WO1998014271A1

WO1998014271A1 - Replaceable elements for a parallel sequential bio-polymer synthesis device

Info

Publication number: WO1998014271A1
Application number: PCT/US1997/017531
Authority: WO
Inventors: Anthony J. Christopher; Richard S. Neves; A. David Boccuti; Charles Ellis; Jose L. Valle
Original assignee: Individual
Current assignee: Individual
Priority date: 1996-10-04
Filing date: 1997-10-03
Publication date: 1998-04-09
Anticipated expiration: 1999-04-04
Also published as: CA2279232A1; AU4802597A

Abstract

Replaceable reagent contact elements (10) are disclosed. The elements are designed for use in a bio-polymer synthesizing device and may be used to synthesize oligonucleotides, polypeptides and other bio-polymers. The elements (10) are particularly useful for the simultaneous synthesis of a number of bio-polymers of the same or different sequence.

Description

REPLACEABLE ELEMENTS FOR A PARALLEL SEQUENTIAL BIO- POLYMER SYNTHESIS DEVICE

FIELD OF THE INVENTION

The present invention relates generally to a parallel sequential bio-polymer synthesis apparatus and, more particularly, relates to replaceable elements for the use in such an apparatus.

BACKGROUND OF THE INVENTION

Since the advent of bio-polymer synthesis methodologies based upon solid supports, the synthesis and use of bio-polymers of defined sequence has played an increasing, and ever more important, role in therapeutics, diagnostic medicine, forensic medicine and molecular biology research. For the purposes of the present invention, the term bio-polymers is seen to encompass peptides, polypeptides, oligo-ribonucleotides, oligo-deoxyribonucleotides, poly-ribonucleotides and poly-deoxyribonucleotides. In view of the increasing importance of synthetically prepared bio-polymers, there is a need in the art for methods and apparatus that permit the rapid synthesis of a large number bio-polymers of defined sequence.

The apparatus known in the prior art are capable of the accurate synthesis of bio-polymers of defined sequences but suffer from the drawback that they do not permit a high throughput of synthesis. These prior art devices are based upon the attachment of an appropriately prepared monomer, either amino acid or nucleotide, to a solid support, or resin, and placing the solid support in a column. Conventionally, the solid support is shaped into the form of a bead although other forms are known. Placement of the solid support beads into a column facilitates the sequential application of reagents to the solid support using known fluid handling technologies previously developed for use in other column based methodologies. This arrangement results in apparatus that are capable of synthesizing bio-polymers of defined sequences. General methodologies useful in the synthesis of bio-polymers are known to those skilled in the art and are taught in numerous prior art references such as U. S. patent no. 4,458,066 to Caruthers et al., U. S. patent no. 4,415,732 to Caruthers et al., and Bray et al., Journal of Organic Chemistry, volume 56, pages 6659-6671, 1991, the disclosures of which are specifically incorporated herein by reference.

Recently, methodologies based on a parallel sequential reactor have been developed. This strategy is exemplified by U.S. Patent 5,288,468, issued to Church et al. (hereinafter Church), the specification of which is specifically incorporated herein by reference. The apparatus of Church is a bio-polymer synthesizer capable of the simultaneous synthesis of a number of bio-polymers, each of a defined, and potentially different, sequence.

The device of Church includes a number of discrete surfaces upon which bio-polymers may be synthesized. The discrete nature of the surfaces permits the simultaneous preparation of a number of bio-polymers, each of which may have a different sequence from those prepared at the same time. The surfaces have a solid support suitable for the synthesis of bio-polymers adhered to them. The device also includes a number of reagent chambers which hold reagents used in the synthetic reactions. By movement of the individual surfaces and the chambers, the device allows individual control over the contact of each surface with each reagent chamber. The movement of a surface into and out of a sequence of reagent chambers results in the proper sequence of reactions for the synthesis of a bio-polymer of desired sequence.

Each discrete surface is attached to the distal portion of a reagent contact element referred to in Church as a reagent tip. Although, in the interest of clarity, the nomenclature of Church will be used when describing the polymer synthesis apparatus, the use of such nomenclature is not to be construed as limiting the reagent contact element in any fashion. Each reagent tip is attached to the end of a rod that is an integral part of a solenoid-piston assembly, with the solenoid action capable of raising or lowering the rod and the attached reagent tip. The solenoid-piston assemblies are arranged in an array of rows above an array of reagent chambers. The reagent chambers may consist of troughs cut in the surface of a block of inert plastic or may be discrete wells. The block can be moved, by a stepping motor, so as to allow a row of solenoid borne reagent tip access to a given reagent trough. When lowered a surface (or reagent tip) dips into the contents of the reagent chamber positioned beneath it.

