Aptamers as molecular recognition elements for electrical nanobiosensors Recent advances in nanotechnology have enabled the development of nanoscale sensors that outperform conventional biosensors . This review summarizes the nanoscale biosensors that use aptamers as molecular recognition elements . The advantages of aptamers over antibodies as sensors are highlighted . These advantages are especially apparent with electrical sensors such as electrochemical sensors or those using field-effect transistors . Introduction With the completion of the Human Genome Project , we have a better understanding of disease-related biomaterials such as DNA , RNA , proteins and small biological molecules . Such knowledge can impact strongly on human health , because the identification of a disease-specific marker at an early stage of a disease can greatly improve the clinical success rate , and improve disease-related mortality . For early detection of disease , it is necessary to locate minute amounts of disease-related proteins , peptides or other small molecules in a patient 's bloodstream or body fluid . Nanotechnology combined with biotechnology is expected to provide paradigm-breaking solutions in this area , as these technologies are extremely sensitive -LSB- 1 -RSB- . Already , nanoscale materials such as quantum dots -LSB- 2 -RSB- and metallic nanoparticles -LSB- 3 , 4 -RSB- , as well as nanodevices such as the nanocantilever -LSB- 5 -RSB- , and nano field-effect transistors -LRB- FETs -RRB- -LSB- 6 -- 9 -RSB- show potential for detecting tiny molecular signals . In this review , we concentrate on nanoscale electrical biosensors with aptamers as molecular recognition elements . Biosensors are devices that can be used to detect the presence of a target analyte . As shown in Fig. 1 , biosensors are built up from a sample delivery component , a molecular recognition subsystem , a transducer changing the binding or reaction of target molecules into a measurable physical signal -LRB- mass , charge , heat or light -RRB- and a signal-processing unit . We are particularly interested here in electrical transducers , because they provide the most promising solutions for point-of-care disease detection . Biosensors using electrical transducers are rapid and convenient , and they do not require heavy optical instruments or expensive measuring devices . Fig. 1A biosensor . MIP molecularly imprinted polymer Aptamers as molecular recognition elements For the highly sensitive detection of a target analyte , the selectivity of the recognition element is vital in sensor performance . Until recently , immunosensors using antibody -- antigen recognition were employed for biodetection . However , highly sensitive and stable biosensors may use different recognition elements . In the 1980s , functional nucleic acids that can specifically bind and regulate viral or celluar proteins with high affinity were discovered -LSB- 10 -RSB- . The major breakthrough for functional nucleic acids occurred in 1990 . First , the presence of small functional RNA molecules -LRB- TAR aptamers -RRB- that can inhibit viral replication -LSB- 11 -RSB- was revealed ; they can effectively bind and inhibit the activity of viral protein , thereby raising the possibility that they may be used as therapeutic agents . In the second study in 1990 , Tuerk and Gold -LSB- 12 -RSB- established the in vitro screening process termed `` systematic evolution of ligand by exponential enrichment '' -LRB- SELEX -RRB- , to identify the RNA ligands with affinity for T4 DNA polymerase and various organic dyes -LSB- 12 -RSB- . Such nucleic acids were termed `` aptamers '' -LSB- from the Latin aptus -LRB- fit or appropriate -RRB- -RSB- by Ellington and Szostak -LSB- 13 -RSB- . The SELEX process is a technique for screening a very large library of oligonucleotides with random sequences by iterative cycles of selection and amplification . Figure 2 shows a schematic diagram of the SELEX process -LSB- 14 -RSB- . The starting pool must be large enough to generate a high probability of producing a desired aptamer -LRB- normally around 1015 different sequences -RRB- . The initial library of random-sequence oligonucleotides , which is obtained through combinatorial chemical synthesis , is incubated with a target of interest . Oligonucleotides showing affinity for the target are partitioned by affinity chromatography or filtration , and then amplified by PCR -LRB- for DNA libraries -RRB- or reverse transcription PCR -LRB- for RNA libraries -RRB- to create a new pool enriched in those oligonucleotides having a higher affinity for the target . As this cycle of selection and amplification is repeated , the abundance of the high-affinity oligonucleotides increases exponentially . Negative selection and counterselection are often employed in order to remove aptamers which bind to supports and molecules similar to the target , respectively . Typically after eight to 15 cycles , cloning and sequencing of the enriched library are carried out , revealing the sequence of oligonucleotides highly specific to the target . Once the sequence information has been obtained , the desired aptamer can be readily produced by chemical synthesis . For a given target molecule , DNA aptamers and RNA aptamers can be generated . However , they are quite different in sequence and three-dimensional structure since DNA lacks the 2 ′ hydroxyl group of the RNA . For example , both the DNA version of the RNA aptamer for ATP and the RNA version of the DNA aptamer for ATP do not recognize ATP -LSB- 15 -RSB- . Fig. 2The systematic evolution of ligand by exponential enrichment -LRB- SELEX -RRB- process -LSB- 14 -RSB- In Table 1 , we summarize the advantages of aptamers over antibodies and the yellow-colored rows detail special advantages of aptamers as recognition elements -LSB- 16 -RSB- . First , aptamers possess high target affinity . It has been generally recognized that aptamer affinity is comparable to or even higher than that of antibodies , even though this is not always true . Unlike antibodies , aptamers can distinguish between targets even if the targets are in the same family . For example , anti-caffeine aptamers have lower affinity -LRB- by a factor of 104 -RRB- for theophylline , despite the two compounds differing by only a single methyl group -LSB- 17 -RSB- . Because aptamers are selected using an in vitro selection process -LRB- SELEX -RRB- , they are cost-effective , have uniform activity , and antitoxin aptamers can be readily synthesized . Antitoxin antibodies are more difficult to prepare , since they are cultured in animal cells . The immobilization of the recognition element can be crucial in biosensors , and this is easier with