Analytical Chemistry, Volume 72, Number 20,
Pages 5092-5096
Synthetic oligonucleotide strands ranging from 5-25 units in length are commonly used as standards, probes and templates in various bioanalytical applications. Until recently their preparation, storage and handling were regarded as unimportant but this work provides valuable information to the contrary. The systematic degradation of oligonucleotide strands during sample preparation is investigated by repeatedly freezing/thawing short strands followed by Matrix-Assisted Laser Desorption Ionization Mass Spectrometric (MALDI-MS) analysis. It is shown here that the longevity of an oligonucleotide strand is dependent on several factors including base composition, solution concentrations, strand length as well as thawing conditions.
Several trends in strand
robustness were established. Our studies reveal that the robustness of strands
is base dependent T-mer>A-mer>C-mer>G-mer. Likewise, an increase in the length of the strands increases the
tendency of a sample to degrade.
Another observation included that samples of mixed bases degrade
according to structural conformations.
All of these observations are attributed to the fact that the samples
undergo degradation during sample/solvent isolation during freezing.
The evolution of MALDI-MS has spurred on its
application as a technique for the detection, analysis and quantification of
synthetic oligonucleotide strands, which are often used as standards, chemical
sensors, templates, probes and even drugs.1-5 As a result much work has been done to
improve MALDI-MS analysis including the optimization of instrumental/tuning
parameters,6-7 the discovery
of new matrices such as ATT,8-10 as well as the investigation of
sensitivity enhancers.11 Fortunately this fundamental work has
permitted the extension of MALDI analysis to even more complicated systems such
as oligonucleotide metabolites12 PCR products13 and
single-nucleotide polymorphisms.14
In addition the technique has been elevated to include sequencing work
through enzymatic digestion15,16 or energetic dissociation.17-19
Despite all of these advances in oligonucleotide
detection by MALDI few investigative studies have been performed on the
solution handling process prior to analysis. The questions arise. What are the
detrimental effects resulting from the everyday storage and handling conditions
of typical strands? Are extra precautions required for different strands?
Unfortunately, several researchers have already noted the degradation of these
commercial samples as a result of time, storage conditions or the
repetitiveness of the freezing/thawing process.20-21 For example, Anchordoquy et al.20 noted
that the fast freezing/thawing cycle was more damaging to a lipid/DNA complex
than a slow freezing/thawing process.
This observation of a typical drug delivery system was not only
informative but also cost effective for the drug manufacturer involved.
Since it is not always possible to store oligos
under ideal conditions (-20C, pH>6) the repetitive freezing/thawing of an
oligonucleotide strand can lead to the undesirable decomposition of a sample
through the loss of a terminal phosphate group, a base or the entire
oligonucleotide unit. This observation
relates to the fact that during the freezing/thawing process the oligonucleotide
and buffer components isolate from the solvent system. As a result this isolation increases the
percentage of similar intermolecular attractions and bond breakages.
To our knowledge no comparative study exists which
evaluates the fundamental ruggedness of various oligonucleotides strands. Therefore, this work represents a systematic
analysis to distinguish the robustness of various oligonucleotide samples.
Degradation results are presented as a function of oligonucleotide base
composition, concentration, length and freezing/thawing conditions. The MALDI
analysis of constant base 5-mers (5¢ AAA AA 3¢, 5¢ CCC CC 3¢ etc.) as well as various strands are
analyzed for their durability during several repetitive freezing/thawing
cycles. The freezing/thawing rates are also studied by applying two methods:
slow freezing at 0C or rapid freezing by placing the sample in liquid
nitrogen.
MALDI-MS lends itself as an excellent technique for
this analysis due to its efficient and effective sample preparation. In
comparison to the traditional native gel electrophoresis analysis, which often
requires hours to days for completion, MALDI only requires minutes from sample
preparation to data collection. This factor is a definite advantage for a
freezing/thawing study because during the cycling process, hundreds of samples
need to be analyzed rapidly. The one
drawback of MALDI analysis is the possibility of fragmentation during the
ionization/desorption process. However, the previous work by Pomerantz et al.
has illustrated that the decomposition of oligo strands during the MALDI event
increases with molecular weight.22 Therefore, to avoid this complication samples of
relatively short length (5-mers to 12-mers) were intentionally chosen to ensure
the absence of degradation products from the ionization/desorption process.