Each reagent tip in a row is, by the action of the solenoids, either dipped into the trough below it or held above the trough and thus not dipped into the trough depending on whether the sequence of the molecule being constructed requires contact with the reagent in the trough. Reagent tips in adjacent rows are positioned above respective adjacent troughs. Dipping of surfaces is controlled individually and simultaneously, thus different reactions, i.e., reactions in different troughs, occur simultaneously.

The device also includes a system of valves, lines and reservoirs to supply the troughs with reagents, a motor to move the trough block, and a computer and interface to control the actions of the solenoids, valves, and trough block. The computer, which is programmed with the sequence of the bio-polymer to be synthesized on each reagent tip, generates instructions for the proper sequence of dipping, trough movement, and valve control, to produce the desired synthetic reaction, i.e., to effect the simultaneous synthesis of specific bio-polymers of defined sequence on specific surfaces.

The reagent tips of Church are made of polypropylene. They are permanently affixed to the end of the solenoid-piston assembly by gluing. A solid support, in the form of a bead, is adhered to the reagent tip by heating the reagent tip until it is just molten, then forcing the heated tip into a shallow container filled with the appropriate bead. When the beads coupled to a given monomer are used for the synthesis of a molecule, that monomer forms the first subunit used in the preparation of the bio-polymer. In the practice of the present invention, any suitable material may be used to construct a solid support. The solid support may fashioned of glass fiber, cellulose, controlled pore glass beads, polypropylene, teflon, cellulose, polyethylene, polysulfones, polyvinylidene difluoride, or any suitable organic or inorganic material known to those skilled in the art. Commercially available solid supports consisting of beads derivatized to incorporate either nucleotide or amino acid monomers may be used. Such solid supports are readily available to those skilled in the art. The reagent tips of Church suffer from several limitations. The method of manufacture limits the number of solid support beads that may be fastened to any one tip. This limits the potential yield of the bio-polymer to be prepared. In addition, the methodology of Church is slow and cumbersome and requires a great deal of hands-on time by the operator in the preparation of reagent tips. This drastically limits the throughput potential of the device.

SUMMARY OF THE INVENTION.

The apparatus of the prior art are limited in utility. It is an object of the present invention to provide a reagent contact element that overcomes the limitations of the apparatus of the prior art.

The apparatus of the prior art are difficult to replace and change. Another object of the present invention is to provide a reagent contact element capable of rapid and simple replacement and change in the bio-polymer synthesis device of the prior art.

The apparatus of the prior art have a severely limited production capability. It is another object of the present invention to provide a reagent contact element configured to permit the synthesis of a larger quantity of bio-polymer by each reagent contact element.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a cross-section of one embodiment of the present invention showing a porous vessel filled with resin attached to the reagent contact element.

Figure 2 is a cross-section of an embodiment of the present invention in which porous membranes are attached to the end of the reagent contact element with solid support resin trapped between the membrane sheets.

Figure 3 is an embodiment of the present invention showing a reagent contact element plugged with a porous filter material and sealed with a porous membrane with solid support resin trapped there between. Figure 4 is a cross section of an embodiment of the present invention showing a porous filter element molded into the reagent contact element.

Figure 5 shows an embodiment of the present invention where a porous vessel is attached to the end of the reagent contact element.

Figure 6 shows an embodiment of the present invention wherein the end of the reagent contact element has been modified with slots so as to permit greater reagent access to the interior of the reagent contact element.

Figure 7 shows an embodiment of the invention incorporating axial and radial locating features.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an interchangeable portion of a chemical reaction system wherein substances are chemically combined by intermittently dipping a solid support into a number of reagents. The invention is a disposable, replaceable reagent contact element that fits to the end of a rod which may be brought into intermittent contact with reagents to produce reactions. The two following features are essential characteristics of each embodiment of the present invention: 1) the reagent contact elements will have sites for chemical reactions; and 2) the reagent contact elements will interface with a rod or other device for bringing the element into contact with reagents.