aptamers than with antibodies , because chemical modification of nucleic acids is simple and straightforward compared with that of antibodies . One of the biggest advantages of aptamers lies in their high stability . Being nucleic acids , aptamers are highly stable , and therefore have unlimited shelf lives . Also , because aptamers can undergo reversible changes of conformation with variations in temperature or salt concentration , aptamer-based sensors are potentially recyclable . The major limitation of aptamers -LRB- especially RNA aptamers -RRB- as molecular recognition elements is degradation by nucleases . However , it was shown that the problem can be overcome through chemical modifications of the sugar at the 2 ′ - position -LSB- 18 -RSB- or use of mirror-image analogs -LSB- 19 -RSB- . Recently chemically modified nucleic acid aptamers for in vitro selections have been reviewed -LSB- 20 -RSB- . Table 1Advantages of aptamers over antibodies -LSB- 16 -RSB- As aptamers show high target affinity and high target specificity , they can be used as therapeutic agents , in affinity columns and as recognition elements in sensors detecting a wide range of targets . There are virtually no limits of targets for aptamers ; small molecules -LSB- 14 -RSB- , proteins -LSB- 15 -RSB- and antibiotics -LSB- 21 -RSB- . The first use of an aptamer as a molecular recognition element in sensors was reported in 1996 , when fluorescent-tagged aptamers were employed for optical detection of human neutrophil elastase -LSB- 22 -RSB- . Since then , various transducers employing aptamers for recognition have been reported . These include the quartz crystal microbalance -LSB- 23 -RSB- , the surface plasmon resonance technique -LSB- 24 -RSB- , evanescent-wave-induced fluorescence -LSB- 25 -RSB- , microcantilevers -LSB- 26 -RSB- , electrochemical sensors -LSB- 27 -- 35 -RSB- and FETs fabricated with single-walled carbon nanotubes -LRB- SWNTs -RRB- -LSB- 36 , 37 -RSB- . In this review , we summarize recent advances in electrochemical and FET types of aptamer sensors , focusing on the high sensitivity and recyclability of the aptamer-containing devices . Electrical aptamer sensors Electrochemical aptamer sensors Typical electrochemical sensors operate by reacting with an analyte of interest and producing an electrical signal proportional to the analyte concentration . Many papers have reported the fabrication of electrochemical aptamer sensors where specific binding of an analyte to an aptamer on an electrode can produce an electrical signal -LSB- 27 -- 35 -RSB- . Some authors use sandwich assays , employing a secondary aptamer for signal enhancement -LSB- 28 , 29 -RSB- . However , a technique that is label-free and fast in response is favored especially for electrical types of biosensors . A difficulty with sandwich assays has recently been removed by Xiao et al. -LSB- 31 -RSB- , who developed a `` signal-on '' architecture . Earlier electrochemical aptamer sensors used a `` signal-off '' architecture -LSB- 30 -RSB- , in which the binding of the target analyte resulted in a decrease in the signal . In such cases , no more than 100 % of the signal can be suppressed by target binding , and hence the dynamic range of the sensor is limited . Also , it is difficult to distinguish a real binding event from a false signal originating from contaminants or sensor degradation . To fabricate the signal-on electrochemical aptamer sensor , Xiao et al. introduced a short DNA sequence tagged with methylene blue -LRB- MB -RRB- that can hybridize with an aptamer . A single-stranded DNA aptamer , which binds to thrombin , was chosen as a model system . Thrombin is a proteolytic enzyme which facilitates blood clotting by converting fibrinogen into fibrin and is also regarded as a tumor marker in the diagnosis of pulmonary metastasis -LSB- 38 -RSB- . Figure 3 shows a schematic diagram of the signal-on aptamer sensor . As shown , the sensor is constructed by immobilizing a thiolated thrombin aptamer to a gold electrode , and an MB-tagged partially complementary DNA is added to form a double-stranded complex . In this configuration , owing to the large persistence length of double-stranded DNA , the MB moiety is far away from the sensor surface , and only a small Faradaic current is observed . The persistence length , defined as the distance over which the direction of a polymer segment persists , provides a measure of elasticity in polymer chains . In the case of double-stranded DNA , the persistence length is about 50 nm , or 150 bp -LSB- 39 -RSB- . Since the thrombin aptamer is much shorter than 150 bp -LSB- 40 -RSB- -LRB- the recognition part is only 15 bp in length -RRB- , the complex would be in a `` standing '' position . When thrombin is introduced to such a complex , the thrombin aptamer binds to thrombin , and the MB-tagged single-stranded DNA approaches the electrode surface , improving the electron transfer with an increase of current . The persistence length of a single-stranded DNA is 1.5 nm in 2 M NaCl solution , and 3 nm in 25 mM NaCl solution -LSB- 41 -RSB- . Fig. 3Thrombin binding with an engineered aptamer . MB methylene blue . -LRB- Reprinted with permission from -LSB- 31 -RSB- , copyright 2005 American Chemical Society -RRB- In the work of Xiao et al. , a threefold signal gain was observed with 260 nM thrombin , while only a 40 % signal decrease was observed using a signal-off sensor . The detection limit of the sensor thus rose to 3 nM as shown in Fig. 4 . Such a high sensitivity is comparable even with that given by sensors using amplification by labeling -LSB- 28 , 29 -RSB- or optical sensors using evanescent-wave-induced fluorescence anisotropy -LSB- 25 -RSB- . Table 2 summarizes the electrochemical aptamer sensors available for thrombin . Fig. 4Electrochemical signal from the signal-on aptamer sensor . -LRB- Reprinted with permission from -LSB- 31 -RSB- , copyright 2005 American Chemical Society -RRB- Table 2The detection limits of an electrochemical aptamer sensor for thrombin Detection limitCharacteristicsReference13 nMUses an MB-tagged `` holding '' DNA sequence -LSB- 31 -RSB- 280 nM/3 .5 nMPeroxidase-labeled thrombin gives an 80 nM detection limit . Biotin-labeled aptamer with horse radish peroxidase labeled streptavidin gives a detection limit of 3.5 nM -LSB- 27 -RSB- 31 μMSandwich assay using two aptamers with different binding sites . Secondary aptamer labeled with glucose dehydrogenase -LSB- 28 -RSB- 410 nMSame as for 3 , but pyrroquinoline quinine glucose dehydrogenase is used for labeling -LSB- 29 -RSB- 56.4 nMMB-tagged thrombin aptamer -LRB- signal-off sensor -RRB- -LSB- 30 -RSB- 60.1 -- 0.15 nMThrombin aptamer with ferrocene moiety -LRB- square-wave