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In Vitro Selection without Intervening Amplification.

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Experimental Section
7 and 8: Freshly prepared [NI(SO,),](ASF,), (1 -2 mmol) was dissolved in SO,
(10 mL) i n a two-legged thick-walled glass vessel at -30°C. The stoichiometric
amount of ligand, also dissolved in SO, (10 mL) was added. A color change to light
green (7) and to yellow (8)indicated immediate reaction, and the mixture was stirred
for 1 h in order to complete the reaction. The product was obtained in quantitative
yield after removal of the solvent.
7: NMR (S0,/CD,CI,/CFC13/TMS/309 K): 6('H) =7.8 (s, br); 6(I9F) = 51.3
(s, br, SF,); IR: vNS= 1508 cm-' (vs, br). Elemental analysis: calcd: Ni 4.87,
S 15.96; found: Ni 4.74, S 16.2.
8: NMR (S0,/CD,CI,/CFC13/TMS/309 K): 6('H) = 8.6 (s, br), 6("F) = 55.1
(s. br, SF,); IR: vNs 1553, 1531 cm-I (vs, br). Elemental analysis (C,,H,,As,F,,N,,Ni,S,): calcd: Ni 5.79, S 14.24; found: Ni 5.84, S 14.3.
Singlecrystals of 7 suitable for structural analysis were obtained after removal of the
solvent and pumping off of excess ligand under vacuum. Single crystals of 8 were
grown by slow removal of SO, from a solution of the complex in SOJdiethyl ether
at ambient temperature
Received: February 14, 1997 [Z10317IE]
German version: Angew. Chem. 1997,109, 1951-1953
Keywords: main group elements
N ligands sulfur
were performed with the SHELXTL program package [18], the refinements
with the program SHELXL-93 [19.20]
[15] P. D. Beer, M. G. B. Drew, P. B. Leeson, K. Lyssenko, M. I. Ogden, J: Chem.
SOC.Chem. Commun. 1995,929.
[16] J. Ribas, M. Monfort, B. Kumar Ghosh, X. Solans, Anger.. Chem. 1994, 106,
2177; Angew. Chem. Int. Ed. Engl. 1994, 33, 2087.
[17] 7 (294 K): ,Y., = 3.993 x lo-' cm3mol; p = 3.06 pB. 8 (294 K): xu = 8 . 6 8 6 ~
cm3mol; p = 4.51 pg. The temperature dependence of ,%, for the complex
cited in reference [15] indicates ferromagnetic behavior There is an indication
of some weak antiferromagnetic coupling at low temperatures that may be due
to interdimer exchange.
[18] Siemens SHELXTL-Plus: Release for Siemens R3 Crystallographic Research
Systems, Siemens Analytical X-Ray Instruments lnc., Madison, WI, 1989.
[19] G. M. Sheldrick, SHELXL-93, Universitat Gottingen, 1993.
[20] Crystallographic data (excluding structure factors) for the structures reported
in this paper have been deposited with the Cambridge Crystallographic Data
Centre as supplementary publication no. CCDC-100215. Copies of the data
can be obtained free of charge on application to The Director, CCDC, 12
Union Road, Cambridge CB2 lEZ, UK (fax: int. code +(1223) 336-033; email:
- multiple bonds - nickel -
[I] B. Buss, P. G. Jones, R. Mews, M. Noltemeyer, G. M. Sheldrick, Angew.
Chem 1979, 91, 253; Angew. Chem. Int. Ed. Engl. 1979, 18, 231.
[2] J. Hanich, P. Klingelhofer, U. Muller, K. Dehnicke, Z . Anorg. Allg. Chem.
1983, 506, 68.
[3] Review: Gmelin Handbook of Inorganic Chemistry, Sulfur-Nitrogen Compound.$, Part 5 , Springer, Berlin, 1990.
[4] Review: K. Dehnicke, U. Muller, Comments Inorg. Chem. 1985, 4, 213.
