Detection of biological macromolecules by NMR-sensitive labels

An apparatus and method for identifying the presence of biological molecules labeled with isotopes that are identifiable and distinguishable by their responses to nuclear magnetic resonance. The method features the use of labels that are: PA1 1) not radioactive or only weakly radioactive, therefore not hazardous to handle, and have a long storage life. PA1 2) small in molecular weight, thus not significantly changing the electrophoresis migration rate of the labeled material. PA1 3) available as several distinguishable labels, thereby enabling simultaneous detection of several classes of molecules.

BACKGROUND OF THE INVENTION 
The ability to detect the presence of a particular biological molecule or 
part of such a molecule at a specific location is an indispensable tool in 
Biological researches and in medicine. Presently, the most frequently used 
method of detection is to label the molecule with a radioactive isotope 
and detect the presence of the label by the radiation (usually beta 
emission) of the label isotope. 
Isotopes that are reliable as labels materials emit high-energy electrons 
at rapid rate. That makes them unsuitable for use in many potential 
applications. This is especially true in situations where the label 
isotopes must be applied to living tissues. Furthermore highly radioactive 
materials are hazardous to handle and have short storage life. 
It is therefore highly desirable to have labeling materials that are only 
slightly radioactive, thus having long storage life; or better yet, not 
radioactive at all. 
There have been serious attempts to define non-radioactive labels. However 
none of them have yet proven as reliable or as sensitive as radioactive 
Phosphorus-32. 
In some applications, it would be extremely useful to have distinguishable 
labels on different parts of biological systems, and be able to tell which 
part is present. An example of such an application is sequencing of DNA. 
One of the essential steps in the sequencing of DNA is the use of an 
elctrophoresis gel column. In this step, the DNA fragments are placed in a 
reservoir at one end of a polyacrylamide column. An electric field 
(typically 1KV over a distance of 50 mm) causes the DNA fragments to 
migrate through electrophoresis towards the anode (at the other end of the 
column). As the molecules of the DNA fragments travel down the column, 
some molecules travel faster than others, molecules of different speeds 
concentrate in discrete bands. After a time period (typically 2 hours), 
the DNA fragments are spread over a column of discrete bands. Within each 
band are DNA fragments of a particular migration speed. If the DNA 
fragments had been labeled with some means of identification (for example 
radioactive isotopes or fluorescent dyes) then their presences can be 
detected. 
One of the most common and most reliable isotope for labeling is 
Phosphorus-32 which emits beta radiation (electrons). DNA fragments that 
are labeled with P-32 are easily detected (for example by x-ray film). 
In the case of DNA sequencing, there are four different Dideoxy 
chain-terminations at the ends of the fragments that need to be 
identified. P-32 alone can only label one of them. It is still common 
practice to run four parallel electrophoresis columns with P-32 labeling a 
different terminations in each column. 
It would be extremely useful to be able to have distinguishable labels on 
different Dideoxy chain-terminations so a single lane gel can be used and 
the identities of the nucleotides can be determined in real time as the 
bands pass by a detector that has the capability of distinguishing the 
identity of the different labels. 
There are other beta emitting isotopes besides P-32, but their radiations 
are spread over a wide spectrum, so one Beta emitter is difficult to 
distinguish from another. 
Other distinguishable labels have been used in attempts on automatic DNA 
sequencing. One such example is the use of several fluorescent dyes that 
re-emit lights of distinguishable colors. But the dyes are large 
molecules, attaching such a label to a DNA fragment changes the migration 
rates, and that causes problems in identification. 
The present invention provides a new possibility for labeling molecules in 
biological studies. The method exploits the response of atomic nuclei to 
Nuclear Magnetic Resonance (NMR). One of the advantages of the present 
invention is the possibility of using several distinguishable labels in a 
system. 
The use of nuclear magnetic resonance (NMR) in biology and medicine has 
become commonplace since the advent of Magnetic Resonance Imaging (MRI) 
machines. These machines use NMR to detect presence of hydrogen nuclei 
(protons) in water and fatty tissues. Since MRI is less invasive than 
X-ray and can detect features that are not observable by X-ray, it has 
become an indispensable tool in many disciplines of medicine. MRI machines 
are very expensive. Part of the expense is the cost of the magnets needed 
to provide the needed field over a large volume (most MRI machines are big 
enough for people to enter into the magnet area), another part of the 
expense is the sophisticated electronics, including computers, to map out 
the protons. 