voltametry or chronopotetiometry used for the measurement -RRB- -LSB- 32 -RSB- 711 nMMB-intercalated thrombin aptamer -LSB- 25 -RSB- MB methylene blue Owing to the unique three-dimensional structure of an aptamer , it is not even necessary to use `` holding '' DNA sequences . Baker et al. -LSB- 34 -RSB- reported the rapid , label-free detection of cocaine in both adulterated samples and biological fluid . The cocaine aptamer engineered by Stojanovic et al. -LSB- 42 -RSB- was used in this work . This aptamer is thought to form a partially unfolded structure , with only one of its three double-stranded stems intact , when target molecules are absent . In the presence of target , the aptamer folds into the cocaine-binding three-way junction structure of Fig. 5a . With this unique approach , Baker et al. reported the detection of 500 μM cocaine in biological fluid , even when contaminants were present . The ability to detect cocaine in undiluted biological fluids -LRB- blood , for example -RRB- is crucial for simple forensic detection technology . The binding of small molecules to an aptamer is highly reversible , so it is possible to regenerate the sensor . Baker et al. -LSB- 34 -RSB- could regenerate the sensor to a degree of 99 % by simply washing a used sensor with buffer . Fig. 5a Binding of coccaine with an MB-tagged aptamer . b Binding of platelet-derived growth factor -LRB- PDGF -RRB- with an MB-tagged aptamer . -LRB- a Reprinted with permission from -LSB- 34 -RSB- , copyright 2006 American Chemical Society ; b reprinted with permission from -LSB- 35 -RSB- , copyright 2007 American Chemical Society -RRB- Regeneration of an aptamer sensor is also possible after protein binding . Lai et al. -LSB- 35 -RSB- reported the detection of picomolar levels of platelet-derived growth factor -LRB- PDGF -RRB- directly in blood serum . PDGF is a protein that regulates cell growth and division , and is very important for blood vessel formation -LRB- angiogenesis -RRB- . While generally undetectable in normal cells , PDGF is overexpressed in a variety of disease such as atherosclerosis , and human tumors including sarcomas and glioblastomas -LSB- 14 , 43 -RSB- . As shown in Fig. 5b , specific binding of PDGF to the aptamer decreases the distance between MB and the electrode and consequently increases the electrical signal . The sensor could be also regenerated by 4-min incubation in 10 % -LRB- w/v -RRB- sodium dodecyl sulfate . In this section , we have shown that engineered aptamers , employed as recognition elements in electrochemical transducers , can greatly improve sensor performance . It is an advantage of aptamers over antibodies that aptamers can be engineered to either enhance or activate signals . Recently , aptazymes -LRB- aptamers with catalytic activities -RRB- have been reported , which combine an enzymatic role with a recognition function -LSB- 44 -RSB- . Carefully designed aptazymes can be developed as sensors that do not require labeling . An electronic -LRB- electrochemical -RRB- sensor approach with carefully designed aptamers offer potential for highly sensitive , selective and reusable sensor platforms . Aptamer sensors with carbon nanotube field-effect transistor transducers Since the first report on the biosensing ability of Si nanowire in 2001 -LSB- 45 -RSB- and the development of the chemical sensor operation of carbon nanotubes in 2000 -LSB- 46 -RSB- , extensive research efforts have been initiated aimed at developing nanotube-based or nanowire-based sensors . Owing to the extremely large surface-to-volume ratio of one-dimensional nanostructures , it is possible to develop sensors with exquisite sensitivity . This is especially true in the case of carbon nanotubes , where all constituent atoms are at the surface . Any small disturbance of or adsorption to the surface could result in a large change in electrical conductance . Already , biosensors fabricated with semiconducting nanowires or nanotubes have been used as sensors that can detect a single virus -LSB- 6 -RSB- , small molecules -LSB- 47 -RSB- or minute amounts of proteins in solution -LSB- 7 -- 9 -RSB- . FET sensors measure a change in the surface potential occurring during the binding between an analyte and a recognition element . Figure 6 shows a schematic diagram of the working principle of carbon-nanotube-based FET sensors . FET sensors `` feel '' the charges when target molecules bind with probe molecules immobilized on the sensor surfaces . Such an effect is often referred to as an `` electrostatic gating effect '' since the bound target molecules behave as an additional gate electrode . In the case of carbon nanotubes , SWNTs behave as p-type semiconductors in ambient atmosphere . In p-type FETs , positive gate voltage decreases the conductance -LRB- depletion -RRB- and negative gate voltages increase the conductance -LRB- accumulation -RRB- from the device . Therefore , if positively charged molecules bind with probe molecules on SWNT-FETs , this event appears as a decrease of conductance , while binding of negatively charged molecules appears as an increase of conductance in SWNT-FETs as denoted in Fig. 6 . Fig. 6The working principle of field-effect transistor -LRB- FET -RRB- sensors based on carbon nanotubes Using FETs for immunosensing was first proposed by Shenk -LSB- 48 -RSB- in 1978 . It was suggested that FETs could be used to detect surface polarization owing to the formation of an antibody and an antigen . This concept failed , however , because small ions in the solution caused a screening effect inhibiting detection of the changes -LSB- 49 -RSB- . Figure 7 shows the charged semiconductor surface in solution . As shown in Fig. 7 , when a charged surface -LRB- this might be a metal electrode , a semiconductor or a charged dielectric -RRB- is brought into contact with an ionic solution , the surface charge potential is dissipated by redistribution of counter ions in the solution . The domains where the variation of electric potential occurs are called electrical double layers , and the depth of an electrical double layer can be determined by calculating the Debye screening length . The Debye screening length is the scale over which mobile charge carriers screen the electric field . In an electrolyte , the Debye screening length can be defined as follows : Fig. 7Electrical double layer at the sensor surface Here the Bjerrum length lB is defined as and is 0.7 nm at standard pressure and temperature . kB is the Boltzmann constant , k is defined as 1/4πɛ0 , ρi is the number density of ions and the zi values are valencies of various ions . The Debye screening length , therefore , is proportional to the inverse square root of the ion number density , i.e. , to ionic strength . For pure water , the Debye screening length is about 1 μm , while it is only 0.3 nm in 1 M NaCl solutions . In 1 M NaCl solution , therefore , two ions or charged species separated by 1 nm do not interact with each other . If we consider the sensor surface , any interactions occurring outside the Debye screening length can not be detected using FET sensors . The biggest disadvantage of FET sensors is that they may not work with blood samples or body fluids , because the ionic strength of such physiological solutions is about 150 mM , which yields a Debye screening length of about 1 nm . Until recently , FET-type sensors have employed an antibody as the recognition receptor . However , the typical size of an antibody is about 10 -- 15 nm -LSB- 50 , 51 -RSB- . In that case , the binding of an antigen with an antibody occurs far outside the Debye screening length in physiological solutions . Molecular recognition elements with high specificity , selectivity and small sizes are essential for applications of FET-type sensors . In this respect , aptamers are highly suitable . Figure 8 shows the typical size ranges of antibodies and aptamers -LSB- 52 -RSB- . Because aptamers are mostly short nucleic acids ranging from 10 to 60 bp in length , the binding of target molecules with aptamers can occur inside the electrical double layer , even in 20 -- 50 mM salt solutions . Fig. 8Size comparison of an aptamer with an antibody . IgG immunoglobulin G. -LRB- Reprinted from -LSB- 52 -RSB- , copyright 2006 Elsevier -RRB- Such a biggest merit of aptamers in FET sensors was first reported by So et al. -LSB- 36 -RSB- using a carbon nanotube biosensor with thrombin aptamers as molecular recognition elements . They also demonstrated the sensitivity and selectivity of aptamers in SWNT-FETs . Anti-thrombin aptamers -LRB- 5 ′ - GGT TGG TGT GGT TGG-NH2-3 ′ -RRB- were immobilized onto the surface of the SWNT-FET , fabricated using a patterned growth technique , with carbonyl diimidazole -- Tween 20 as the linking molecules -LRB- Fig. 9a -RRB- . The authors observed an abrupt decrease in conductance upon reaction with thrombin , while almost no changes were observed with elastase -LRB- Fig. 9b -RRB- . Elastase is a member of the thrombin family and has almost the same molecular weight and isoelectric point as thrombin . Moreover , the sensor could be regenerated by simple washing with 6 M guanidine hydrochloride solution . The authors regenerated the sensor more than five times without significant loss of activity . As shown in Fig. 9c , the authors measured the sensitivities of SWNT-FET sensors with aptamers as recognition elements . Before each measurement , the sensor was `` reset '' by washing it with guanidine hydrochloride solution . The lowest detection limit was around 10 nM thrombin , and the sensor signal was saturated at about 100 nM thrombin . Fig. 9a Single-walled carbon nanotube -LRB- SWNT -RRB- FET sensor with aptamers as molecular recognition elements . b Selectivity of an anti-thrombin aptamer-functionalized SWNT-FET . c Sensitivity of an anti-thrombin aptamer-functionalized SWNT-FET . The sensor was reset by washing it with 6 M guanidine hydrochloride solution before each measurement . CDI carbonyl diimidazole . -LRB- Reprinted with permission from -LSB- 36 -RSB- , copyright 2005 American Chemical Society -RRB- Maehashi et al. -LSB- 37 -RSB- have recently addressed the size issue of recognition elements in FET-based sensors . In their work , immunoglobulin E -LRB- IgE -RRB- aptamer -LRB- 5 ′ - NH2-GCG CGG GGC ACG TTT ATC CGT CCC TCC TAG TGG CGT GCC CCG CGC-3 ′ -RRB- and monoclonal anti-IgE antibody -LRB- IgE-mAb -RRB- were immobilized on carbon nanotubes , with the aid of the linking molecule 1-pyrenebutanoic acid succinimidyl ester . Figure 10 a and b shows the responses of the aptamer-functionalized and antibody-functionalized sensors , respectively , when they were exposed to known concentrations of target IgE in 10 mM phosphate-buffered saline -LRB- PBS -RRB- . As shown in Fig. 10 , the aptamer-functionalized sensor exhibited small changes with only 250 pM target protein molecules , while a negligible change in conductance was observed in the antibody-funtionalized sensor even with 140 nM IgE . In 10 mM PBS , the Debye screening length would be roughly 3 nm , and the binding of IgE with the aptamer would be safely inside the electrical double layer . With IgE-mAb , however , there is little hope that binding can be detected in the buffer solution used for the experiment , because the antibody molecule is about 10 nm long -LSB- 53 -RSB- . Fig. 10The sensor signal measured from a an aptamer-functionalized SWNT-FET and b an antibody-functionalized SWNT-FET . IgE immunoglobulin E , PBS phosphate-buffred saline . -LRB- Reprinted with permission from -LSB- 37 -RSB- , copyright 2007 American Chemical Society -RRB- As discussed , aptamers may allow a wider range of analytes in FET sensors owing to their small size . SWNT-FET sensors with aptamers as recognition elements showed high sensitivity and selectivity , and could be readily regenerated . Highly sensitive carbon nanotube sensors combined with small , economic , highly selective and stable aptamers could provide cost-effective point-of-care testing devices . Concluding remarks This review has focused on the advantages of electrochemical and FET sensor types with aptamers as recognition elements . Through use of aptamers for recognition , no labeling is required for electrochemical sensors , and signal-on architecture that is only possible with aptamers has made possible the sensitivity improvements . Also , as pointed out already , sensors can be recycled because of the reversibility of aptamer configurations . Alternatively , bound proteins may be simply washed off , without damaging the aptamers . In immunosensing , by contrast , it is practically impossible to remove bound antigens from antibodies without damaging the antibodies , because both antibodies and antigens are proteins in nature . Aptamers have proved their superiority over antibodies in nano-FET sensors . When no signal can be measured with an antibody -- antigen pair because of the large size of the antibody , the binding of small aptamers to various targets can occur inside the electrical double layer where the nanotube can `` feel '' the change . Owing to the rapid aging of society , there is great demand for paradigm-breaking biosensors to detect the onset of disease with speed , convenience and accuracy . In a few more years , electrical nanosensors , with engineered aptamers , will become prominent in the market . They will be small in size , high in sensitivity and competitive in price .