All oligonucleotides
(Midland Certified Reagent Company-Midland, TX) were dissolved in Millipure
water to yield the appropriate concentrations ranging from 1-50M. From each
prepared solution 100 L was transferred into a 1.5-mL centrifuge tube. The
oligonucleotide solutions were then repeatedly frozen/thawed by one of the
following techniques: during the first procedure, which tested the robustness
of the sample strands under stressed situations, the solutions were frozen in
liquid N2 for 1 minute and then thawed in water bath (65°C) for 5
minutes. The second technique provided
a comparison of typical, everyday laboratory conditions to our simulated,
accelerated conditions. This method was
performed under the following less harsh environment. The samples were frozen in
laboratory freezer (0°C) for 5 hours. Followed
by thawing on the laboratory bench top (31°C) for 1 hour.
After ten freezing/thawing
cycles 1 L of sample was aliquoted from the centrifuge tube
and prepared for MALDI analysis. Thawing caused during shipping or from the
daily freezer removal was not included as part of cycling process since their
effects are considered minimal. Each
sample was repeated in triplicate to ensure reproducibility.
All spectra were acquired on
the PerSeptive Biosystems Voyager-DE Biospectrometry Workstation (Framingham,
MA) The instrumental conditions were as follows: linear mode and negative ion
detection. In this mode a negative
20,000-acceleration voltage is used with the delayed extraction option activated
at 50 ns.
After every ten
freezing/thawing cycles 1L of sample was removed from the centrifuge tube and
mixed with the appropriate matrix. The 10-mg/ml solution of the matrix
6-aza-2-thiothymine (Sigma Chemical Co.,
St. Louis, MO) was prepared daily in 50/50 solution of acetonitrile and 0.1M
ammonium citrate. ATT was chosen for
this study since it is the established matrix for short oligonucleotide
analysis.22 Matrix and analyte
solutions were spotted on the MALDI sample stage in a 2:1 ratio (2L of matrix to 1L of analyte) and allowed to air dry. After visual inspection a water wash was
performed on the MALDI spot to remove excess salt when necessary.23 All spectra were acquired
by averaging 100 shots acquired by rastering across the sample spot. Data was analyzed using the software program
Grams 386.
Influence of Base Composition on Decomposition
Figure 1 represents the decomposition of 5AAA AA 3 after undergoing 0, 20 and 70 repetitive
freezing/thawing cycles respectively. After this process the samples exhibited
decomposition peaks (labeled in the figures as “*”) as a result of 1) the loss
of the terminal phosphate group 2) the loss of a base or 3) the loss of a
complete oligonucleotide unit. Initially each sample was prepared and analyzed
directly after receipt from the vendor and prior to the freeze/thaw cycling
(cycle=0) This is an important step in the procedure because it determined the
threshold energy of the laser irradiance and also established the absence of
failure sequences as an undesirable residual product of the oligonucleotide
synthesis. Once determined the laser irradiation was not deviated therefore the
possibility of ion production from prompt fragmentation was discounted.
The comparison of Figures 1 and 2 illustrates the
degradation of two separate 5-mers of different base compositions (5 AAA AA 3 versus 5 TTT TT 3). The
observed trend indicates that the adenosine (A) sections of the oligo strands
are more susceptible to decomposition than the thymidine (T) sections of a
strand. In general, the breakdown of the A-mer is evident after only twenty
freeze/thaw cycles. It is interesting
to note the immediate decomposition of the A-mer during the first 20 cycles
followed by very little degradation during the following 50 cycles. This observation indicates a trend for the
decomposition to occur during an early phase of the freezing/thawing cycling. In contrast, the T sections are not susceptible
to decomposition, even after 70 cycles
Table
1 lists the oligo sequences used in this section of the study as well the
percentage of strand intact after various cycling periods. This value serves as a robustness
measurement for the various 5-mer strands as illustrated in equation 1:
% Intact = (molecular +
decomposition ions/molecular ion)n cycles x 100 (1)
It is important to measure the decomposition peaks
relative to the molecular ion since previous work has proven an influence of
base composition on mass spectrometric detection as a result of ionization
efficiency.24 Note that the overall trend established from this
data was that the robustness of T-mer>A-mer>C-mer>G-mer. This observation is attributed to the interaction
of each of these strands as it undergoes isolation from the aqueous solvent
system. This relationship follows the
tendency for the various available sites in the strand to undergo hydrogen
bonding characteristic of helical duplexation, intermolecular or intramolecular
bonding. More specifically the adenine-thymidine base pair experiences hydrogen
bonding at two sites whereas the guanine-cytosine base pair undergoes bonding
at three sites. The larger the number
of hydrogen bonds cites a sample contains the greater the decomposition it
experiences.
It is interesting to note that this trend does not
resemble the independently established trend of ionization efficiency for
nucleic acid bases as they undergoes mass spectrometric desorption ionization.