[5] Reviews: H. W. Roesky, K. K. Pandey, Adv. Inorg. Chem. Rudiochem. 1983,26,
337; K. K . Pandey, Prog. Inorg. Chem. 1992,40,445; M. Herberhold, Nachr.
Chem. Tech. Lab. 1981,29,365; T. Chivers, F. Edelmann, Polyhedron 1986,5,
1661; P. F.Kelly, F D. Woolins, ibid. 1986, 5, 607.
[6] B. Buss, W. Clegg, G. Hartmann, P. G. Jones, R. Mews, M. Noltemeyer, G M.
Sheldrick, J. Chem. Soc. Dalton Trans. 1981, 61 ; U. Behrens, R. Hoppenheit,
W. Isenberg, E. Lork, J. Petersen, R. Mews, Z . Naturforsch. B 1994,49, 238.
[7] J. Petersen, E. Lork, R. Mews, unpublished results.
[S] 0. Glemser, H. Meyer, A. Haas, Chem. Ber. 1%5,98,2049.
[9] 0. Glemser. J. Wegener, R. Hofer, Chem. Ber. 1972, 105, 474.
[lo] 0. Glemser, W. Koch, Z . Naturforsch. B 1968, 23, 745.
Ill] D. B Beach, W. L. Jolly, R. Mews, A. Waterfeld, Inorg. Chem. 1984,23,4080.
Chem. Commun. 1979, 278.
[12] R. Mews, J. Chem. SOC.
[13] X-ray crystal structure analysis of 7 (Cl,H3,As,F,,N,,NiS6), M , =1205.44;
crystal dimensions 0.8 x 0.4 x 0.3 mm3; orthorhombic, space group Pbca,
u=1303.2(3). b=1263.5(3), c = 2499.3(5)pm, V=4.115(2)nm3, Z = 4 ,
=1.946 Mgm-', p = 2.511 mm-'. A single-crystal suitable for X-ray
analysis was fixed to a glass fiber with Kel-F oil and measured at - 100°C on
a Siemens P4 four-circle diffractometer (graphite monochromator, Mo,. radiation, 6420 scan). Of the 5876 measured reflections (2.39"<8<27.5") 4687
independent reflections remained after averaging. The structure was solved by
direct methods. The refinement calculations converged at wR, = 0.1316
(refinement against F 2 ) for all 4687 reflections and 267 variables (R1 = 0.0514
for 2851 reflections with 1>2a(I)). Heavy atoms were assigned individual
anisotropic deflection parameters. Hydrogen atoms were calculated with a
riding model and common isotropic temperature factors. A difference Fourier
synthesis indicated no residual electron density beyond +730 enm-3 and
-929 enm-'. The structure solution and preparation of the drawings were
performed with the SHELXTL program package [18], the refinements with the
program SHELXL-93 [1920]
M , = 2026.43;
[I41 X-ray crystal structure analysis of 8 (C,,H,,As,F,,N,,Ni,S,),
crystal dimensions 0 6 x 0.4 x 0.3 mm3; triclinic, space group, a = 1190.8(2),
b = 1375.3(3), c = 2313.7(5) pm, a =77.40(3), 6 =75.51(3), y = 64.50(3)",
V = 3.2847(11)nm3, 2 = 2, pEIlcd
= 2.049 Mgm-', p = 3.018 mm-I. A single-crystal suitable for X-ray analysis was fixed to a glass fiber with Kel-F oil
and measured at - 100 "C on a Siemens P4 four-circle diffractometer (graphite
monochromator, Mo,, radiation, 4 2 0 scan). 12076 reflections were measured
(2.62" <0<25'). The structure was solved by direct methods. Due to twinning
in the crystal, some reflections lay so close to each other that they appeared
with greater than normal intensity. These reflections had to be removed from
the data set before the final refinement. 912 of the 12076 reflections were
deleted under the following criteria: > F:' and F: > 30(F:). 10379 reflections
were obtained after averaging of the remaining 11164 intensities. The final
refinement calculations converged at wR, = 0.2296 (refinement against F 2 )for
10379 reflections and 905 variables (R1 = 0.0873). Heavy atoms were assigned
individual anisotropic deflection parameters. Hydrogen atoms were calculated
with a riding model and common isotropic temperature factors. A difference
Fourier synthesis indicated no residual electron density beyond + 1872enm-'
and -885enm-? The structure solution and preparation of the drawings
Angew. Chem. Int. Ed. En,pl. 1997,36, No. 17
In Vitro Selection without Intervening
Joseph Smith and Eric V. Anslyn*
Aptamers to medically important targets have potential value
as drug leads."] Currently, aptamer selection procedures are
typically limited to unmodified DNA and RNA. When modified oligomers are incorporated, the selection procedure is terminated after just one round of amplification since polymerases
will not tolerate most chemically modified mononucleotides.[']
However, if the modified nucleotides are not part of the random
region that is amplified by the polymerase chain reaction (PCR)
after each selection, they would not disturb the amplification.