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O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O

aptamers
nanobiosensors
electrochemical aptamer sensors


Formation of gutingimycin : analytical investigation of trioxacarcin A-mediated alkylation of dsDNA Formation and fragmentation of recognition complexes between trioxacarcin A and various DNA sequences were examined by temperature-dependent UV and CD spectroscopy , HPLC analysis , and ESI mass spectrometry with regard to reaction conditions , intermediates , products , mechanism , and sequence specificity . Cleavage of the trioxacarcin -- DNA complexes provided the natural product gutingimycin by guanine abstraction . The resulting DNA with an abasic site was further cleaved into a DNA fragment with a furanyl unit at the 3 ′ - end and an oligonucleotide with a phosphorylated 5 ′ - end . Introduction Trioxacarcins are complex natural antitumor antibiotics with anti-malaria activity -LSB- 1 -- 3 -RSB- . The first trioxacarcins were isolated from streptomycetes in 1981 by Tomita -LSB- 1 , 2 -RSB- and their structure was elucidated by Shirahata -LSB- 4 -RSB- . Gutingimycin -LRB- 1 -RRB- was isolated in 2004 from a marine streptomycete together with further trioxacarcin derivatives and named according to the ancient name of Göttingen -LSB- 3 , 5 , 6 -RSB- . It turned out to be an interesting natural product because of its mode of formation . The structure of gutingimycin -LRB- 1 -RRB- , elucidated by X-ray analysis , suggested that gutingimycin -LRB- 1 -RRB- results from nucleophilic attack of the N7 atom of a guanine subunit in DNA at the epoxide of trioxacarin A -LRB- 2 -RRB- -LSB- 5 -RSB- . Natural antibiotics like hedamycin and altromycin B are able to intercalate into the dsDNA followed by alkylation of guanine at the N7 position because of their epoxide functionality -LSB- 7 -- 9 -RSB- . Like hedamycin , altromycin B or kapurimycin A3 -LSB- 10 , 11 -RSB- , trioxacarcin A -LRB- 2 -RRB- contains an epoxide . Therefore , it has been proposed that gutingimycin -LRB- 1 -RRB- is not built up from low-molecular-weight precursors -LSB- 5 -RSB- . As for other cytotoxic natural products , it is likely that alkylation of trioxacarin A -LRB- 2 -RRB- is followed by depurination and DNA strand scission at the abasic site -LSB- 7 , 12 -- 14 -RSB- . Saito obtained derivatives structurally related to gutingimycin -LRB- 1 -RRB- by reaction of kapurimycin A3 with various oligonucleotides . At 0 °C a stable ionic intermediate is formed ; on heating to 90 °C this dissociates into a depurinated oligonucleotide and a kapurimycin derivative -LSB- 15 , 16 -RSB- . Hedamycin binds to dsDNA by intercalation and alkylation with a distinct preference for the central guanine in the sequence d -LRB- CGT -RRB- or d -LRB- CGG -RRB- -LSB- 8 , 17 , 18 -RSB- . The covalent hedamycin-DNA adduct was isolated by Sheil and characterized by ESI MS -LSB- 19 , 20 -RSB- . From previous studies it was known , that trioxacarcin A -LRB- 2 -RRB- also forms a stable complex with dsDNA , but did not react with deoxyguanosine even at higher temperatures -LSB- 1 -RSB- . The aim of this study was to investigate the conditions and mechanism of the trioxacarcin-mediated alkylation of dsDNA by applying high-performance liquid chromatography and high-resolution electrospray mass spectrometry together with temperature-dependent UV and CD spectroscopy . In particular , the influence of the DNA sequence on binding and adduct formation and the identification of intermediates and cleavage products like gutingimycin -LRB- 1 -RRB- and DNA fragments is described in detail . Experimental Methods and materials The DNA oligomers d -LRB- AATTACGTAATT -RRB- -LRB- 4a -RRB- , d -LRB- CAATTATAATTG -RRB- -LRB- 4b -RRB- , d -LRB- AATTXGXAATT -RRB- -LRB- 5a -- d -RRB- , and the complementary oligonucleotides d -LRB- AATTGGGAATT -RRB- -LRB- 7 -RRB- and d -LRB- TTAACCCTTAA -RRB- -LRB- 8 -RRB- were purchased from IBA -LRB- Göttingen , Germany -RRB- . An oligomer length of eleven or twelve nucleotides was sufficient to provide stability at room temperature -LRB- Tm = 22 -- 35 °C -RRB- . Trioxacarcin A -LRB- 2 -RRB- and trioxacarcin B -LRB- 3 -RRB- were obtained by fermentation of Streptomyces sp. isolate B8652 -LSB- 6 -RSB- . The reactions were carried out in aqueous phosphate buffer -LRB- 10 mmol L − 1 Na2HPO4/NaH2PO4 , 0.1 mol L − 1 NaCl , H2O , pH 7.0 -RRB- with 5 μmol L − 1 dsDNA and trioxacarcin A -LRB- 2 -RRB- in a 1:2 ratio . UV spectroscopy Melting curves were measured on a Perkin -- Elmer Lambda 10 UV -- visible spectrophotometer in connection with a Perkin -- Elmer PTP-1 Peltier system following the temperature program : 0 °C -LRB- 15 min -RRB- → 90 °C -LRB- 180 min -RRB- → 0 °C -LRB- 180 min -RRB- . dsDNA -LRB- 30 nmol -RRB- was dissolved in 1.5 mL phosphate buffer -LRB- 10 mmol L − 1 Na2HPO4/NaH2PO4 , 0.1 mol L − 1 NaCl , H2O , pH 7.0 -RRB- , trioxacarcin A -LRB- 2 -RRB- -LRB- 60 nmol -RRB- in 1.5 mL phosphate buffer was added and the measurement was performed at a wavelength of λ = 260 nm in a quartz cuvette -LRB- 1 cm -RRB- . CD spectroscopy CD spectra were obtained on a Jasco J-810 spectropolarimeter at room temperature -LRB- data mode : CD , band width 1.0 nm , slit width : auto , sensitivity : 5 mdeg , time const. : 4.0 s ; step resolution : 0.1 nm , scan speed : 50 nm min − 1 , five accumulations -RRB- . dsDNA -LRB- 30 nmol -RRB- was dissolved in 1.5 mL phosphate buffer -LRB- 10 mmol L − 1 Na2HPO4/NaH2PO4 , 0.1 mol L − 1 NaCl , H2O , pH 7.0 -RRB- , trioxacarcin A -LRB- 2 -RRB- -LRB- 60 nmol -RRB- in 1.5 mL phosphate buffer was added and the measurement was performed in a quartz cuvette -LRB- 1 cm -RRB- . Kinetic measurements A solution of trioxacarcin -- DNA -LRB- 30 nmol -RRB- adduct in phosphate buffer -LRB- 600 μL , 10 mmol L − 1 Na2HPO4/NaH2PO4 , 0.1 mol L − 1 NaCl , H2O , pH 7.0 -RRB- was heated to 80 °C for 4 h. Samples of 60 μL were taken after 0 , 15 , 30 , 45 , 60 , 75 , 90 , 120 , and 240 min and analyzed by HPLC . The area under the curve of the trioxacarcin -- DNA adduct peak was plotted versus time to determine the time constant as the slope of the regression line . High-performance liquid chromatography HPLC separations