Typically, the loss of the nucleic acid base is the primary fragmentation
reaction that occurs during the ionization process. During this process it has
been suggested that the N-glycosyl bond is weaker for the purine nucleotides
than for the pyrimidine nucleotides resulting in an overall trend of stability
to be (A, G>C>>T) This ionization/desorption trend is contrary to our
results and allows us to conclude that our observations are strictly dependent
on the solution chemistry and not a reflection of the desorption/ ionization
process.
The ruggedness of bases under freezing/thawing
conditions (T-mer>A-mer>C-mer>G-mer) however shows some similarities
to the trend of bases to withstand cleavage from the backbone in an acidic
medium due to hydrolysis of the N-glycosyl linkage (T>>C>G, A). Since
our medium was neutral, this was not a detrimental consideration. To address the issue of which trend is more
detrimental to an oligonucleotide strand (freezing/thawing versus acidic
medium) future studies could be conducted in a more acidic medium and analyzed.
Influence of Solution Concentration on Decomposition
The samples 5 AAA AA 3 and 5 CCC CC 3 were chosen to represent a pyrimidine and purine
while conducting the study on concentration dependence. Figure 3
shows the decomposition of the 5 AAA AA 3 as a) 50µM solution and b) 1 µM
solution after 20 harsh cycles. The
general trend demonstrates that the lower the oligomer concentration, the more
susceptible a short strand is to decomposition. For example, after 70 cycles
the 50M A-mer is 46% intact whereas the 1M sample is only 23% intact. (Spectra are not
shown). The C-mer exhibits a similar
trend with a 21% percent intact value after 50 cycles and a 19% intact value
after 70 cycles.
The results from this section of the study lead to
the conclusion that the analyte is undergoing a separation and isolation from
the remainder of the solvent system while freezing. It also supports that fact
that degradation is caused during this isolation process. As the analyte begins to isolate together, the
more concentrated (50M) solution has a larger component of the
oligonucleotide aggregating together than the 1µM solution. Therefore, the
detrimental influence of the solvent system is smaller for the 50µM sample than
for the 1µM sample. Since the lower concentration (1µM) has a larger solvent:
analyte ratio the harmful effects of the solvent system are more pronounced at
the lower concentration. No significant difference are noted for the purine
versus pyrimidine dependence on concentration since the A-mer and C-mer appear
to degrade equally as quickly at lower concentrations. All of this information can prove is
valuable to researchers who store oligonucleotide strands as standards: short
strands are best stored at low concentrations.
Figure 4 illustrates the effect of repetitive
freezing/thawing on larger mixed base strands at various concentrations. This
figure shows the sample 5 GGG GGA AAA A 3 as it quickly degrades to less than
4% after 20 cycles at the 50 M sample (Figure 4a). Fifty cycles of freezing and thawing are required for similar
results on the 1M sample (Figure 4b). Although this tendency differs from the conclusions drawn in
Figure 3 the observation is attributed to the mixed base composition and
increased length of the strand. The
more complex structure causes steric hindrance and prevents the efficient
aggregation in the solvent system, thereby allowing the detrimental effects of
the solvent system to prevail.
Influence of Strand Length on Decomposition
The spectra displayed in Figure 5 demonstrate the
influence of strand length on the decomposition of a thymidine region of an
oligonucleotide strand and Table 2 provides the experimental calculations. The overall trend that is revealed from
these studies follows the basis that as oligonucleotide length increases the
likelihood of its degradation during freezing/thawing also increases. To
illustrate this Figure 5a shows that after 90 cycles, the 5-mer experienced
little degradation (only 2%) unlike the 12-mer of the same makeup that
fragmented to 40% of its original value.
This observation is attributed to the increased number of hydrogen
bonding interactions that occur for a longer strand and supports the
conclusions drawn from Figure 4. In
analyzing these results it becomes apparent that the length is equally as
important as base composition in determining the robustness of a sample.
Influence of Mixed Bases on Decomposition
All of the 12-mers presented in Figure 6 consist of
longer strands of mixed bases (20µM) that have undergone 50 freezing/thawing
cycles. From Figure 6c we notice that
the TC-mer did not decomposed.
Unexpectedly, the GA-mer degraded more than the GC-mer, which violated
the trend, established from the base composition studies (Table 1.) This
observation, however, is explained by the fact that the GC-mer is
self-complimentary and is undergoing a structural conformation that makes it
more effective in shielding itself from hydrogen bonding interactions within
the solvent system.
Comparison of Unusual Freezing/Thawing to Routine Handling
Most synthetic samples do not experience the unusual
freezing/thawing conditions (Liquid N2/65C) that our samples experienced. Therefore, a comparison study of freezing
thawing rates was performed to compare our results with average conditions
(freezer/countertop) and is shown in Figure 7.
In comparison, the laboratory bench/freezer approach yielded results
similar to our liquid nitrogen method.