Unfortunately, using such a method would require reincorporation of the modified oligomers into the library after each selection cycle, making the method quite tedious. This limitation
would be circumvented if the screening process did not involve
amplification during each selection cycle, but instead relied on
a single amplification of the random region after the final round
of selection. If the modified region was separated from the random region by a PCR primer (Scheme l), then the successful
Scheme 1. Representation of the DNA aptamers found
aptamer could be resynthesized in a manner similar to that used
to produce the original library. In addition, such a recursive
selection without intervening amplification should yield highaffinity aptamers in a fraction of the time required for in vitro
selection. Although we do not describe the use of modified nucleotides here, we introduce three requirements for in vitro selection without intervening amplification and demonstrate its viability.
[*I Prof. E. V. Anslyn, Dr. J. Smith
The Department of Chemistry and Biochemistry
The University of Texas at Austin
Austin, TX 78712 (USA)
Fax: Int. code +(512)471-8696
[**I We gratefully acknowledge financial support for this work from the National
Science Foundation, the Texas Advanced Tech&ology Program, Sloan and
Dreyfus Foundation Awards to E. V. A., and a University of Texas Fellowship
to J. S.
0 WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1997
0570 0833/97/3617-1879 $17.50+ Sol0
We envisioned three minimal conditions that must be met
before in vitro screening of oligonucleotide libraries can be
achieved without intervening amplification. First, the number
of copies of ligand-binding sequences must be great enough to
survive iterative small losses during the entire process. Second,
the ratio of signal to noise (ratio of ligand-binding to nonspecific binding sequences) must increase with every round of
screening until the noise is totally overwhelmed. Since the signal
is not amplified, the noise must be capable of being repetitively
reduced until signal strength is greater than noise level. The
third condition is that the amplification after the last selection
round must be sufficiently effective so that polymerases can
make enough copies of the few surviving sequences for eventual
identification. As an initial test of the concept, iterative screenings for aptamers to ATP were performed without amplification. We used ATP since it has been previously targeted;I3] hence
we could compare our new method to the literature method for
in vitro selection. Steps from the new method and the traditional
method are schematically summarized in Figure 1.
Pool of
Random Oligonucleotides
PCR Amplification
(+) selection
of 4.2 x lo7 cpm. After the first selection round, the ATP-eluted
oligonucleotides had an activity of near lo5 cpm, less than
0.25 % of the original quantity loaded. Since almost all the sequences lost during selection are likely oligonucieotides that do
not bind the target, this serves as an estimate of noise. After the
second cycle, the activity of the ATP-eluted DNA had dropped
to about 600 cpm. Retained noise was again reduced over 99%.
For later cycles no measurable radioactivity above background
could be detected. Since the amount of signal was unknown, we
could not determine at this point in the selection whether the
second condition had been met.