were performed with an Äkta purifier from Amersham Pharmacia Biotech , a UV900 UV detector operated at 260 nm , a NewGuard RP-8 Brownlee Precolumn -LRB- 15 × 3.2 mm , 7 μm -RRB- , and an Aquapore RP-300 Brownlee Column -LRB- 220 × 4.6 mm , 7 μm -RRB- from Perkin -- Elmer . Samples were eluted within 45 min with a flow rate of 1 mL min − 1 by applying a two-stage gradient -LRB- 5 -- 20 % B in 20 min and 20 -- 45 % B in 25 min -RRB- . Eluent A : 0.1 mol L − 1 triethylammonium acetate buffer -LRB- H2O , pH 7.0 -RRB- . Eluent B : 0.1 mol L − 1 triethylammonium acetate buffer -LRB- 80 % acetonitrile and 20 % water , pH 7.0 -RRB- . Electrospray mass spectrometry To a solution of 120 nmol dsDNA in phosphate buffer -LRB- 10 mmol L − 1 Na2HPO4/NaH2PO4 , 0.1 mol L − 1 NaCl , H2O , pH 7.0 -RRB- a fivefold excess of trioxacarcin A -LRB- 2 -RRB- was added . The resulting mixture was incubated several hours at 0 °C and the trioxacarcin -- DNA adduct formed was isolated by HPLC . High-resolution mass spectrometry was performed using an Apex IV 7 T FTICR MS -LRB- Bruker Daltonics , Billerica , MA , USA -RRB- equipped with an Apollo electrospray ion source which was used in the negative ion mode -LRB- capillary voltage 4.2 kV , end plate voltage 3.8 kV -RRB- . Analytes were dissolved in a water -- methanol -LRB- 50:50 -RRB- solution and introduced by a syringe pump at a flow rate of 2 μL min − 1 . Nitrogen was used as drying gas at 100 °C . Hexapole accumulation was set between 0.2 s to 3.0 s and mass spectra were acquired within the mass range m/z 400 to 2000 with a resolution of about 100,000 at m/z 600 . The capillary exit voltage was − 80 V , − 100 V , − 140 V , or − 170 V. Deconvolution and mass calculation for the corresponding ions were done using Xmass 6.1.2 from Bruker . Results and discussion In order to investigate the sequence dependency of guanine abstraction from dsDNA by trioxacarcin A -LRB- 2 -RRB- , the two self complementary DNA oligomers d -LRB- AATTACGTAATT -RRB- -LRB- 4a -RRB- and d -LRB- CAATTATAATTG -RRB- -LRB- 4b -RRB- were investigated with the reactive guanine located in the center or at the end of the double strand . The position of guanine was varied to determine the influence of the helix topology on alkylation . Four additional non-self complementary oligonucleotides with the general sequence d -LRB- AATTXGXAATT -RRB- -LRB- 5a -- d -RRB- together with the corresponding complementary strands were examined with respect to sequence specificity . The sequences were limited to only one guanine in the double strand in order to limit alkylation to a single site . It was placed in the central position investigating all possible alterations of the neighboring nucleobases using adenine and thymine . Fig. 1Gutingimycin -LRB- 1 -RRB- , trioxacarcin A -LRB- 2 -RRB- , and trioxacarcin B -LRB- 3 -RRB- A first approximation of the interaction of trioxacarcin A -LRB- 2 -RRB- with double-stranded DNA can be obtained from temperature dependent UV-spectroscopic measurements of double-strand stabilities , because double-strand recognition , formation of a covalent adduct , nucleobase excision , or strand scission are expected to affect DNA stability . A mixture of double-stranded oligonucleotide d -LRB- AATTACGTAATT -RRB- -LRB- 4a -RRB- with two equivalents trioxacarcin A -LRB- 2 -RRB- gave a stability of Tm = 41 °C which is significantly higher than the double strand transition of oligomer 4a without addition of the natural product -LRB- Tm = 34 °C -RRB- -LRB- Fig. 2 -RRB- indicating a DNA interaction with the trioxacarcin A -LRB- 2 -RRB- . For this interaction intercalation and groove binding of the aglycon is more likely than covalent linkage of 2 , because double-strand stability obtained for d -LRB- AATTACGTAATT -RRB- -LRB- 4a -RRB- with trioxacarcin B -LRB- 3 -RRB- is similarly increased -LRB- Tm = 42 °C -RRB- even though trioxacarcin B -LRB- 3 -RRB- is not able to alkylate DNA . In the cooling cycle the UV melting curve of the 4a/2 mixture provided only a stability of Tm = 28 °C . This destabilization is most likely because of nucleobase excision initiated at higher temperatures to yield gutingimycin -LRB- 1 -RRB- and a DNA double strand with apurine site which might further lead to DNA strand scission . Fig. 2UV-melting curve of d -LRB- AATTACGTAATT -RRB- -LRB- 4a -RRB- with and without trioxacarcin A -LRB- 2 -RRB- -LRB- 5 μmol L − 1 , 1:2 -RRB- In contrast , for the oligomer with the terminal guanine 4b the differences in double-strand stability were not significant comparing the self pairing -LRB- Tm = 31 °C -RRB- with the 1:2 mixture of oligomer 4b and natural product 2 in the heating -LRB- Tm = 29 °C -RRB- or cooling cycle -LRB- Tm = 29 °C -RRB- -LRB- Fig. 3 -RRB- . Apparently , for DNA recognition of trioxacarcin A -LRB- 2 -RRB- the double helix topology is required which is not sufficiently assured at the terminus of the helix . Therefore , the guanine placed at the terminal position can not act as a nucleophile without the help of helix recognition . This is in agreement with guanosine itself not being a nucleophile for trioxacarcin A -LRB- 2 -RRB- -LSB- 1 -RSB- . Fig. 3UV-melting curve of d -LRB- CAATTATAATTG -RRB- -LRB- 4b -RRB- with and without trioxacarcin A -LRB- 2 -RRB- -LRB- 5 μmol L − 1 , 1:2 -RRB- The modification of DNA 4a by trioxacarcin A -LRB- 2 -RRB- was also indicated by CD spectroscopy -LRB- Fig. 4 -RRB- . The spectra of 4a and the mixture of 4a with 2 measured at room temperature are quite similar , whereas the CD spectrum of the 4a/2 mixture heated to 90 °C differed by a shift and the intensity of the Cotton effect around 270 , 250 , and 220 nm . Fig. 4CD-spectra of self pairing d -LRB- AATTACGTAATT -RRB- -LRB- 4a -RRB- with -LRB- grey -RRB- and without -LRB- black -RRB- trioxacarcin A -LRB- 2 -RRB- -LRB- 5 μmol L − 1 , 1:2 -RRB- . The dashed line indicates the spectrum obtained after interim heating to 90 °C For a more detailed picture of trioxacarcin A -LRB- 2 -RRB- adduct formation with dsDNA , high-performance liquid chromatography was applied determining the components present after mixing at room temperature and after heating to 90 °C . The HPLC chromatogram of the mixture of oligonucleotide 4a with