For many biological applications, including automatic sequencing of DNA, 
neither a large detection area nor a high-resolution image is needed. For 
such applications, inexpensive permanent magnets can be used instead of 
large super-conducting magnets, and detection instruments compact enough 
to fit into small laboratories can become a reality. 
Another common application of NMR is in spectroscopy. There the spatial 
resolution is not critical, but the NMR output is scanned over a wide 
spectrum at very high spectral resolution. 
For many biological applications where isotopes with the suitable NMR 
properties are used as labels, successful detection of the label isotope 
requires neither the spatial resolution of MRI nor the spectral resolution 
of spectroscopy. It can therefore be a much simpler machine. 
Technologies developed for MRI and for spectroscopy can obviously be 
"borrowed", when applicable, to improve the detection of NMR-active labels 
in biological molecules. 
Whereas MRI uses almost exclusively hydrogen as the NMR-active isotope, and 
spectroscopy uses mostly hydrogen and carbon-13; there are many other 
isotopes that are suitable for used as NMR-active labels in biological 
applications. 
For applications to automated DNA sequencig, it would be highly desirable 
to be able to simultaneous identify all four bases at the end of a DNA 
chain--Adenine, Guanine, Thymine, and Cytosine (A,G,T & C). The present 
invention provides a possibility for using the following combination of 
labels: 
______________________________________ 
A labeled with P-31 only, 
G labeled with P-31 + H-3, 
T labeled with P-31 + F-19 
and C labeled with P-31 + F-19 + H-3. 
______________________________________ 
Reasons for the above choice are described in greater details below, in the 
"Detailed Descriptions" section. 
SUMMARY OF THE INVENTION 
The present invention provides an apparatus and method for identifying the 
presence of biological molecules labeled with isotopes that are 
identifiable and distinguishable by their responses to nuclear magnetic 
resonance. The method features the use of labels that are: 
1) not radioactive or only weakly radioactive, therefore not hazardous to 
handle, and have a long storage life. 
2) small in molecular weight, thus not significantly changing the 
electrophoresis migration rate of the labeled material. 
3) available as several distinguishable labels, thereby enabling 
simultaneous detection of several classes of molecules.

DETAILED DESCRIPTION 
An apparatus for detecting the presence of NMR-sensitive isotopes is shown 
in FIGS. 1 and 1a. The apparatus comprises a magnet 1 with pole pieces 2 
to generate a stable and homogeneous magnetic field in the sample area 3. 
It is usually desirable to have the magnetic field as strong as possible. 
NMR would be very difficult to observe in any field less than one thousand 
Gauss (1KG). The magnet 1 may be a permanent magnet, an electromagnet, or 
a superconductor magnet. Permanent magnets are the least expensive, they 
also require the least maintenance, and provide a very stable field of 
strength up to around 15K Gauss; electromagnets can provide more field 
strength, but they consume power and usually require cooling in addition 
to feedback circuits to keep the field stable; Superconductor magnets 
provides the highest field strength (100K Gauss), but they are presently 
most expensive and must be kept at liquid Helium temperatures. 
Positioned around the sample area 3 is a set of coils 4 for applying a 
radio frequency (RF) input magnetic field to the sample in a direction 
perpendicular to the constant magnetic field. The magnetic field 
oscillating at the NMR frequency excites the nuclei to a higher energy 
state, the energy is later reemitted as an output signal. 
Another set of coils 5 (oriented perpendicular to both the constant 
magnetic field and the input RF field) is provided for detecting NMR 
output from the sample. The output from coils 5 is fed to a RF receiver 
(not shown in FIGS. 1 & 1a). In the coordinates system shown in FIGS. 1 
and 1a, the axis of the input coils 4 is along the x axis, the axis of the 
output coils 5 along the y axis, and the constant field is along the z 
axis. 
The output coils 5 shown in FIGS. 1 & 1a are thin and rectangular in shape 
so they will pick up only NMR signals from a small range of positions 
along the x-axis. The output coils 5 are oriented substantially 
perpendicular to the input coils 4. The positions and orientations of the 
input and output coils are carefully adjusted so that the output coils 5 
pick up as little of the RF signal directly from the input coils 4 as 
possible. 