The results of leaving the sample on the bench top and returning it to
the freezer 8 times over a 40 hour period (2.5 hours in the freezer: 2.5 hours
on the benchtop) yielded a 55% intact strand.
By referring to Table 1 a similar result would be obtained at
approximately 50 cycles under the liquid nitrogen conditions. Therefore, the slower freezing and thawing
performed in typical laboratory setting is more harmful to the oligonucleotide
samples than our simulated conditions.
The robustness of several
oligonucleotide strands was analyzed during simulated freezing/thawing
processes. The results indicated that
the characteristic robustness of a strand was dependent on base composition (T-mers>A-mers>C-mers>G-mers)
as well as length (5-mers were more sturdy than 12-mers for the T-mer
strands). Concentration was another
consideration, which was illustrated to be influential since a 50-µM concentration
of the 5¢ AAA AA 3¢ solution illustrated less decomposition than
a 1 M solution. Experiments were
also performed to compare typical laboratory conditions to our unusual
freezing/thawing conditions.
This research was supported by a grant from the Office of the Vice-President for Academic Affairs at University of the Sciences in Philadelphia. We also thank Dr. Michael Nedved and Dr. Mark Plucinsky of Johnson & Johnson for their generous assistance and instrument time.
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Table 1. The robustness of the
various 5-mer strands is determined from the percent of strand intact after 10
cycles of freezing in liquid nitrogen and thawing in 65°C bath.
Table 1.
Percentage of Intact
Oligonucleotides
|
||||
|
Freezing/ Thawing Cycles |
5’ TTT TT 3’ |
5’ AAA AA 3’ |
5’ CCC CC 3’ |
5’ GGG GG 3’ |
|
10 |
100 % |
90 (+/-0.4)% |
56 (+/-0.6)% |
48 (+/-0.4)% |
|
20 |
100 % |
74 (+/-0.6)% |
32 (+/-0.8)% |
31 (+/-0.7)% |
|
30 |
100 % |
68 (+/-0.7)% |
30 (+/-0.4)% |
35 (+/-0.8)% |
|
40 |
99 (+/-0.2)% |
66 (+/-1.2)% |
23 (+/-0.9)% |
19 (+/-1.1)% |
|
50 |
98 (+/-0.2)% |
66 (+/-2.5)% |
21 (+/-0.9)% |
18 (+/-1.3)% |
|
60 |
98 (+/-0.2)% |
67 (+/-3.4)% |
19 (+/-1.9)% |
4.0 (+/-0.9)% |
|
70 |
98 (+/-0.3)% |
56 (+/-5.6)% |
19 (+/-2.4)% |
4.2 (+/-1.5)% |
Table 2. The robustness of the T-mer as a function of various strand
length the 5-mer versus the 12-mer.
Table 2.
Percentage of Intact Oligonucleotides
|
||||
|
STRAND LENGTH |
20 cycles |
50 cycles |
70 cycles |
90 cycles |
|
5-mer |
100 % |
98 (+/-0.3)% |
98 (+/-0.2)% |
96 (+/-0.4)% |
|
12-mer |
67 (+/-0.7%) |
55 (+/-0.9)% |
52 (+/-0.8)% |
40 (+/-0.8)% |
FIGURE LEGENDS
Click here to view actual figures.
Figure 1. MALDI mass spectra of 50 mM solution of 5’ AAA AA 3¢ acquired after 0, 20 and 70 cycles of freezing
in liquid nitrogen folllowed by thawing
in 65°C bath. ATT was used as the matrix.
Figure 2. MALDI mass spectra of 50 mM solution of 5’ TTT TT 3¢ acquired after 0, 20 and 70 cycles of
freezing in liquid nitrogen folllowed by
thawing in 65°C bath.
Figure 3. MALDI mass spctra of the strand 5¢ AAA AA 3¢ a) 50mM and b) 1mM of the strand 5¢ AAA AA 3¢ after undergoing 20
freezing/thawing cycles.
Figure 4. MALDI mass spectra of the strand of a) 50mM and b) 1mM solution of 5¢GGG GGA AAA A 3¢ analyzed after 20 cycles of
harsh freezing and thawing.
Figure 5. MALDI mass spectra of a)
5¢ TTT TT 3¢ and b) 5¢ TTT TTT TTT TTT 3¢ after 90 cycles of freezing/thawing
conditions.
Figure 6. MALDI mass spectra of a 50mM of the mixed strands a) 5¢ GG GGG AAA AA 3¢, b) 5¢ GG GGG AAA AA 3¢ and c) 5¢ TT TTT CCC CC 3¢ after 50 cycles of decomposition.
Figure 7. MALDI mass spectra of
the 5´ AAA AA 3’ after freezing and
thawing for an eight hour period on the laboratory bench top and in the
freezer.