trioxacarcin A -LRB- 2 -RRB- before starting the heating cycle provided three peaks -LRB- Fig. 5a -RRB- . Next to DNA -LRB- 4a -RRB- and trioxacarcin A -LRB- 2 -RRB- , an additional compound was indicated which was assigned to the stable covalent complex between the oligonucleotide and the natural product trioxacarcin A -LRB- 4a + 2 -RRB- . The compound corresponding to peak -LRB- 4a + 2 -RRB- was isolated and characterized by high-resolution ESI MS as the DNA -- trioxacarcin A adduct . After heating to 90 °C additional peaks appeared in the oligonucleotide area together with gutingimycin -LRB- 1 -RRB- and trioxacarcin B -LRB- 3 -RRB- generated by epoxide ring opening of trioxacarcin A -LRB- 2 -RRB- with water -LRB- Fig. 5b -RRB- . The assignment of all HPLC peaks was confirmed by co-injection and mass spectrometry . Fig. 5HPLC chromatogram of a mixture of oligonucleotide 4a and trioxacarcin A -LRB- 2 -RRB- : a at room temperature ; b after heating to 90 °C ; next to DNA 4a further oligonucleotides and oligonucleotide fragments are eluting between 11 and 17 min Examining the trioxacarcin A -LRB- 2 -RRB- interaction with oligomer d -LRB- CAATTATAATTG -RRB- -LRB- 4b -RRB- carrying the guanine at the terminal position , the HPLC chromatogram turned out to be quite similar to the HPLC chromatogram obtained after interaction with DNA 4a . Nevertheless , the yield of the respective DNA -- trioxacarcin adduct -LRB- 4b + 2 -RRB- was significantly lower and gutingimycin -LRB- 1 -RRB- was only obtained in traces after heating -LRB- Fig. 6 -RRB- . As indicated in Table 1 , adduct formation of trioxacarcin A -LRB- 2 -RRB- with oligonucleotide 4a is about five times more efficient than binding to DNA 4b . Sterically , the nucleophilic attack should be easier at the end of the double helix , but the ease of adduct formation turned out to be higher in the central position of the double strand . Therefore , trioxacarcin A -LRB- 2 -RRB- seems to take advantage of dsDNA recognition by intercalation or groove binding prior to nucleophilic attack . This recognition behavior is also described for the natural products hedamycin and altromycin B of the pluramycin family -LSB- 7 , 8 , 21 , 22 -RSB- . Fig. 6HPLC chromatogram of a mixture of the oligonucleotide 4b and trioxacarcin A -LRB- 2 -RRB- : a at room temperature ; b after heating to 90 °C Table 1Examined oligomers and adduct formationDNA oligomers Adduct formula -LSB- Madduct − 6H -RSB- 6 − a calcd . / found mass -LSB- Madduct + Na − 7H -RSB- 6 − a calcd . / found massAdduct -LRB- % -RRB- bAATTACGTAATT4aC161H202N43O90P11752 .32 / 752.33755.99 / 755.9953 CAATTATAATTG4bC161H202N43O90P11752 .32 / 752.39755.99 / 756.0010 AATTAGTAATT5aC152H190N40O84P10704 .15 / 704.16707.81 / 707.8313 AATTTGAAATT5bC152H190N40O84P10704 .15 / 704.16707.81 / 707.825 AATTTGTAATT5cC152H191N37O86P10702 .65 / 702.65706.31 / 706.3116 AATTAGAAATT5dC152H189N43O82P10705 .65 / 705.66709.31 / 709.321 a Adduct ions up to the sevenfold charged ion were observed ; the relative intensities varied from oligomer to oligomerb Conversion rate given by the trioxacarcin A to adduct proportion The isolated adduct of oligonucleotide 4a with trioxacarcin A -LRB- 2 -RRB- is stable at room temperature for more than 24 h and at 56 °C for at least 4 h . At 80 °C within 4 h , decay to gutingimycin -LRB- 1 -RRB- was observed obeying first-order kinetics -LRB- Fig. 7 -RRB- . Fig. 7Kinetic analysis based on a decay of the d -LRB- AATTACGTAATT -RRB- -- trioxacarcin A adduct -LRB- 4a + 2 -RRB- -LSB- 23 -RSB- In order to get preliminary information about sequence selectivity in trioxacarcin A -LRB- 2 -RRB- recognition and alkylation , all adenine and thymine combinations were investigated as the neighboring nucleobases of a central guanine . The four oligonucleotides 5a -- d reacted with trioxacarcin A -LRB- 2 -RRB- to yield the corresponding adducts , which were characterized by high-resolution ESI MS. The corresponding HPLC profiles of oligomers 5a -- d with trioxacarcin A -LRB- 2 -RRB- before and after heating to 90 °C were almost similar , except for the peak height of the generated adducts . The conversion rate was calculated from the peak area proportion of trioxacarcin A -LRB- 2 -RRB- to the adduct . As indicated in Table 1 , the conversion rate from trioxacarcin A -LRB- 2 -RRB- to the corresponding DNA adducts varied from 1 to 16 % . Based on non-self complementary double strands 5a -- d and results obtained by HPLC and ESI MS , adduct formation seems to be preferred when thymine is placed next to guanine with thymine neighboring the 3 ′ - site having a greater impact . It still needs to be investigated if this result holds only for thymine in comparison with adenine or if this is a more general preference for pyrimidines over purines neighboring the trioxacarcin-substituted guanine . The alkylation of oligomers with more than one guanine was investigated with the double strand formed by the complementary strands d -LRB- AATTGGGAATT -RRB- -LRB- 6 -RRB- and d -LRB- TTAACCCTTAA -RRB- -LRB- 7 -RRB- which provide a quite stable double strand -LRB- Tm = 35 °C -RRB- . In a mixture of 6 + 7 with six equivalents of trioxacarcin A -LRB- 2 -RRB- the double strand stability decreased to Tm = 29 °C -LRB- heating cycle -RRB- whereas double strand formation was no longer detected after heating to 90 °C . This leads to the conclusion that trioxacarcin A -LRB- 2 -RRB- addition and DNA degradation is accelerated with three neighboring guanines . The high tendency for adduct formation of trioxacarcin A -LRB- 2 -RRB- to dsDNA 6 + 7 at 0 °C was indicated by an HPLC signal at 26.0 min nearly the size of the remaining oligomer 7 -LRB- Fig. 8a -RRB- . Even after 24 h signals appeared at 30.8 and 31.6 min that were assigned to DNA 6 functionalized with two equivalents of trioxacarcin A -LRB- 2 -RRB- . The amount of double substitution increased significantly within 72 h reaction time -LRB- Fig. 8b -RRB- . Fig. 8HPLC chromatograms of a mixture of the DNA double strand d -LRB- AATTGGGAATT -RRB- -LRB- 6 -RRB- + d -LRB- TTAACCCTTAA -RRB- -LRB- 7 -RRB- and trioxacarcin A -LRB- 2 -RRB- : a 24 h at 0 °C and b 72 h at 0 °C Further insight into the trioxacarcin A -LRB- 2 -RRB- - initiated