It may be difficult, by mechanical adjustments alone, to reduce the 
direct-coupled noise (unwanted signal coupled directly from the input 
coils 4 to the output coils 5) to a low enough level for satisfactory 
detection of the NMR output. When necessary, the residual direct-coupling 
noise, may be further reduced electronically, by adding to the output a 
part of the input signal with the amplitude and phase adjusted (when no 
NMR nuclei is present in the sample area) to null out all the 
direct-coupling noise. This electronic nulling circuit is shown as an 
attenuator 31, a phase-shifter 32 and adding circuits 33 in FIG. 3. 
FIG. 2 shows the output coil 5 positioned along an electrophoresis column 
6. The electrophoresis column 6 is similar to the type used for sequencing 
DNA. At the top of the column 6 is a cathode 8 and a reservoir 7 where the 
DNA samples are initially positioned. At the bottom of the column 6 is an 
anode 9. An electric voltage source 10 maintains an electric field between 
the anode and the cathode. Typically, the column 6 would be a 
polyacrylamide gel column, about 50 mm long. A typical voltage across the 
electrodes 8 and 9 is around 1 kv. Bands of DNA fragments 11 are detected 
as they migrate pass the output coil 5. 
The NMR output signal from the output coils is amplified by an amplifier 34 
(FIG. 3); and the direct-coupling noise is removed by electronic nulling. 
The signal is then multiplied (with multiplier 35) to a component of the 
input RF signal (this component of the input signal is adjusted, by means 
of an attenuator 36 and a phase-shifter 37, to be in-phase with the output 
signal). The product is then integrated, by integrator 38, over a period 
of time (typically, 1 second). Upon integration, the random noise averages 
out to zero; but the in-phase product of the NMR input and output signals 
integrates out to a detectable value, and may be displayed by a recorder 
39. 
The technique for enhancing signal-to-noise ratio by multiplying the input 
and output signals and integration over time is well known among 
electronic engineers as "coherent detection". It is one of the simplest 
methods to extract usable data from the very weak NMR output signals. 
Continuous wave (CW) RF signals as weak as 10.sup.-9 volt can be 
successfully detected by this technique. More sophisticated methods for 
processing NMR signals have been developed: For example, Pulsed Fourier 
Transform techniques for NMR spectroscopy and Computer Image 
Reconstruction techniques for MRI provide higher performances than the 
relatively simple method described here. These more advanced techniques 
can obviously be "borrowed" when their higher performances are required 
and their much higher costs can be tolerated. 
It is desirable to use as label nuclei isotopes that have the highest 
sensitivity to NMR (thus providing the strongest output signals). Among 
the isotopes that are most sensitive to NMR are Hydrogen-1, Hydrogen-3, 
and Fluorine-19. 
Hydrogen-1, among all stable isotopes, has the highest NMR sensitivity. 
That is fortunate for MRI applications. However, for some other biological 
studies (such as sequencing of DNA), hydrogen-1 is probably not a 
particularly good label material because the molecules to be studied are 
usually surrounded by a large quantity of water, so any label of 
hydrogen-1 would be confused with the large background of hydrogen-1 in 
water. 
Fluorine-19 is the second most sensitive stable isotope for NMR, it is the 
most abundant isotope in natural Fluorine, and it is not radioactive. 
Fluorine is not present in any appreciate amount in most biological 
systems, but it can easily replace hydrogen in many organic molecules so 
it is an excellent candidate for use as an artificially-introduced label 
material. 
Hydrogen-3 (Tritium) has the highest sensitivity for NMR, it is weakly 
radioactivity, emitting low-energy beta particles and having a half-life 
of over twelve years (by comparison, Phosphorus 32, a frequently-used 
radioactive label, emits 100-times-more-energetic beta particles, and has 
a halflife of about two-weeks). Hydrogen-3 is chemically identical to 
ordinary hydrogen, that can be a very important advantage when the labeled 
molecules has to go through some chemical reactions before detection. 
Artificial introduction of a NMR sensitive labeling isotope to a biological 
molecules is quite similar to the introduction of radioactive labels. 
Techniques for such processed are well-developed, and label materials such 
as Fluorouracil and Tritium-labeled deoxynucleotide Triphosphates (H.sup.3 
dNTP's) are easily available. 