The reagent contact elements may be made of a chemically inert material which incorporates a solid support resin onto which molecules may be synthesized or reacted. The inert portion of the reagent contact element may be formed of any suitable material as long as such material is chemically inert to the reagents required for the subsequent synthesis reactions. In addition to being chemically inert, the material used for reagent contact elements must also have a low water absorption. The reagent contact elements of the present invention may be made from a variety of materials such as polypropylene, teflon, cellulose, polyethylene, polysulfones, polyvinylidene difluoride or glass as long as the material possesses the characteristics of non-reactivity and low water absorption. In a preferred embodiment, the reagent contact elements are made of polypropylene. The reagent contact elements of the present invention are removably attached to rods which are integral parts of the solenoid-piston assemblies of the prior art. The rods are used to transport the reagent contact elements into reactive media or fluids. The reagent contact element is fictionally retained upon the rod. The reagent contact element defines a cylindrical space that is in fluid communication with the exterior of the reagent contact element. This permits use of the reagent contact element in a device where the rod delivers fluid through the reagent contact element as well as in a device wherein the rod has no fluid connections. It may be desirable to convey reagents through the reagent contact element and into the reaction mixture. Alternatively, it may be desirable to convey an inert gas through the reagent contact element in order to expel reagents from the reagent contact element and/or agitate a reagent mixture into which the reagent contact element is submerged.

The reagent contact elements can contain a variety of solid support "seed" molecules. For the purposes of this application seed molecules is seen to encompass amino acids, nucleotides and molecules with reactive functionalities. While generally the seed molecules will be monomeric nucleotides or amino acids, one skilled in the art will recognize that it is possible, and in some cases desirable, to use a defined sequence bio-polymer as the seed molecule. This embodiment will be useful when it is desired to synthesize a number of molecules that share some but not all sequences. For specialized uses, it may be desirable to have a reagent contact element to which various molecules may be attached using the reactive functionalities.

The solid support molecules may be of any type known to those skilled in the art. In a preferred embodiment the solid support molecule is controlled pore glass beads. The solid support molecules may be attached to the reagent contact element by a variety of methodologies. In one embodiment the solid support is attached to the reagent contact element using hot melt glue. Alternatively, the solid support may be heat welded or ultrasonically welded to the reagent contact element. In a preferred embodiment the solid support is ultrasonically welded to the reagent contact element. In other embodiments, the solid support is attached to the reagent contact element by trapping the solid support between porous sheets as shown in Figure 2. The porous sheets may be made of any material so long as the material is not reactive with the reagents required for synthesis. The porous sheets may be attached to the reagent contact element by glue, heat welding, or ultrasonic welding.

In another embodiment, a porous filter plug is inserted into the reagent contact element to define a cavity at the distal end of the reagent contact element as shown in Figure 3. The cavity is filled with solid support material and then the end of the contact element is sealed with the porous sheet. Alternatively, a porous filter element may be molded into the body of the reagent contact element as shown in Figure 4.

In another embodiment, seed molecules are incorporated into the material making up the reagent contact element. These seed molecules may be monomers or multimers, nucleotides or amino acids, appropriately blocked for use in synthesis. Alternatively, the active site may be a reactive functionality suitable for subsequent reaction so as to incorporate a monomer into the reagent contact element.

In another embodiment the solid support is attached directly to the reagent contact element. This may be accomplished by means of glue, such as hot melt glue or silicone glue, or by heat welding or ultrasonic welding. In an alternative embodiment, a membrane that has active sites may be attached to the reagent contact element. For example, the DNA synthesis membrane described in U.S. Patent 4,923,901 to Koester et al., the specification of which is specifically incorporated herein by reference, may be attached to the reagent contact element. When membranes are used one or more membranes may be attached to a single reagent contact element so as to provide more reactive sites.

The reagent contact elements are designed to fit in an array of holes in a tray for easy insertion/removal from an array of pins. The array of holes in the tray may be fashioned so as to be substantially identical to the array of pins in the parallel sequential bio-polymer synthesizer. Both the array of pins and the array of holes in the tray may be fashioned so as to be compatible with devices currently in use in the art. For example, the spacings of the pins in the apparatus and the holes in the tray may be the same as that in a standard 96- well microtiter plate. The reagent contact elements may be delivered as individual reagent contact elements in a tray or alternatively the reagent contact elements may be attached to one another in an array rather than loose in the tray, i.e., the reagent contact elements may be molded into one piece. The reagent contact elements thus molded may be used all at once or alternatively one or more reagent contact elements may be removed from the molded array and used separately.

