Download Gene targeting by hybridization-hydrolysis process

August 2004
Gene targeting by hybridization-hydrolysis process
By Jean-Michel Lélias
Introduction
We have developed a simple procedure* to
significantly deplete or eliminate specific gene
transcripts from a complex population of cDNA
molecules that represent individual mRNA species.
Single stranded cDNA molecules are mixed with an
excess of oligonucleotides corresponding to the
reverse and complement sequences of specific
cDNA targets in regions where the gene sequences
contain an appropriate restriction site. When the
oligonucleotides anneal to their targets, the
hybridization reconstitutes the short double
stranded structure necessary to be specifically
recognized and hydrolyzed by a restriction
endonuclease while all other single stranded
molecules containing only one strand of the
restriction site sequence are not affected (Figure 1).
As a consequence of this very specific cut, the two
extremities of the cDNA molecules targeted are
separated and it becomes possible to discriminate
them from the rest of the complex population. The
technology was applied during cDNA library
construction
to
dramatically
reduce
the
representation of specific transcripts among the
cDNA clones.
Figure 1
Schematic representation of the process
oligonucleotide
cDNA
endonuclease
Oligonucleotides anneal to their cDNA targets in
regions that contain the restriction site. This specific
hybridization reconstitutes the double stranded DNA
structure to be hydrolyzed by the endonuclease.
Specific hydrolysis of targeted cDNA molecules
We only tested a set of enzymes active at temperatures that minimize non-specific hybridization of singlestand DNA. The endonuclease Bsa JI (5’-CCNNGG-3’) was selected because of its full activity at 60°C and
the high frequency of its restriction site in mRNA sequences. The specificity of this enzyme to hydrolyze
short double-strand structures reconstituted by oligonucleotides annealed to their corresponding single
stranded cDNA molecules is illustrated in Figure 2. This experiment was carried out with cDNAs obtained
from mRNA molecules synthesized in vitro from a recombinant clone corresponding to the human beta actin
gene. After one hour of digestion, shorter cDNA fragments, resulting from hydrolysis, were only observed
when a mixture of three different oligonucleotides corresponding to different regions of the gene was added
to the reaction in the presence of the enzyme.
M
1
2
3
Figure 2
Analysis of cDNAs after one hour treatment
Radiolabeled cDNA samples were loaded on a 1% agarose
gel. After electrophoresis, the gel was dried and exposed to
X-ray film. Lane M: Radiolabeled 1 kb DNA ladder. Lane 1:
Human beta actin cDNA after one hour in the reaction
conditions with the corresponding oligonucleotides but
without the enzyme. Lane 2: The same cDNA and conditions
with the enzyme Bsa JI but without the oligonucleotides.
Lane 3: The same cDNA and conditions in the presence of
both the enzyme and the oligonucleotides.
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Elimination of specific cDNA targets during library construction
The following data were obtained by integrating the hybridization-hydrolysis process with a cloning
1
technique that involves a step where the cDNAs are single stranded. In this example (Figure 3), each
single stranded cDNA molecule needs to contain both a specific primer sequence at one end and a poly(G)
stretch at the other end to be inserted into the cloning vector. Hydrolysis by Bsa JI in the presence of
specific oligonucleotides is performed after the d(G)-tailing of the cDNA before the annealing step into the
cloning vector.
Figure 3
Schematic representation of the cloning technique used for library construction
The cDNAs are reverse transcribed from mRNA molecules by using a synthetic oligonucleotide, 5’-CCCGGG(T) 24 -3’,
as a primer. The mRNAs are removed by alkaline hydrolysis, and the remaining single-strand cDNA molecules are
(dG)-tailed. The cDNAs are then annealed with the adapter 5’- (A) 24 CCCGGGAGCT -3’, and the plasmid pT3 that has
been linearized with Pst I, (dC)-tailed and further digested with Sac I. Once the single stranded region is ligated and
repaired, it produces recombinant molecules in which the inserts are bound at their 5’ termini by a poly(C) and at their
3’ termini by a Sac I site (reconstituted by the cohesive ends of the primer/adapter and vector). These molecules are
finally transformed into E. coli to obtain the ampicillin-resistant clones.
To demonstrate the feasibility of applying this technology to any cDNA target, independently of the
abundance of the corresponding transcripts in cells, different human genes were chosen as examples to
represent three main classes of abundance.2 Actin gamma 1 (ACTG1), actin beta (ACTB) and ribosomal
protein L3 (RPL3), with respectively 13938, 10729 and 8508 sequences found in the GenBank® database,
were selected to represent the class of the most abundant transcripts. Eukaryotic translation initiation factor
3 subunit 2 (EIF3S2), RuvB-like 2 (RUVBL2) and apoptosis antagonizing transcription factor (AATF), with
respectively 1096, 708 and 421 sequences, were chosen to represent the intermediate class of abundance.
Neutrophil cytosolic factor 4 (NCF4), activating transcription factor 2 (ATF2) and general transcription factor
IIE polypeptide 1 (GTF2E1), with respectively 99, 73 and 40 sequences, were chosen to represent the class
of the lowest abundance.
Each mRNA sequence contained between 6 and 20 Bsa JI sites. Three oligonucleotides were synthesized
for each one of these genes to cover specifically three different regions to be hydrolyzed during
hybridization. One set of primers was also designed for each cDNA target to specifically detect their
presence by polymerase chain reaction (PCR) in the resulting libraries.
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Figure 4
Analysis of cDNAs before and after treatment
Multiple experiments have been carried out
for different periods of time and with different
combinations of oligonucleotides. The best
results were observed after 18 hours of
incubation with a set of nine oligonucleotides
targeting three genes simultaneously (three
hydrolysis sites per gene). Similar results
were observed when targeting larger sets of
genes (up to 27 oligonucleotides at a time).