One advantage of the use of NMR for labeling molecules in Biological 
systems is the possibility for using more than one distinguishable label 
isotopes in a system. Different label isotopes can be easily distinguished 
by their NMR frequencies (in general, NMR has very narrow bandwidth and 
different isotopes in the same magnetic field have widely different NMR 
frequencies.) The NMR frequency of a nucleus in a given magnetic field is 
also affected by the surrounding molecular structure: but this effect 
(usually less than 0.2%) is very small compared to the differences of 
frequencies between different isotopes. In most cases, the NMR frequency 
of the isotope in the labeled molecule (or a similar molecule) can be 
determined and compensated for before the experiment. The frequencies and 
sensitivities of most common isotopes are available from handbooks (such 
as the Handbook of Chemistry and Physics). 
For applications to automated DNA sequencing, it would be highly desirable 
to be able to simultaneous identify all four possible bases at the end of 
a DNA chain--Adenine, Guanine, Thymine, and Cytosine (A,G,T & C). In order 
to provide four distinguishable labels, one needs one more label isotope 
(in addition to Hydrogen-3 and Fluorine-19). 
Phosphorus-31 is the "natural phosphorus", it is not radioactive, and its 
sensitivity to NMR (at constant magnetic field) is about one-sixteenth 
that of Hydrogen-1. In spite of its much lower sensitivity, Phosphorus-31 
is potentially an excellent label material for DNA because Phosphorus is 
present naturally in the DNA chains. 
With the above listed bases and suggested label isotopes, one possible 
combination for labeling is: 
______________________________________ 
A labeled with P-31 only, 
G labeled with P-31 + H-3, 
T labeled with P-31 + F-19 
and C labeled with P-31 + F-19 + H-3. 
______________________________________ 
Of course other combinations, or combinations using other isotopes can also 
be used for labeling. 
There are other isotopes (besides H-1, H-3, and F-19) that have higher NMR 
sensitivity than Phosporus-31. Some of them may also turn out to be good 
materials for NMR labeling. However, it is not clear whether the advantage 
of being more sensitive to NMR alone would make them better choice than 
Phosphorus which is already in DNA. Some of these isotopes are difficult 
to attach by chemical means (for example Helium-3). Some may have 
potentially-unacceptable effects on the biological properties of the 
labeled molecules (most metals--Lithium-7, Sodium-23, Aluminum-27, and 
several heavier metals are all questionable as labels for biological 
systems). Boron is commonly used in the buffer in electrophoresis gel 
columns, so the use of Boron-11 as label may be troubled by the presence 
of the same nuclei in the background. Carbon-13, less sensitive to NMR 
than P-31, is also questionable as label because of the large quantity of 
carbon atoms in all biological systems. 
When more than one labeling isotopes are used with the apparatus of FIGS. 1 
and 1a, the input RF field at the input coil 4 may be the sum of the 
signals at all the NMR frequencies of the labeling isotopes used. The 
different label isotopes can be distinguished by feeding the NMR output 
into several coherent detectors as shown in FIG. 4, each detector will 
detect NMR output at only one of the input frequencies. 
The present invention can be used in other Biological applications (besides 
sequencing of DNA), for example, determining the sequences in RNA. In 
these other applications, the details of the apparatus and procedures may 
differ from those mentioned above; but the basic idea of using 
NMR-sensitive labels remains the same. 
The apparatus described so far detects the output signals from the test 
samples' NMR response to a continuous wave (CW) input. NMR is also 
observable in other forms, for example: 
1) NMR absorption; the absorption of RF signal from the input coils can 
also be observed and be used to detect the presence of the NMR label 
nuclei. 
2) Pulsed input, Pulse Fourier-transform techniques are often used in 
spectroscopy. 
3) Multiple output observations; the output is observed at many positions, 
and an image of the NMR source is reconstructed from the relative 
amplitudes and phases of the output at the different observation points. 
This method is used in MRI machines. 
Any of these or other variations or combinations of them may be used 
instead of the apparatus and method described above, and the application 
will still fall within the scope of the present invention. 
The present invention provides a method for identifying the presence of 
biological molecules which have been labeled with isotopes that can be 
detected by their responses to nuclear magnetic resonance. The method 
features the use of labels that are: 
1) not radioactive or only weakly radioactive, therefore not hazardous to 
handle, and have a long storage life. 
2) small in molecular weight, thus not significantly changing the 
electrophoresis migration rate of the labeled material. 
3) available as several distinguishable labels, thereby enabling 
simultaneous detection of several classes of molecules.