The third condition was met using the "rare-DNA PCR"
protocol created specifically for the amplification of DNA with
a low number of copies.151The effective range of the optimized
amplification protocol is reported to be from 1 to 20000 molecules of DNA as the initial substrates. Also, rare-DNA PCR has
been used to quantitate less than ten molecules of herpes simplex
viral DNA in a reaction that also had 100 ng of contaminating
nonviral DNA as background.[61Although use of a single amplification at the end of an in vitro selection protocol requires
more cycles than if each selection cycle is followed by amplification, the former has the advantage that it should introduce less
replication bias in the final populations than the latter.C71
After the rare-DNA protocol was performed (see the Experimental Section), the amplified DNA was cloned and ten clones
were sequenced. Three sequences had duplicates. There were
obvious homologies among the three with two consensus regions. Figure 2 shows the base pairing of these sequences based
Figure 1. Schematic representation of the new and traditional in vitro selection
methods. The "no PCR' sign is only meant to imply a lack of PCR during the
selection cycles since a final PCR step is still required. 0 = ATP, (-) selection
indicates an acetate-agarose precolumn, and (+) selection indicates an ATPagarose column. dsDNA = double-stranded DNA, ssDNA = single-stranded
The first condition of recursive selection without amplification was satisfied with the synthesis of a library of DNA
oligonucleotides containing an 18-nucleotide random region,C4]
which after labeling and purification yielded roughly 17800
copies per distinct sequence. Six cycles of selection were then
performed without any intervening amplification (see the Experimental Section).
As mentioned in reference to the second condition, noise reduction is of paramount importance in this method. Therefore,
the radioactive decay (in counts per minute (cpm)) was measured after each selection cycle to monitor the progress of the
procedure (Table 1). The initial library had a total activity
Kd = 15-20 phn
Figure 2. Base pairings based on the proposed general sequence for an ATP DNA
aptamer[3]. The sequences in bold are thought to form two stacked guanine
quartets. The italicized sequences are parts of the fixed primer regions. The methods
of isocratic elution[8], membrane diafiltration[lb 31, and equilibrium gel filtration[9] were used to determine the dissociation constant for the ATP-aptamer
complex. 0 indicates a non-Watson-Crick base pair.
Table 1 Review of the six cycles of selection. cv = column volumn, fb = folding
pH 7.5)
buffer ( 3 0 0 m ~KCI, 5mM MgCI,, 2 0 m Tris,
3 f
6 f
30 pM
ATP in fb
800 pL)
2c v
+ 5 cv
3 mL,
5 mM ATP in fb
4.2x 10'
(300 pL)
I xi05
I cv
i 2 cv
3 mL,
10 mM ATP in fb
0 WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1997
on Huizenga's and Szostak's postulated general sequenceC3Ifor
an ATP DNA aptamer. Indeed, according to the number of
flanking base pairs stabilizing the guanine-rich regions of the
sequences, the order of ATP affinity found correlates with the
predictions of Huizenga and Szostak.
The original method of Huizenga and Szostak proceeded
through several rounds of screening, amplifying, isolating
ssDNA, radiolabeling, and purifying before a DNA population
was developed with moderate affinity to ATP.13]After 17 clones
had been sequenced, no consensus sequence was found. One
clone was chosen for a series of truncation experiments wherein
the ATP-binding domain was localized to a region 42 nucle-
0570-0833/97/3617-1880$17.50+ .50/0
Angew. Chem. Inr. Ed. Eng/. 1997, 36, No. 17
otides in length. In vitro “evolution” was done on this region at
30% mutagenesis. and four more rounds of in vitro selection
followed before this second population was cloned. From these
sequences, a consensus region was discovered. Certainly though
this work is a pioneering achievement in the field, it is an example of how the conventional protocol is significantly more
involved than that presented here.
In summary, a novel in vitro selection protocol has been designed to take advantage of a combinatorial library of small size
that has multiple copies of every distinct sequence. The method
condensed the many days of a typical screening strategy to less
than two days. This was a proof-of-concept experiment that
showed that the new method succeeds by creating a large number of copies of individual sequences in the initial random pool,
consistently reducing the level of nonspecific binding sequences
per selection round, and effectively amplifying the few surviving
Since only the original synthesized sequences were used for all
the screenings, the technique should allow for the iterative
in vitro selection of modified oligonucleotides that previously
could not undergo this powerful process.“01 Hence, this method
should significantly increase the power of the in vitro selection
method and is the direction that we are currently investigating.
