Method for determining a nucleic acid

Details Method for determining a nucleic acid Method for determining a nucleic acid

1997-11-21

2001-05-01

Riley, Jezia (Department: 1656)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S810000, C436S501000, C536S023100, C536S024100, C536S024300, C536S024310, C536S024320, C536S024330, C536S025300

Reexamination Certificate

active

06225052

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to methods for determining a nucleic acid in a sample with a probe molecule using electron transfer from an electron donor to an electron acceptor. The invention is further directed to compounds useful in such methods and to compounds useful for the preparation of the probe molecules.
BACKGROUND OF THE INVENTION
Determinations of nucleic acids is becoming increasingly important as a tool for diagnosis in the health fields. For example, the presence of nucleic acids from organisms, like viruses, usually not present in the human body, can be determined using probes for the infecting nucleic acids. Further, any changes in the genome which may have a potential influence on the metabolism and the state of health of the individual can be determined. Such changes may have occurred by mutation or other means. Nucleic acid determination has made further progress with the introduction of nucleic acid amplification procedures, like the polymerase chain reaction (PCR).
The presently known nucleic acid assays can be divided into two types, the heterogeneous and the homogeneous assays. In heterogeneous assays, the nucleic acid is determined by binding to a nucleic acid probe which is labeled for detection or by incorporation of labeled mononucleoside triphosphates and subsequent immobilization of the so-labeled nucleic acid to a solid phase. This is preferably done by using a solid phase bound capture probe, a format which provides the advantage that any excess amount of labeled probes or mononucleotides can easily be separated from the solid phase bound labeled nucleic acid. The homogeneous type of nucleic acid assay uses the interference between two labels. In a first method, the two labels are linked together and the event of hybridization initiates cleavage of the linkage between the two labels. (The labels are chosen such that they elicit a signal as soon as they are separated.) In a second method, the distance between the labels is changed by hybridization events. In this case, the labels may be located on one probe or on two separate probes having the capability of hybridizing to the analyte nucleic acid such that the labels can interact with each other.
Electron transfer between donors and acceptors is to be subdivided into two categories. In the first category, the donors (Do) and acceptors (Ac) are bound to the DNA duplex by non-covalent forces, such as van der Waals', electrostatic and hydrogen bonding. In the second class are systems where Do and Ac are covalently linked to the DNA. The earliest demonstration of the first approach was reported by Fromherz and Rieger in 1986, who studied photoinitated electron transfer (PET) from intercalated ethidium to surface-associated methyl viologen (Fromherz, P.; Rieger, B. J. Am. Chem. Soc. 1986, 108, 5361). Electron transfer products were demonstrated by direct observation of the reduced viologen acceptor. However, no special effect of the DNA, other than to provide a high effective concentration of the donor and acceptor, was observed. In 1992, Harriman and Brun reported PET from ethidium and acridine donors to diazapyrenium acceptors under conditions where the redox components were intercalated (Brun, A. M.; Harriman, A. J. Am. Chem. Soc. 1992, 114, 3656). Multiexponential electron transfer kinetics were attributed to Do-Ac separations of 3, 4, and 5 base pairs. The B value derived in that study (0.88 Å
−1
) is comparable to that determined for Do-Ac systems in proteins, where stacked &pgr;-electron systems are not available for mediating electron transfer. Barton, Barbara and co-workers studied transition metal complex donors and acceptors which are intercalated into DNA and found that quenching of the donor fluorescence as well as recovery of the ground state absorption proceeds at rates which are independent of the number of bound acceptors, suggesting a very shallow distance dependence for electron transfer through the DNA duplex (&bgr;<0.2 Å
−1
) (Arkin, M. R.; Stemp, E. D. A.; Holmlin, R. E.; Barton, J. K.; Hörmann, A.; Olson, E. J. C.; Barbara, P. F. Science 1996, 273, 475). However, cooperative binding of the donor and acceptor molecules, which would account for the loading-independent kinetics, could not be completely ruled out in that system.
One of the problems associated with the use of non-covalently bound donor and acceptor molecules in these studies is the inability to control precisely the location of the redox components relative to one another when they are bound to the DNA. In one extreme, the intercalation locations will be controlled statistically, leading to a distribution of Do-Ac separation distances. At another extreme, binding will be cooperative, leading to short distances between Do and Ac over a wide range of concentrations.
Covalent linkage of Do and Ac to the 5′-ends of complementary oligonucleotides has led to systems with better defined Do-Ac separation distances. Barton, Turro and co-workers reported fluorescence quenching that occurs in less than one nanosecond for a system containing linked Do and Ac metal complexes intercalated near the ends of a 15 base pair duplex (Murphy, C. J.; Arkin, M. R.; Jenkins, Y.; Ghatlia, N. D.; Bossmann, S.; Turro, N. J.; Barton, J. K. Science 1993, 262, 1025). A rate this fast indicates that the distance dependence of electron transfer through DNA is extremely shallow, but this interpretation must be regarded with caution pending a clear demonstration of redox products. In contrast, there is a report of a covalently linked system having Do and Ac metal complexes at the opposite ends of an 8 base pair duplex which shows electron transfer on a microsecond time scale (Meade, T. J.; Kayyem, J. F. Angew. Chem. Int. Ed. Engl. 1995, 34, 352). In this case the redox components were not intercalated within the helix so the rate of electron transfer may simply reflect the time required to orient the donor and acceptor in order to obtain sufficient electronic coupling through the &pgr;-electron stack before long distance electron transfer can occur.
At this time, there are many unresolved questions regarding the ability of duplex DNA to mediate electron transfer. None of the systems cited above has unambiguously demonstrated the rate or efficiency of electron transfer between donor and acceptor moieties held at a fixed distance of separation in a DNA/DNA duplex.
In a modification designed to detect hybridization of nucleic acids in homogeneous solution, Tyagi and Kramer (Tyagi, S.; Kramer, F R. Nature Biotechnology 1996, 14, 303) describe a doubly substituted single-stranded DNA construct that possesses a stem-loop (i.e. hairpin) structure. The construct contains a fluorescer covalently linked to one terminus of the strand and an energy transfer quencher of the fluorescer at the opposite terminus. When unconjugated, this single stranded chain exists predominantly in hairpin conformation that constrains the fluorescer and quencher to be relatively close in space. When in this structural form, excitation of the fluorescer with actinic light leads to reduced emission because the fluorescing excited state transfers its energy to the nearby quencher. However, when this single-stranded structure hybridizes with a second strand complementary to its loop region, the distance between the fluorescer and quencher is increased and, consequently, the efficiency of fluorescence increases. The change in fluorescence intensity is an indicator that hybridization has occurred.
The modification described by Tyagi and Kramer offers several advantages for homogeneous real-time assays for hybridization. However there are certain disadvantages to the system they report. First, the indication of hybridization relies on energy transfer quenching of the fluorescer. This requires that the quencher have a lower excited singlet energy than the fluorescer, and this can cause difficulties in selecting a quencher whose absorption spectrum does not overlap with that of the fluorescer. Second, the nature of the hairpin

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