With reference to Figure 1 the reagent contact element 10 is shown frictionally attached to a rod 12 of the parallel sequential bio-polymer synthesis apparatus. In one embodiment the rod may be equipped with a circumferential protrusion 14. In this embodiment the reagent contact element 10 is equipped with a groove 16 adapted to frictionally engage the circumferential protrusion 14 so as to ensure a positive attachment to the rod 12. Optionally, the rod 12 may be equipped with an ejection sleeve 18 so as to facilitate the automatic removal of the reagent contact elements at the end of the synthesis reaction.

The reagent contact elements may be equipped with a flange 20. When the ejection sleeve 18 is activated it pushes against flange 20 so as to disengage the reagent contact element from the rod. In addition, flange 20 serves to retain the reagent contact element in a tray when the reagent contact elements are dispensed in a tray. Figure 1 shows the reagent contact element submerged in a reagent solution 22 that is conveyed in trough 24. In the embodiment shown, the reagent contact element comprises a porous vessel 26 containing solid support resin and the porous vessel 26 is submerged in the reagent mixture 22.

With reference to Figure 2, an alternative embodiment is shown wherein reagent contact element 10 is provided with two porous membranes 28 between which solid support 30 is trapped.

Figure 3 shows an embodiment of reagent contact element 10 wherein a filter element 32 is inserted into reagent contact element 10 so as to define a cavity in the distal portion of reagent contact element 10. The filter element 32 frictionally engages the side walls of reagent contact element 10 so as to maintain its position relative to the distal end of the reagent contact element 10. The cavity defined by the filter element and the side walls of reagent contact element 10 is then filled with solid support 30 and the end closed off with membrane 28. Figure 4 shows an alternative embodiment wherein filter element 34 is molded from the side walls of reagent contact element 10 and forms an integral part of the reagent contact element. The molded filter element 34 defines a cavity at the distal portion of reagent contact element 10. The cavity is then filled with solid support 30 and closed off with membrane 28.

Figure 5 shows an alternative embodiment of the present invention wherein the most distal portion of reagent contact element 10 has been modified so as to permit the attachment of a hanging porous vessel 36. Solid support material is enclosed within the hanging porous vessel. The hanging porous vessel 36 may be fabricated of any suitable material i.e., one that is nonreactive with the reagents used in the synthetic process and has a low water absorption as well as the requisite strength.

Figure 6 shows an embodiment of the invention wherein the most distal portion of reagent contact element 10 has been equipped with one or more slots 38. The slots 38 promote entrance of the reagents into the reagent contact element and facilitate drainage of the reagents from the reagent contact element. One skilled in the art will appreciate that the slots 38 may be incorporated into any other embodiment of the instant invention. Figure 6 shows an embodiment of the invention wherein a porous vessel defined by two porous sheets 28 enclose solid support material 30.

Figure 7 shows an embodiment of the invention wherein the proximal portion of the reagent contact element 10 has been modified so as to incorporate axial and radial locating features. In the embodiment shown, the reagent contact element 10 has been modified to define a slot 40 which engages a pin 42 on rod 12. Other methods of insuring the accurate placement of the reagent contact element on the rod are known to those skilled in the art and are seen to be within the scope of the present invention.

The reagent contact elements of the present invention can be manufactured by making up separate porous vessels then attaching the porous vessels to the inert portion of the reagent contact elements. The fabrication of the inert portion of the reagent contact element is within the skill of the ordinary practitioner in the art. The porous vessels may be attached by gluing, heat welding or ultrasonic welding to the reagent contact element. In preferred embodiments, the porous vessels are ultrasonically welded to the reagent contact element.

