentially retained. The spine of the fibril was
a twisted pair of interdigitated, antiparallel b-sheets that
were formed by the Q10 insertions, suggesting that protein refolding
is not required to create fibrils. See Sambashivan et al. [1]
for complete details about the experiments and controls used
in this study.
| More About the RNaseAlert® Lab
Test Kit & QC System |
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Sambashivan et al. [1] used Ambion’s
RNaseAlert® technology (patent pending) to assay biological
activity in their structural biology research, a very specialized
application. However, RNaseAlert is also useful in everyday
experiments for fast, sensitive detection of RNase contamination
that could interfere with RNA-based experiments (Figure
1). The RNaseAlert tests are sensitive to several RNases,
including RNase A, RNase T1, RNase I, micrococcal nuclease,
S1 nuclease, mung bean nuclease, and Benzonase®.
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| Figure
1. Schematic of RNaseAlert® Procedure. |
RNaseAlert uses a novel RNA substrate
tagged with a fluorescent reporter molecule (fluor) and
a quencher to detect RNase activity. In the absence of
RNases, the physical proximity of the quencher dampens
fluorescence from the fluor. RNases cleave the RNA substrates,
and the fluor becomes spatially separated in solution from
the quencher, emitting a bright green signal when excited
by light of the appropriate wavelength. Fluorescence can
be readily detected by eye upon illumination on a UV box,
or with a filter-based or monochromator-based fluorometer.
Since the fluorescence of the RNaseAlert Substrate increases
over time when RNase activity is present, results monitored
with a fluorometer can be evaluated kinetically.
The RNaseAlert Lab Test Kit is formatted
for small sample numbers, while the RNaseAlert QC System
is designed for high throughput assays using 96 well plates.
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