Experimental Section
The DNA library[4] (31 mg) was labeled at the Yend with [y-”P]ATP, purified by
gel chromatography, and suspended in 300 mL of folding buffer (300 mM KCI, 5 mM
MgCI,. 20mM Tris. pH 7 5 ) . After cooling down to room temperature following
denaturation at 75 C, the ”P-labeled DNA was loaded onto an acetate-agarose
precolumn (300 pL), which was attached directly to a 2.5mM ATP-agarose column
(800 pL, Sigma). The precolumn was washed with 600 pL of buffer, and the eluted
DNA was allowed to equilibrate on the ATP-agarose column for 10 min. The
precolumn was discarded after a single use as were all subsequent columns. After
equilibration, the ATP-agarose column was washed with 4 mL of folding buffer to
elute unbound or weakly bound oligonucleotides. The retained DNA was eluted
with 3 mL of the ATP elution buffer (5mM ATP in folding buffer) and collected in
500 pL fractions.
In order to perform another round ofselection, the ATP had to be removed. Hence,
the eluted fractions were collected directly into Microcon-3 microcentrifuge devices
(3000 D cutoff, Amicon) After membrane diafiltration. about 98% of the total
ATP was removed. The filtered fractions were then pooled, and folding buffer was
added until a final volume of 10 mL was attained. Theconcentration ofcontaminating ATP concentration was 3 0 p for
~ the DNA sample, which was over 80 times
more dilute than that of the 2 . 5 m ~ATP-agarose column. Each cycle of selection
started with a new set of stacked affinity columns, i.e. a precolumn attached to a
ligand column. The screening cycles for the ATP aptamers are summarized in
Table 1
The rare-DNA PCR was performed as follows: On the last cycle the DNA was
eluted from the ATP-agarose column with 3 mL of 1OmM ATP in 20mM Tris,
pH 7.5 This last fraction was precipitated twice from ethanol, and the PCR reagents
(50mM KCI, 8mM MgSO,, lOmM (NH,),SO,, 20mM Tris, pH 8 8 at 25”C, 200pM
dNTPs, 0.1 YOTriton X-100,20 units of Deep Vent (exo-) DNA polymerase, 0.5 pg
5’-primer, 0.5 pg 3’-primer) were added. Thermal cycling (94’C for 45 s; 42-C for
90 s: 60 C for 45 s . 45 cycles) was done in a microcentrifuge tube that had first been
irradiated with UV light. A positive control containing a dilute solution (-20000
molecules) o f a 52-mer. and a negative control containing no DNA also underwent
the same amplification protocol. Gel electrophoresis after amplification showed
DNA in all lanes except the negative control.
Received: February 27, 1997 [Z10170IE]
German version: Angen. Chem. 1997, 109, 1956-1958
Keywords: aptamers * combinatorial chemistry - in vitro selection . nucleotides - polymerase chain reaction
[I] L. C. Bock, L C. GI-iffin, J. A. Latham, E. H. Vermaas, J. J. Toole, Nature
1992,335. 564 566: J. R. Lorsch, J. W. Szostak, Biochemistry 1994,33,973982: D. Smith. G. P. Kirschenheuter, J. Charlton, D. M. Guidot, J. E. Repine,
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Brorhcmrsrrj, 1994.33,10450--10456;C. Tuerk, S. MacDougal, L. Gold, Proc.
Nuti. Acud. Sci. U S A 1992,8Y, 6988-6992; C. Tuerk, S MacDougal-Waugh,
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J. Am. Chem. Soc. 1992,114,3990-3991; M. Famulok, ;hid 1994,116,16981706; J. G. Connell, M. Illangesekare, M. Yarus, Biochemistry 1993,32, 5497;
M. Sassanfar, J. W. Szostak, Nature 1993, 364, 550-553: a ) M. G Wallis, U.
von Ahsen, R. Schroeder, M. Famulok, Chem. Biol. 1995,2.543-552; b) R. D
Jenison, S.C. Gill, A. Pardi, B. Polisky, Science 1994, 263, 1425-1429; J. F.