The porous vessels may be made by forming a well or indentation in a sheet of porous material then filling the well with solid support material and then sealing the top of the well with a second piece of porous material. Alternatively, solid support material may be trapped between two flat sheets of porous material. One skilled in the art will readily appreciate that these two methods will provide for the incorporation of various amounts of solid support material thereby permitting adjustment of the scale of the reaction to suit individual synthetic applications. The porous vessels will be designed so as to maximize the amount of the reaction sites at the lower end of the reagent contact element to minimize the reagent usage in the trough. For example, in the embodiment of Figure 5, the hanging bag may be configured so as to trap the majority of the solid support material at the most distal portion of the bag. This can be accomplished by creating a seal 44 at some point along the length of the bag so as to trap the material into the distal portion.

The reagent contact elements are constructed of a size to fit into ninety-six well microtiter plates while in an array in a tray. This permits placing reagent into a microtiter plate and dipping the array of reagent contact elements into the reagents. This will be useful for the simultaneous cleavage of completed polymers from the solid support and transfer of the polymer into a microtiter plate for subsequent screening. For example, in the case of an oligonucleotide, the wells of the microtiter plate may be filled with a cleavage reagent and the reagent contact elements may be placed in a tray and dipped into the reagent.

The reagent contact elements are designed so as to interface firmly in a frictional fit with the rods 12. For example, the reagent contact elements may be configured with a groove 16 and the rods with a circumferential protrusion 14 designed so as to engage the groove as shown in Figure 1. Alternatively, the rod and the interior of the reagent contact element may be manufactured with complimentary tapers so that the reagent contact element may be slid onto the end of the rod and firmly engage the rod.

The reagent contact elements of the present invention may be distributed in trays. The trays may be both color coded for easy identification by users and have a unique feature such as a whole or bar code, etc. for automatic identification by the instrument. The trays will be designed such that the reagent contact elements may be made color coded for different starting seed molecules that are attached at the end of the reagent contact element. The reagent contact elements may be made of translucent material for better view of the reagents used during the synthetic process. The reagent contact element is of such shape and length so as to bring the solid support into appropriate position in a trough or microtiter plate. The reagent contact element is equipped with axial and radial locating features which interface to the rod for precise reagent contact element positioning. These features may be slots or grooves in the reagent contact element and corresponding pins or protrusions in the rod.

This invention has been described in terms of specific embodiments set forth in detail, but it should be understood that these are by way of illustration only and that the invention is not limited to the specifically recited embodiments. Modifications and alterations will be readily apparent to those skilled in the art and these modifications and alterations are within the scope of the invention. Accordingly, these modifications and alterations of the disclosed invention are considered to be within the scope of the invention.

Claims

What is claimed is:

1. A replaceable reagent contact element for a bio-polymer synthesis apparatus, comprising: a body; and seed molecules attached to said body.

2. A replaceable reagent contact element according to claim 1, wherein said seed molecule comprises a nucleotide or nucleoside.

3. A replaceable reagent contact element according to claim 2, further comprising axial and radial locating elements.

4. A replaceable reagent contact element according to claim 3, wherein said body defines a cylindrical space.

5. A replaceable reagent contact element according to claim 4, wherein said body defines a groove adapted to frictionally engage a circumferential protrusion.

6. A replaceable reagent contact element according to claim 4, wherein said body comprises a flange.

7. A replaceable reagent contact element according to claim 4, wherein said body defines at least one slot.

8. A replaceable reagent contact element according to claim 4, further comprising a porous filter.

9. A replaceable reagent contact element according to claim 8, wherein said porous filter is inserted into the cylindrical space so as to define a cavity in the distal portion of the cylindrical space, said seed molecules being located within the cavity, and a porous membrane attached to the body so as to retain the seed molecules within the cavity.

10. A replaceable reagent contact element according to claim 4, further comprising at least one porous membrane attached to said body.

11. A replaceable reagent contact element according to claim 10, wherein said porous membrane defines a space and said seed molecules are located within the space.

12. A replaceable reagent contact element according to claim 10, wherein said seed molecules are attached to said membrane.

13. A replaceable reagent contact element according to claim 10, wherein two porous membranes are attached to said body so as to define a space and said seed molecules are located within the space.

14. A replaceable reagent contact element according to claim 1, wherein said seed molecule comprises an amino acid.

15. A replaceable reagent contact element according to claim 14, further comprising axial and radial locating elements.

16. A replaceable reagent contact element according to claim 15, wherein said body defines a cylindrical space.

17. A replaceable reagent contact element according to claim 16, wherein said body defines a groove adapted to frictionally engage a circumferential protrusion.