substitution reaction and the DNA cleavage products were obtained by additional ESI MS experiments stepwise increasing the capillary-skimmer dissociation energy in the ion source -LSB- 24 -- 30 -RSB- . Like increasing the temperature this also initiated fragmentation of alkylated DNA followed by strand cleavage . The adduct of trioxacarcin A -LRB- 2 -RRB- with oligomer 5d was unequivocally assigned by ESI MS with a low capillary exit voltage of − 80 V providing exclusively ions of the DNA -- trioxacarcin complex 5d + 2 -LRB- Fig. 9a -RRB- : -LRB- -LSB- M − 3H -RSB- 3 − , -LSB- M − 4H -RSB- 4 − , -LSB- M − 5H -RSB- 5 − , and -LSB- M − 6H -RSB- 6 − -RRB- . By applying capillary -- skimmer dissociation by raising the capillary exit voltage to − 140 V the signals for the DNA -- trioxacarcin A adduct were also found -LRB- Fig. 9c -RRB- . Nevertheless , the adduct ions were less intense . One of the peaks with the highest intensity appears at m/z 1026.37 ; this is assigned to the single negatively charged ion of gutingimycin -LRB- 1 -RRB- comparable to the thermal cleavage product observed already by HPLC . Depurination is known to result in oligonucleotide strand scission -LSB- 19 , 20 , 27 -RSB- . Furthermore , it has been reported that collision-activated dissociation of negatively charged oligonucleotide ions starts with cleavage of a base followed by cleavage of the 3 ′ C -- O bond of the sugar unit from which the base was lost -LSB- 31 -RSB- . In accordance , two ESI MS peaks were correlated to the doubly charged w5-fragment ion at m/z 781.64 -LRB- calculated 781.63 -RRB- and the doubly charged -LRB- a6-B -RRB- - type fragment ion at m/z 821.66 -LRB- calculated 821.64 -RRB- of the oligonucleotide 5d . The accompanying smaller peaks to the right of w52 − and -LSB- a6-GT -RSB- 2 − were assigned to the corresponding -LSB- M + Na − 3H -RSB- 2 − ions . These fragments might mechanistically be explained by the initiation of strand cleavage at C3 ′ leading to a furanyl unit at the 3 ′ - end of d -LRB- AATTA -RRB- and a phosphoryl group at the 5 ′ - end of the second fragment d -LRB- AAATT -RRB- -LSB- 31 -RSB- . An additional small peak m/z 801.90 -LSB- 10 − 4H -RSB- 4 − can be assigned to the depurinated oligonucleotide 10 -LRB- Scheme 1 , together with its sodium peak m/z 807.40 -LSB- 10 + Na − 5H -RSB- 4 − -RRB- . Nevertheless , oligonucleotide 10 with a double bond between C1 ′ and C2 ′ -LRB- Scheme 1 -RRB- has a calculated mass -LSB- 10 − 4H -RSB- 4 − of m/z 801.89 and -LSB- 10 + Na − 5H -RSB- 4 − of m/z 807.38 corresponding to the mass of the intermediate fragment . This assignment is clearly supported by the corresponding deconvoluted mass spectra -LRB- Figs. 9b and d -RRB- . Fig. 9a ESI mass spectrum of trioxacarin-DNA d -LRB- AATTAGTAAATT -RRB- 5d + trioxacarcin -LRB- 2 -RRB- adduct , capillary exit potential − 80 V ; b Respective deconvoluted spectrum of trioxacarin-DNA 5d adduct ; c ESI mass spectrum of trioxacarin-DNA d -LRB- AATTAGTAAATT -RRB- 5d + trioxacarcin -LRB- 2 -RRB- adduct , capillary exit potential − 140 V ; d Respective deconvoluted spectrum of trioxacarin-DNA 5d adductScheme 1Proposed cleavage reaction of DNA -- trioxacarcin A adduct The fragments observed for depurination and DNA strand scission of trioxacarcin -- DNA adducts are in agreement with the results from Sheil reporting similar oligonucleotide fragmentation in ESI MS -- MS experiments for various hedamycin -- DNA adducts -LSB- 19 -RSB- . Based on the ESI MS fragments detected by cleavage of the trioxacarcin -- DNA adduct -LRB- 5d + 2 -RRB- the following products and intermediates for the fragmentation of the trioxacarcin -- DNA adduct are proposed -LRB- Scheme 1 -RRB- : Depurination is generating gutingimycin -LRB- 1 -RRB- and the oligonucleotide with an abasic site 10 dissociating in fragment 8 with a furanyl unit at the 3 ′ - end -LRB- w5-type fragment -RRB- and fragment 9 with a phosphorylated 5 ′ - end -LRB- a6-B-type fragment -RRB- . The phosphoester 9 seems to be a perfect leaving group for generation of a furan . The results described for the trioxacarcin -- DNA 5d adduct were supported by the mass spectra of the trioxacarcin adducts with oligonucleotides 4a -- b and 5a -- c . The signals assigned to adducts , abasic sites , fragments , and gutingimycin were also observed for these oligomer adducts . With low dissociation energy in the ion source -LRB- capillary exit potential − 80 V -RRB- the adduct peak was obtained for the self complementary double strand 4a with guanine in the central position almost exclusively , whereas fragmentation already occurred in the case of oligomer 4b with the terminal guanine obtaining the gutingimycin signal as the most prominent . Trioxacarcin A adducts with oligonucleotides 5a -- c always provided the strongest ESI MS signals for the adduct peaks using the lowest dissociation energy ; nevertheless , the peak intensities corresponding to the other fragments varied . The trioxacarcin adducts with oligomers 5a and 5d were the most stable . Conclusions The formation of stable adducts of the natural product trioxacarcin A -LRB- 2 -RRB- with guanine-containing dsDNA was investigated by a combination of temperature-dependent UV and CD spectroscopy , high-performance liquid chromatography , and high-resolution mass spectrometry . Recognition and non-covalent binding of trioxacarcin A -LRB- 2 -RRB- to dsDNA seems to be a prerequisite for nucleophilic attack of the guanine to form a covalent complex . Preliminary evidence indicates that trioxacarcin A-DNA interaction has sequence specificity with greater reactivity observed for guanines 5 ′ to thymidines . Cleavage of the trioxacarcin -- DNA adducts can be initiated at higher temperatures or by applying capillary-skimmer dissociation by raising the capillary exit potential in mass spectrometry experiments leading to the formation of the bioactive secondary metabolite gutingimycin and DNA strand scission . As DNA cleavage products , a fragment with a furanyl unit at the 3 ′ - end was identified next to an oligonucleotide with a phosphorylated 5 ′ - end .

O O B O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

electrospray mass spectrometry
gutingimycin
trioxacarcin a