For each one of these experiments no
significant difference was ever observed
when comparing the overall profiles of the
complex cDNA populations and their
resulting libraries before and after treatment.
Figure 4 shows some of these comparative
results obtained with cDNA samples that
were synthesized from a complex population
of RNA in vitro (corresponding to a pool of
different human cDNA libraries previously
established). The fact that the cDNA profiles
look a priori the same, with or without
hydrolysis by Bsa JI, suggests that the
hybridization-hydrolysis process is very
specific to the few cDNA molecules targeted
with the corresponding oligonucleotides and
does not significantly interfere with other
single-strand molecules.
A
M
1
B
2
M
1
2
A: Analysis of the radiolabeled cDNAs before cloning:
Lane M: Radiolabeled 1 kb DNA ladder. Lane 1: cDNA sample
without treatment. Lane 2: The same cDNA after 18 hours of
hydrolysis by Bsa JI in the presence of the oligonucleotides.
B: Analysis of the resulting libraries after Not I digestion:
Lane M: 1 kb DNA ladder. Lane 1: The average insert size of
the library obtained without treatment ranges from 1 to 3 kb.
Lane 2: The exact same average insert size is observed in the
library obtained after 18 hours of treatment.
All cDNA libraries obtained after each experiment had a significant number of recombinant clones (>106 cfu).
Their corresponding DNA was precisely quantified for further PCR application. Figure 5 shows a multiplex
PCR analysis carried out with the two libraries obtained before or after targeting three genes that represent
the most abundant class of transcripts (ACTG1, ACTB and RPL3). One gene from the intermediate class of
abundance (EIF3S2), which was not targeted by hydrolysis in this experiment, was analyzed in parallel as
an internal control. The results demonstrate the specificity and the efficiency of the procedure by showing a
similar abundance level for the internal control in both libraries while the targeted gene levels are decreased
by at least 1000 fold in the library obtained after treatment (the average of 15 cycles difference observed
between the two samples represents over 32000 fold if each PCR cycle were optimal and multiplicative by a
factor of two).
Figure 5
Multiplex PCR analysis for the 3 genes corresponding to the class of the most abundant transcripts
M
10 cycles
-------------+
15 cycles
-------------+
20 cycles
-------------+
M
25 cycles
-------------+
30 cycles
-------------+
35 cycles
-------------+
H3
H2
---------- Internal control
H1
The characterization of the genes was monitored by specific amplification of short products differing from each other by their
size. H1, H2 and H3 represent the PCR products corresponding to the different genes (from the most abundant to the less
abundant in this class). Aliquots were taken every 5 cycles for semi-quantitative analysis by agarose gel electrophoresis.
Lanes M: 1 kb DNA ladder. Lanes (-): Library obtained without treatment. Lanes (+): library obtained after 18h incubation with
Bsa JI and the oligonucleotides.
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Figure 6
Multiplex PCR monitoring the cDNA targets from the intermediate and lowest classes of abundance
M
10 cycles
-------------+
15 cycles
-------------+
20 cycles
-------------+
M
25 cycles
-------------+
30 cycles
-------------+
35 cycles
-------------+
-------- Internal control
M3
M2
M1
L2
------- Internal control
L1
L3
H2 and H3 were used as internal controls. M1, M2 and M3 represent the different genes chosen in the intermediate class of
abundance while L1, L2 and L3 represent those chosen in the class of lowest abundance. Aliquots were taken every 5 cycles
for semi-quantitative analysis by agarose gel electrophoresis. Lanes M: 1 kb DNA ladder. Lanes (-): Library obtained without
treatment. Lanes (+): library obtained after 18h incubation with Bsa JI and the oligonucleotides.
Similar results were obtained (Figure 6) when the technology was applied to less abundant transcripts, but
since the starting number of cDNA molecules targeted was much lower, the process became efficient
enough to completely eliminate the representation of these genes in the resulting libraries (no PCR products
were detectable after 35 cycles of amplification).
Conclusion
A new technology used to specifically target any transcript from a complex population of single-strand cDNA
molecules was applied to dramatically decrease the abundance of selected genes in cDNA libraries. This
innovative procedure offers new alternatives to previous efforts focused on normalizing the amounts of
cDNA molecules representing each expressed gene,2,3 and could potentially be applied to a much broader
range of techniques currently used in molecular biology. One example would be to use this process with
phagemid libraries amplified as circular single stranded DNA molecules and targeted with specific
oligonucleotides by the hybridization-hydrolysis process. After treatment, the second strand DNA would be
synthesized by using a vector-specific sequence common to all recombinant molecules (upstream of the
cDNA inserts). The targeted recombinant molecules would have been hydrolyzed and by consequence
linearized (open molecules essentially single stranded) while all the other would have become double
stranded circular DNA molecules, which are the only molecules that could be efficiently transformed in a
bacterial host strain to create the resulting library. Many other techniques based on selecting single stranded
cDNA molecules could incorporate this process to selectively deplete specific genes. This technology
creates new opportunities to better control the gene representation in a complex population of molecules by
providing a powerful tool to precisely pick and choose the gene representation through selective elimination.
REFERENCES
1.
2.
3.
Lélias, J.-M. et al. (1993) Proc. Natl. Acad. Sci. USA 90(4): 1479-83.
Soares, M. B. et al. (1994) Proc. Natl. Acad. Sci. USA 91(20): 9228-32.
Lélias, J.-M. et al. (1998) Strategies 11(2): 29-32.
* Patent pending. For all information please contact Jean-Michel Lélias at Genofi LLC, 1030 Calle Cordillera, Suite 107,
San Clemente, CA 92673 (e-mail: [email protected]).
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