18. A replaceable reagent contact element according to claim 16, wherein said body comprises a flange.

19. A replaceable reagent contact element according to claim 16, wherein said body defines at least one slot.

20. A replaceable reagent contact element according to claim 16, further comprising a porous filter.

21. A replaceable reagent contact element according to claim 20, wherein said porous filter is inserted into the cylindrical space so as to define a cavity in the distal portion of the cylindrical space, said seed molecules being located within the cavity, and a porous membrane attached to the body so as to retain the seed molecules within the cavity.

22. A replaceable reagent contact element according to claim 16, further comprising at least one porous membrane attached to said body.

23. A replaceable reagent contact element according to claim 22, wherein said porous membrane defines a space and said seed molecules are located within the space.

24. A replaceable reagent contact element according to claim 22, wherein said seed molecules are attached to said membrane.

25. A replaceable reagent contact element according to claim 22, wherein two porous membranes are attached to said body so as to define a space and said seed molecules are located within the space.

26. A replaceable reagent contact element according to claim 1, wherein said seed molecule comprises a reactive functionality.

27. A replaceable reagent contact element according to claim 26, further comprising axial and radial locating elements.

28. A replaceable reagent contact element according to claim 27, wherein said body defines a cylindrical space.

29. A replaceable reagent contact element according to claim 28, wherein said body defines a groove adapted to frictionally engage a circumferential protrusion.

30. A replaceable reagent contact element according to claim 28, wherein said body comprises a flange.

31. A replaceable reagent contact element according to claim 28, wherein said body defines at least one slot.

32. A replaceable reagent contact element according to claim 28, further comprising a porous filter.

33. A replaceable reagent contact element according to claim 32, wherein said porous filter is inserted into the cylindrical space so as to define a cavity in the distal portion of the cylindrical space, said seed molecules being located within the cavity, and a porous membrane attached to the body so as to retain the seed molecules within the cavity.

34. A replaceable reagent contact element according to claim 28, further comprising at least one porous membrane attached to said body.

35. A replaceable reagent contact element according to claim 34, wherein said porous membrane defines a space and said seed molecules are located within the space.

36. A replaceable reagent contact element according to claim 34, wherein said seed molecules are attached to said membrane.

37. A replaceable reagent contact element according to claim 34, wherein two porous membranes are attached to said body so as to define a space and said seed molecules are located within the space.

38. A replaceable reagent contact element for a bio-polymer synthesis apparatus, comprising: a body, wherein said body comprises a reactive functionality.

39. A replaceable reagent contact element according to claim 38, further comprising axial and radial locating elements.

40. A replaceable reagent contact element according to claim 39, wherein said body defines a cylindrical space.

41. A replaceable reagent contact element according to claim 40, wherein said body defines a groove adapted to frictionally engage a circumferential protrusion.

42. A replaceable reagent contact element according to claim 40, wherein said body comprises a flange.

43. A replaceable reagent contact element according to claim 40, wherein said body defines at least one slot.

44. A method of synthesizing a biopolymer, which comprises:

providing a replaceable reagent contact element, the element comprising seed molecules;

contacting a reagent solution with the replaceable reagent contact element such that the seed molecules contact the reagent solution, the reagent solution containing molecules to be attached to the seed molecule;

attaching the molecules in the reagent solution to the seed molecules; and

repeating the contacting and attaching steps until a desired biopolymer is formed.

45. A method according to claim 44, wherein the seed molecule comprises a nucleotide or nucleoside.

46. A method according to claim 44, wherein the seed molecule comprises an amino acid.

47. A method according to claim 44, wherein the seek molecule comprises a reactive functionality.

48. A method of synthesizing a biopolymer which comprises:

providing a replaceable reagent contact element, the element comprising a reactive functionality;

contacting a first reagent solution containing a first molecule with the replaceable reagent contact element such that the reactive functionality contacts the reagent solution;

attaching the first molecule to the reactive functionality;

contacting the replaceable reagent contact element with at least one additional reagent solution containing at least one additional molecule;

attaching the additional molecule to the first molecule; and

repeating the contacting and attaching steps with additional reagent solutions containing additional molecules until a desired biopolymer is formed.

49. A method according to claim 48, wherein the first molecule is a nucleoside or nucleotide.

50. A method according to claim 48, wherein the first molecule is an amino acid.