Residues for binding third strands to complementary nucleic...

Details Residues for binding third strands to complementary nucleic... Residues for binding third strands to complementary nucleic...

1995-06-07

2002-07-30

Fredman, Jeffrey (Department: 1655)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100, C536S024300, C536S024310, C536S024330, C514S049000, C435S005000, C435S006120, C435S091200

Reexamination Certificate

active

06426407

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to compositions of matter capable of serving as residues for specific binding of third strands to double-stranded complementary nucleic acids of any base-pair sequence.
2. Description of Related Art
Definitions
Before describing related art, it may be useful to define certain terms to be used throughout the specification in describing the present invention:
Canonical base—any one of the standard nucleic acid bases, adenine-A, guanine-G, cytosine-C, thymine-T, uracil-U.
Canonical base pair—the complementary or Watson-Crick base pairs, AT/U and GC, formed from canonical bases.
Canonical base triplet—a base triplet formed by the interaction of a canonical third strand base and a canonical base pair.
Direct base pair—a target base pair with its purine base located in a homopurine strand of a target duplex sequence, i.e., AT/U, GC.
Inverted base pair—a target base pair with its pyrimidine base located in a purine-rich strand of a target duplex sequence, i.e., U/TA, CG. An inverted base pair therefore interrupts the continuity of a homopurine strand sequence.
Triplex motif—triplex stereochemistry as determined by the predominant bases of the third strand binding to a target Watson-Crick duplex with purine-rich, pyrimidine-rich strands and by the orientation of the third strand relative to that of the purine-rich strand of the target. Note that the mode of third strand base or residue H-bonding to the target base pair is characteristic of each motif.
N-residue—a synthetic third strand residue designed to bind with specificity to a particular direct target base pair or inverted target base pair.
Oligonucleotides (third strands) can bind to double-stranded nucleic acids to form triple-stranded helices (triplexes) in a sequence specific manner (Beal and Dervan,
Science
251: 1360 (1991); Beal and Dervan,
Nucleic Acids Res
., 20:2773 (1992); Broitman and Fresco,
Proc. Natl. Acad. Sci. USA
, 84:5120 (1987); Fossella, et al.,
Nucleic. Acids Res
. 21:4511 (1993); Letai et al.,
Biochemistry
27:9108 (1988); Sun, et al.,
Proc. Natl. Acad. Sci. USA
86:9198 (1989)).
The third strand binding code (a complementarity principle) dictates the sequence specificity for binding third strands in the major groove of double-stranded nucleic acids to form a triple-stranded helix or triplex. The code provides the specificity of third-strand binding for design of gene-based therapeutics that bind specifically to target nucleic acid sequences with little or no non-specific binding to non-target sequences.
Third-strand binding differs from the familiar Watson-Crick complementarity principle (A:T/U and G:C) for the double-stranded helix in two major respects: (1) the third-strand binding code is degenerate, and (2) third strands bind only to double-strands which contain a sequence of adjacent (or run of) purine bases (A or G) in one of the strands, which here will be called the center or core strand. The third-strand binding code is illustrated in the Table 1 below.
TABLE 1
Third Strand Base
Center
Strand
Purine
A
U/T
I
G
C
A
+
+
+
−
−
G
−
−
+
+
+
In the center of the table, a “+” means the bases are complementary or correspondent, and a “−” means they are not complementary or not correspondent. The bases are: A=adenine (purine); G=guanine (purine); C=cytosine (pyrimidine); T=thymine (pyrimidine); U=uracil (pyrimidine in RNA); I=inosine (purine nucleoside with the universal third-strand binding base hypoxanthine).
A serious practical limitation for stable third-stand binding dictated by the code in Table 1 is the necessity for runs of purines in the center target strand of typically 10 or more bases interrupted by only one or two pyrimidines (hereafter called “purine-rich” sequences or targets). While runs of sufficient length are present in many of the genes and the non-gene DNA (or RNA) of eukaryotes and prokaryotes and their viruses, they are not frequent enough for widespread diagnostic and therapeutic uses. It is therefore desirable to be able to target a duplex nucleic acid segment with a mixed purine and pyrimidine composition in the center strand.
There are a number of “motifs” which further define third-strand binding to purine-rich targets, still in conformity with the third-strand binding code. The motifs define the base-compositional features of the third strand and whether the third-strand binds parallel or antiparallel to the purine-rich target strand (polarity). Motifs thereby define the hydrogen-bonding (H-bonding) schemes of the third-strand bases to the target base pairs. In consequence, the motifs also determine target specificity and nearest neighbor effects on binding. There are five motifs that describe third-strand binding (Sun and Helene,
Current Opinion in Structural Biology
. 3:345 (1993)). Table 2 summarizes the five motifs, which constitute a subset of constraints to the binding code. Thus, the motifs provide further instructions for defining the sequences of different third-strands that can alternatively bind with specificity to the same target. The Table also gives examples of selected analog bases which may be substituted for the standard or canonical A, G, T/U and C third-strand bases.
TABLE 2
Third-Strand
Binding
Some Analog
Bases/Strand Polarity
Code
Substitutions
Pyrimidine/parallel
T:AT
me
5
C
+
for C
+
C
+
:GC
propyne
5
C
+
for C
+
propyne
5
U for T
Purine/parallel (A-rich
A:AT
2,6 DAP for A
1
targets)
G:GC
Purine/antiparallel (G-rich)
A:AT
2,6 DAP for A
1
targets)
G:GC
T and G/parallel (high
T:AT
7-deaza-2′-deoxy-
nearest neighhor
G:GC
xanthosine for T
frequencies for AA, GG in
center strand)
T and G/antiparallel (high
T:AT
propyn
5
U for T
nearest neighhor
G:GC
frequencies for AG, GA in
center strand)
In the Binding Code column, the colon indicates third-strand binding of the base to the left of the colon, to at least the center purine base on the immediate right of the colon. The + superscript indicates that the base is in the protonated form when it binds (the energy of binding provides the energy for protonation). 2,6 DAP stands for 2,6 diaminopurine. “Parallel” or “antiparallel” refers to the relation of third-strand polarity in the triplex relative to the purine-rich target strand.
In designing third strands, there are other considerations that can affect the stability of the resulting triplex. Third strands composed of A and G bases, for example, have the potential for several kinds of self structure. Very G-rich third strands tend to form either hairpin or linear helices stabilized by G-tetrads (Fresco and Massoulie,
J. Am. Chem. Soc
., 85:1352 (1963); Zimmerman, et al.,
J. Mol. Biol
., 92:181 (1975); Gern, et al.
Biochemistry
, 34:2042 (1994)). With more equal portions of A and G bases, linear or hairpin duplexes with AG, AA and GG base-pairs form, as well as the tetraplexes, which have melting temperatures (T
m
) in the same ranges as do dissociations of such third-strands from target duplex. Such third-strand self structures will obviously weaken third-strand binding by altering the equilibrium from triplex toward the duplex plus self-structured third strand. The processes and compositions of this invention can be utilized to devise third strands with reduced tendency for self-structure.
U.S. Pat. Nos. 5,034,506, 5,166,315 and 5,405,938 are directed to various polymers said to be effective to bind to Watson-Crick base pairs. In contrast to the present invention, however, those patents do not precisely model the stereochemistry of the canonical base triplets; nor do they precisely model the stereochemistry of their designed residues. Instead, they are directed to flexible non-native backbones that, upon triplex formation, are possibly capable of assuming locations acceptable for triplex formation. One concern with this approach is that backbones of greater flexibility than native sugar-phosphate backbones may suffer unacceptable negati

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