Milligan, M. D. Matteucci, J. C Martin, J Med. Chem. 1993. 36,1923-1937;
J. D. Ecker, S. T.Crooke, Biotechnology 1995, 13, 351 - 360.
121 Some exceptions include 2’-amino- and 2’-fluoro substitutions on sugar (H.
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1-pentynyl substitution on pyrimidine (J. A. Latham. R. Johnson. J. J. Toole,
Nucleic Acids Res. 1994, 22, 2817-2822).
[3] D. E. Huizenga, J. W. Szostak, Biochemistr? 1995, 34, 656 665.
[4] Oligonucleotides were purchased HPLC-purified from Operon. The initial sequence was S’-GAATTCCAGATCTCT-(lSN)-GATATCAGGATCCCA-3’.
The two primers were 5’-GAATTCCAGATCTCT-3’ and 5’-TGGGATCCTGATATC-3’. These sequences incorporated EcnR I and BamH I digestion enzyme restriction sites.
[S] D. M. Coen in Current Profocols in Molecular BLology (Eds. F. M. Ausubel, R.
Brent, R. E. Kingston, D D. Moore, J. G. Seidman. J. A. Smith, K Struhl).
Current Protocols, New York, 1994, Chapter 15.4.
[6] J. P. Katz, E. T. Bodin, D. M. Coen, J. Viral. 1990, 64. 1690-1694.
[7] S . A. Kauffman, The Origrns o f o r d e r : SelJOrganizatron undSelecrion in Evolution, Oxford University Press, New York, 1993. Chapter 3.
[8] D. H. Freeman, And. Chem. 1972,44,117-120; B. M. Dunn, I. M. Chaiken,
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[9] J. P. Hummel, W. J. Dreyer, Biochim. Biophys. Acta 1962, 63. 530
[lo] Another method for the use ofmodified nucleotides in the Sregion of a primer
has been described recently: J. Burmeister, G. von Kiedrowski, A. D. Ellington,
Angen. Chem. 1997, 109. 1379-1381; Angeu. Chem. Int. Ed. Engl. 1997, 36,
The Synthesis of Enantiopure
o-Methanoprolines and o-Methanopipecolic
Acids by a Novel Cyclopropanation Reaction:
The “Flattening” of Proline **
Stephen Hanessian,* Ulrich Reinhold, and
Gabriella Gentile
Proline occupies a prominent position in the hierarchy of
natural amino acid constituents of mammalian proteins.“ As
part of a peptidic motif, its unique structure results in secondary
amide bonds, leading to important conformational and functional consequences.[21 For example, the well-known cis- trans
isomerism in prolylamides is associated with vitally important
biological phenomena and functions, such as protein folding,[31
hormone regulation,[41r e c ~ g n i t i o n ) ~and
] transmembrane signalingc6]to mention a few. The importance of cis-trans conformational changes is manifested by the role that peptidyl prolyl
isomerases such as the immunophilins play in immunoregulation.[’] Proline has also figured prominently as a component of
therapeutic agents,[*] in drug design,[g1and in probing enzyme
activity.“ ‘1
Conformationally constrained analogues of proline have
been used extensively in connection with peptidomimetic research.“ ’] Although 2,3- and 3,4-methanoprolines have been
Prof. Dr. S. Hanessian, Dr. U. Reinhold, G. Gentile
Department of Chemistry
Universiti: de Montrkal
C. P 6128, Succ. Centre-ville
Montreal, QC, H3C 357 (Canada)
Fax: Int. code +(514)343-5728
[**I We thank NSERC for generous financial assistance through the Medicinal
Chemistry Chair program. We thank Dr. Michel Simard for the X-ray analyses. U. R. acknowledges a DFG Research Fellowship from the Deutsche
Forschungsgemeinschaft. G. G. thanks the University of Siena and the Italian
C. N. R. for a summer fellowship.
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without, intervening, selection, amplification, vitro
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